Intermediary Metabolism of Mycobacteria - NCBI

BACTERIOLOGICAL REVIEWS,_Mar., 1972, p. 65-108 Vol. 36, No. 1Copyright ( 1972 American Society for Microbiology Printed in U.S.A.

Intermediary Metabolism of MycobacteriaT. RAMAKRISHNAN,1 P. SURYANARAYANA MURTHY,2 AND K. P. GOPINATHANMicrobiology and Pharmacology Laboratory, Indian Institute of Science, Bangalore 12, India

INTRODUCTION ................................................. 66GROWTH OF MYCOBACTERIA ........ .......................... 67CARBOHYDRATE METABOLISM ........ ........................ 67Assimilation of Glycerol and Glucose ....... ...................... 67Glycolytic and Oxidative Pathways ....... ........................ 67Metabolism of Other Sugars ......... ............................ 68Endogenous Metabolism ............ ............................. 69Tricarboxylic Acid Cycle.................. 69

LIPID METABOLISM .............. .............................. 70Fatty Acid Oxidation .............. .............................. 70Biosynthesis of Fatty Acids .......... ............................ 70

Saturated straight-chain fatty acids ....... ..................... 70Unsaturated straight-chain fatty acids ...... ................... 72Branched-chain fatty acids ......... ........................... 72

Phospholipids................................................... 74Cord Factor..................................................... 75Mycosides A and B............................................... 75Lipopolysaccharide ............................................. 75

Effect of isoniazid on lipid metabolism ...... .................... 75Other Lipids.75

ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLA-TION ...................................................... 75

Preparation, Properties, and Composition of the Particulate (Mem-brane) Fraction ............... .............................. 75

Respiratory Chain............................................... 76Phosphorylation ................................................. 78

Sites of phosphorylation .......... ............................. 78Coupling factors............................................... 78Intermediates of oxidative phosphorylation at the naphthoquinone

level of the respiratory chain ........ ......................... 79Electron Transport in Other Mycobacteria ...... .................. 79Regulation of Electron Transport and Coupled Phosphorylation 79

OTHER ASPECTS OF CARBON METABOLISM ..... .............. 80Aromatic Compounds .............. .............................. 80Hydrocarbon Utilization ........... .............................. 80Oxidation of Phenolic Compounds ........ ........................ 80

NITROGEN METABOLISM .......... ............................. 80Nitrate Metabolism ................ .............................. 80Amino Acids and Amides ........... ............................. 81General ....................................................... 81Asparagine and aspartic acid ........ .......................... 81Glutamine and glutamic acid ........ .......................... 82Alanine ....................................................... 82Lysine ........................................................ 82Isoleucine and L-valine ............ ............................. 82Acetylated amino acids ........... ............................. 83

Paraaminobenzoic Acid ............ .............................. 83Carboligase ..................................................... 83

iOn sabbatical leave at: Department of Molecular Biol- 2Present address: Department of Biochemistry, Val-ogy, University of California, Berkeley, Calif. 94720. labhbhai Patel Chest Institute, University of Delhi, Delhi-7,

India.

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RAMAKRISHNAN, MURTHY, AND GOPINATHAN

PROTEIN SYNTHESIS ...........................................Ribosomes ......................................................

NUCLEIC ACID METABOLISM ..................................General .........................................................DNA and RNA ..................................................Purines and Pyrimidines .........................................

VITAMINS AND COENZYMES ...................................Nicotinic Acid and Nicotinamide Coenzymes ......................Other B Vitamins ...............................................Vitamin K ......................................................

MISCELLANEOUS ENZYME SYSTEMS ..........................MECHANISM OF ACTION OF ANTITUBERCULAR DRUGS.

Isoniazid ........................................................Other Antitubercular Agents .....................................

GENETICS OF MYCOBACTERIA .................................General .........................................................Lysogenic Conversion ............................................Transduction ....................................................Transfection ................................................Conjugation .................................................Mapping ........................................................

CONCLUDING REMARKS ........................................LITERATURE CITED .............................................

8384858585858686878788888889909090919292929294

INTRODUCTIONThe last comprehensive review devoted to

this topic was made in 1951 by Edson (81), andthe present-review is designed to cover workdone after that year. Since that time, however,two special reviews have appeared, one on bio-chemical properties of virulent and avirulentstrains of Mycobacterium tuberculosis byBloch (37) in 1960 and the other on the en-zyme systems in saprophytic and avirulentstrains of mycobacteria by Goldman (113) in1961. Metabolism in experimental tuberculosisand the structure of the cell wall and immuno-logical aspects of mycobacteria are not in-cluded, because, although a wealth of informa-tion has recently accumulated in these areas,they do not legitimately come within the scopeof this review.The genus Mycobacterium encompasses a

variety of organisms ranging from such patho-genic species as M. tuberculosis and M. para-tuberculosis and their mutants, avirulent,drug-resistant, etc., to the apparently nonpath-ogenic species such as M. phlei, M. smegmatis,and M. lacticola. The common propertieslinking these organisms include the similarityof the basic structure of peptidoglycans, myco-sides, and mycolic acids of their cell walls, thedegree of homology found among their deoxyri-bonucleic acids, the limited auxotrophy ofmost of them, and, with rare exception, theiracid-fastness. Only about half a dozen labora-tories in the world have been studying themetabolism of the pathogenic typical myco-

bacteria, and this is all the more surprisingsince tuberculosis is generally conceded to betoday the most important communicable dis-ease in the world. Hence, for the developmentof effective antimicrobials, basic data on themetabolism of the organism responsible ismost important.The three species of the genus Mycobac-

terium concerned in the tuberculosis of mam-mals and birds are M. tuberculosis, M. bovis,and M. avium, known as the human, bovine,and avian strains, respectively, and denotingchiefly the relative distribution of the bacte-rium in the respective hosts. The role of the"atypical" mycobacteria, such as M. intracel-lulaire, M. kansasii, and M. scrofulaceum, inthe spectrum of chest diseases is extremelyimportant. The problem of pulmonary infec-tion with atypical acid-fast mycobacteria hasbeen receiving considerable attention of late,since the number of reports of well-docu-mented cases of pulmonary disease, apparentlycaused by atypical mycobacteria, has beensteadily increasing for many years. In general,they exhibit poor susceptibility to antimi-crobial drugs, and many of them produce pig-ments during growth. Except for a report (99)on determination of dehydrogenase, practicallyno information is available on the metabolismof pathogenic atypical mycobacteria.Regarding M. leprae, various laboratories are

still trying to standardize growth conditionsfor this bacterium in a laboratory medium, sothat, if this is successful, biochemical tech-

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INTERMEDIARY METABOLISM OF MYCOBACTERIA

niques can be applied to unravel the mysteryof this organism. In recent years, however,there has been partial success in growing somestrains in a defined medium. Available meta-bolic information on this bacterium will becited at appropriate places in this review.A section is devoted to the mode of action of

the currently available antitubercular drugsbecause of its importance in both fundamentaland applied aspects. And, since the biochemistcannot ignore our developing knowledge ofgenetics, the genetic aspects of mycobacteriahave been dealt with briefly. For a fuller re-view of this aspect, reference is made here to aconvenient summary of knowledge emergingfrom the recent symposium edited by Juhaszand Plummer (181) on host-virus relationshipsin mycobacteria, nocardia, and actinomy-cetes.

GROWTH OF MYCOBACTERIAOne important reason that progress in the

work on the metabolism of M. tuberculosis hasbeen tardy is the slow growth of the organismon laboratory media; several investigators haveattempted to devise better media for itsgrowth. Some of the conditions for increasingthe rate of growth of M. tuberculosis are theuse of shake culture, an increase in the size ofinoculum, substitution of glutamate for aspara-gine as nitrogen source, and use of glycerol ascarbon source (44). A few growth factors havealso been reported for the slow-growing speciesof mycobacteria. Yamane (425) has isolatedfrom egg-yolk a crystalline growth-enhancingfactor which is heat-stable and is required inconcentrations of 0.01% and above. Rama-krishnan, Indira, and Sirsi (298, 299) reportedthe isolation of a heat-stable polysaccharidegrowth-promoting factor for M. tuberculosisfrom coconut water; this substance has anti-genic similarity to a component in the bacillus(249). Better known are the iron-chelatinggrowth factors, mycobactins from mycobac-teria themselves-a topic recently covered in aextensive review by Snow (336). Mycobactinspromote the growth of M. paratuberculosis andare unique in the sense that they are highlyspecific within a bacterial group. A recent re-port (155) that L-asparagine (which is com-monly used as the nitrogen source for thegrowth of M. tuberculosis) contains an isofla-vanoid as impurity and that the stimulatoryproperties ascribed to L-asparagine may be dueto this isoflavanoid is interesting and shouldencourage further work on the effect of variousisoflavanoids on the growth of slow-growingmycobacteria.

CARBOHYDRATE METABOLISM

Assimilation of Glycerol and GlucoseGlycerol is the primary carbon source em-

ployed in the culture of mycobacteria, thoughthe organisms can also use glucose. M. phleicells grown on glycerol medium and glucosemedium have identical rates of growth asjudged by protein and deoxyribonucleic acid(DNA) synthesis, but glycerol-grown cellshave, per unit volume of medium, consistentlya greater weight than glucose-grown cells (364).The increased weight of glycerol-grown cells isattributable to an increased lipid and polysac-charide content. There is also a basic differ-ence in the kinetics of uptake and utilizationof glucose and glycerol by M. phlei (365). Withglycerol, the rates of uptake, respiration, andassimilation are saturated at low substrateconcentration, whereas with glucose they donot show saturation even at high concentra-tions. These quantitative differences in theutilization of glycerol and glucose can accountfor the increased content of lipid and polysac-charide found in glycerol-grown M. phlei andprobably also in M. tuberculosis. By em-ploying "4C-labeled glycerol and analyzing thedistribution of the label, Edson et al., (82) con-cluded that, in M. butyricum, glycerol is de-graded to a-ketoglutarate and carbon dioxidethrough pyruvate. The presence of glycerol 3-phosphate dehydrogenase has been demon-strated in M. butyricum (156) and in M. bovisBCG (406). Further, 8-day-old cultures of thelatter organism grown on a rotary shaker donot possess any glycerol dehydrogenase ac-tivity. The phosphorylation of glycerol fol-lowed by dehydrogenation is therefore consid-ered to be the main route of glycerol metabo-lism in M. bovis BCG. Indira and Rama-krishnan demonstrated the presence of a nico-tinamide adenine dinucleotide phosphate(NADP)-dependent glycerol 3-phosphate dehy-drogenase and glycerol dehydrogenase in a 14-day culture of M. tuberculosis H37Ra (160)and a nicotinamide adenine dinucleotide(NAD)-dependent glycerol 3-phosphate dehy-drogenase in M. tuberculosis H37Rv (161). It ispossible that the appearance of glycerol dehy-drogenase activity in the former experimentsis due to the low oxygen tension, as has beenreported for Streptococcus faecalis (166). AnNADP-dependent glycerol 3-phosphate dehy-drogenase has also been noted in M. smeg-matis (326).

Glycolytic and Oxidative PathwaysThe glyceraldehyde 3-phosphate formed

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from glycerol is apparently metabolized fur-ther by conventional pathways. Qualitativelythese pathways in the virulent and avirulentstrains appear to be identical.Evidence for a functional glycolytic system

in the M. tuberculosis H37Ra. has been pre-sented (31), and the key enzymes of both theglycolytic and the hexose monophosphateshunt (HMP) pathways have been shown to bepresent in the glucose-grown cells of H37Rastrain (160, 161) and in the H37Rv strain (162,354). Le Cam, Madec, and Bernard (224), how-ever, could not detect glucose 6-phosphatedehydrogenase and 6-phosphogluconate dehy-drogenase in glycerol-grown cells of M. tuber-culosis, H37Ra, and they suggested that inthese organisms glycolysis is predominant overthe HMP pathway. In glycerol-grown cells ofM. phlei, however, the enzymes of both path-ways could be detected. The presence of aninhibitor in extracts of H37Ra strain whichmasks the activity of phosphofructokinase ac-tivity has also been reported (31).

Cell-free extracts of M. tuberculosis H37Rv,BCG, and M. avium exhibit aldolase activity(19). The enzyme activity is maximal in theextracts of H37 Rv, and the optimum pH is 8.6in the case of all the strains. Co2+, Fe2+, Zn2+,Ni2+, and Mn2+ as well as metal chelators ifrhibit the enzyme activity. Since the organismsgrow well in the presence of Fe2 , Zn2+, andMn2+, the significance of their inhibition ofaldolase is not clear. The individual enzymesof the glycolytic pathway and shunt in myco-bacteria have not been extensively purified.The effect of age of the culture on the activi-

ties of some enzymes of glycolysis and hexose-monophosphate pathways has been studied inM. phlei and M. tuberculosis H37Ra (224) andin M. smegmatis (326). In M. smegmatis, theactivity of all the enzymes with the exceptionof glycerol 3-phosphate dehydrogenase de-creases from the second day to the sixth dayand tends to increase again from the eighthday. In M. phlei and M. tuberculosis, however,the activities show an irregular pattern.The metabolism of the end product of gly-

colysis, i.e., lactate, has been worked out indetail in mycobacteria. The presence of lactateoxidative decarboxylase (which oxidizes lac-tate to acetate, carbon dioxide, and water) hasbeen reported in M. smegmatis, M. phlei, andM. stercoris and in M. tuberculosis H37Rv (67).This enzyme has been crystallized from M.phlei (357) and M. smegmatis (344). The en-zyme has a molecular weight of 30,000 to40,000, contains 6 to 8 moles of flavine mono-nucleotide (FMN) per mole of protein as its

prosthetic group, and catalyzes the oxidationof only L-lactate and a-hydroxy-n-butyrate. Itis inhibited competitively by D-lactate.Recent years have been marked by efforts to

use relatively simple isotope techniques todetermine the balance of alternative respira-tory pathways. To discover whether the ina-bility of the avirulent strain to multiply in tis-sues, where generally low oxygen tension pre-vails, is attributable to its more predominantlyoxidative metabolic pathways, Ramakrishnan,Indira, and Maller (300) studied the oxidationof glucose by M. tuberculosis H37Rv andH37Ra strains through the labeling of CO2formed from uniformly labeled glucose as wellas glucose _1_14C and glucose-6-' 4C. The glucosedissimilation in the avirulent strain takes placeto an equal extent via the glycolytic and oxida-tive pathways, whereas in the avirulent strain,the dissimilation is predominantly via the gly-colytic pathway, which accounts for 85% of theglucose utilized. By using essentially similarmethods, O'Barr and Rothlauf (271) estimatedthat about 97% of glucose is metabolized bydrug-susceptible M. tuberculosis through theglycolytic pathway, whereas strains resistantto streptomycin, p-aminosalicylic acid, andisoniazid metabolize glucose through thispathway to a slightly smaller extent. Moredetailed studies (159), also using glucose-2- 4Cand analysis by chemical degradation of thepyruvate formed, showed that, even in the avir-ulent strain, the glycolytic pathway domi-nates to the extent of about 74% of the glucosemetabolized. It must be admitted that, basedon the work of Katz and Wood (187), themethods used in the earlier-mentioned studiesare not free from errors in interpretation.

Metabolism of Other SugarsAlthough mycobacteria have been reported

to prefer glucose or glycerol over other carbo-hydrates as carbon source, they apparentlypossess enzymes to metabolize the latter.Kimura and Sasakawa (195) reported that anenzyme preparation from M. avium with pol-yol dehydrogenase activity utilized myo-inos-itol, D-mannitol, glycerol, propanediol-1, 2, andethylene glycol in the presence of NAD, but itis not clear whether this enzyme is identicalwith glycerol- or glycerol 3-phosphate dehydro-genase. Hey-Ferguson and Elbein (148) foundthat M. smegmatis grows much more rapidlyon mannose and fructose than on glucose andthat cells grown on these sugars have an en-zyme, D-mannose ketol isomerase. The enzymehas been purified 60-fold; it catalyzes the con-version of D-mannose to D-lyxose and fructose.

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The occurrence of a fl-D-galactosidase has beenreported in M. smegmatis (167). A transgluco-sylase which catalyzes the synthesis of treha-lose 6-phosphate from glucose 6-phosphate anduridine diphosphate-glucose has been studiedin M. tuberculosis H37Rv and H37Ra (236).The avirulent strain H37Ra contains also an

inhibitor which acts on the enzyme of both theavirulent and virulent strains. The inhibitionis noncompetitive. The inhibitor is an oligori-bonucleotide containing between six and ninepurine bases but no pyrimidine bases and hasbeen given the trivial name of mycoribnin.During purification, the transglucosylase losesits sensitivity to mycoribnin but gains sensi-tivity if the bicarbonate concentration of thepreparation is reduced to about 0.01 mM; bi-carbonate and mycoribnin are probably boundto the same site on the enzyme (237). Thetransglucosylase from brewer's yeast whenprepared free from bicarbonate is also inhib-ited by mycoribnin.

Lornitzo and Goldman (238, 239) havestudied the occurrence in M. tuberculosisH37Ra of a number of related polysaccharidescontaining 6-0-methyl D-glucose, as they may

act as precursors to polysaccharides containing6-0-methyl D-glucose and other methylatedsugars (239). Cell-free extracts of the organismcontain an enzyme which catalyzes thetransfer of the methyl group of S-adenosylme-thionine to a-glycerol phosphate, forming 3-methoxy 1, 2-propanediol (a-glyceryl methylether). The methoxy group is thus introducedat the triose level for incorporation into hex-oses and other sugars.A new type of kinase-inorganic polyphos-

phate: D-glucose 6-phosphotransferase-hasbeen reported in M. phlei (355). The enzymehas been partially purified and some of itsproperties have been studied; however, its rolein the overall metabolism of glucose, if any,has not been worked out.

Endogenous MetabolismThe endogenous metabolism of M. phlei has

been linked to the presence of glucose-6-phos-phate dehydrogenase functioning with reducedNADP (NADPH)-2, 6-dichlorophenol-indo-phenol reductase (353). The necessary sub-strate and coenzyme, i.e., glucose-6-phosphateand NADP, were found to be contained in thecell-free extract, and the only required addi-tion to activate the system is a suitable elec-tron aceptor. The substrates for endogenousmetabolism are identified as the phosphateesters of the glycolytic pathway, energy-richphosphates, and nucleotides in M. smegmatis

(306). The endogenous respiration in M. tuber-culosis is stimulated by uncoupling agents likedinitrophenol and azide, and inhibited by sulf-hydryl binding reagents and arsenite, indi-cating that the stored endogenous substratesare utilized via pathways similar to glycolysisand the tricarboxylic acid cycle (351). Furtherwork on the characterization of the endogenoussubstrates, especially in M. tuberculosis, wouldbe highly desirable.

Tricarboxylic Acid CycleThe first evidence for the occurrence of the

tricarboxylic acid cycle in mycobacteria wasprovided by Youmans, Millman, and Youmans(430), who showed that acetone-dried cells ofM. tuberculosis H37Rv oxidize all the interme-diates of the cycle. Goldman (106-111) has par-tially purified isocitric dehydrogenase, malicdehydrogenase, condensing enzyme (108),pyruvic oxidase, and pyruvic dehydrogenasesystem from M. tuberculosis H37Ra. For de-tails, the reader is referred to the review byGoldman (113). The individual enzymes of thetricarboxylic acid cycle and the glyoxylate by-pass are present in sonic extracts of the H37Rvstrain, and, except for malate dehydrogenase,all the other dehydrogenases investigated areNADP- but not NAD-dependent (350). A pow-erful reduced NAD (NADH) oxidase (121, 161,350) present in the extracts prevents even theNAD reduction unless it is carried out at a pHunfavorable to the activity of the oxidase. It isprobable that the presence of NADP-requiringdehydrogenases in M. tuberculosis guaranteesa constant high concentration of NADPH as areducing agent for its metabolic processes,especially for the biosynthesis of lipids, andthe presence of NADH oxidase in the organismensures that sufficient NAD is available as anoxidizing agent in these processes. In M. tu-berculosis H37Rv, NAD is present in doublethe concentration of NADP, whereas the re-verse is true for the corresponding reducedcompounds (121). The NAD content of H37Rais similar to the H37Rv strain, but NADP,NADH, and NADPH are present in almostequal proportions in H37Ra (223). M. tubercu-losis appears to be similar to M. phlei withrespect to its pyridine nucleotide coenzymecontent (399) in that both contain NAD andNADP, unlike M. butyricum which containsonly NAD in the oxidized form (183). The ratioof NAD to NADP is 6.0 in M. phlei and 2 to 3in M. tuberculosis (121, 223).The operation of the glyoxylate cycle in cell-

free extracts of M. tuberculosis H37Ra has alsobeen demonstrated, and the enzymes of the

VOL. 36, 1972 69


cycle, viz., isocitrate lyase and malate synthe-tase, have been purified (117). A glycine dehy-drogenase catalyzing the reductive aminationof glyoxylate to glycine has also been purifiedfrom this strain. The specific activity of all theenzymes of the tricarboxylic acid cycle in-creases with age of the culture up to 14 daysand decreases after 21 days; however, the ac-

tivity of isocitrate lyase increases continuouslywith age up to 28 days (265).

In addition to those already reported for M.tuberculosis H37Ra, some of the individualenzymes of the tricarboxylic acid cycle havebeen purified from other species of mycobac-teria. The occurrence of malate dehydrogenaseand malic enzyme in different species of my-

cobacteria has been investigated by ParvinKhan, Pande, and Venkatasubramanian (282).NAD-dependent malate dehydrogenase is highin M. phlei, considerably less in M. tubercu-losis H37Rv, and not at all detectable in M.smegmatis 607. Although NAD-dependentmalate dehydrogenase is not detectable in an-

other strain of M. smegmatis, NADP-de-pendent malate dehydrogenase is present inboth the strains (326). On the other hand,malic enzyme, which is present in all the spe-

cies of mycobacteria studied, is highest in M.smegmatis 607; this enzyme has been purified100-fold (283). The enzyme requires NADPand bivalent metal ions like Mn2+ or Mg2+ foractivity and is inhibited by sulfhydryl bindingagents. Substrate concentrations above 2.5 mminhibit the enzyme, but this inhibition can bereversed by increasing the Mg2+ concentration.Kimura and Tobari (196, 370) purified malatedehydrogenase from M. avium and showedthat the enzyme requires flavine adenine dinu-cleotide (FAD) and phospholipid for its ac-

tivity; phospholipid is apparently necessary tomake this particulate enzyme active.The intact cells of M. tuberculosis H37Rv

grown in vitro on laboratory media and in vivoin the lungs of infected mice show succinicdehydrogenase activity, but not succinoxidaseactivity (322). On the other hand, extracts ofonly the in vitro-grown bacilli have succinoxi-dase activity. Since permeability for succinateis apparently not a problem for these orga-

nisms, the conclusion drawn was that, in thepathogenic state, M. tuberculosis as present intuberculous animals is deficient in certaincomponents of the terminal respiratorysystem. A confirmation of this thesis by a sys-

tematic analysis of the electron transportsystem in this strain would be most inter-esting. Bekierkunst and Artman reported (34)that cell-free extracts of M. tuberculosis

H37Rv grown in the lungs inhibited lacticdehydrogenase of BCG extracts and succinoxi-dase activity of lung tissues, in agreement withthe earlier reports (323, 324) that the proper-ties of lung-grown organisms are different insome respects from those grown on laboratorymedia. However, the nature of the inhibitor inlung-grown tubercle bacilli has been estab-lished and identified as nicotinamide adeninedinuclease activity (20).Resting cells of mycobacteria grown on glu-

cose or glycerol do not oxidize most of the in-termediates because of permeability barrier.The intact cells of M. smegmatis grown onfumarate and acetate oxidize these intermedi-ates rapidly, and the organism may possesspermeases for fumarate and acetate (83). How-ever, no direct evidence for any permease hasbeen reported in mycobacteria.The fixation of carbon dioxide into malonate

by M. avium has been demonstrated (218); theenzyme, fractionated with ammonium sulfate,requires coenzyme A and Mn2+ for activity.

LIPID METABOLISMThe chemistry of the lipid constituents and

the lipid composition of different species ofmycobacteria are outside the scope of this re-view. The reader is referred to the several re-views on these aspects (27, 28, 225, 227) andthe paper by Goren (123a).

Fatty Acid OxidationThe work of earlier investigators gave some

clues that the catabolism of fatty acids inmycobacteria is through fl-oxidation, but firmevidence in favour of (-oxidation of fatty acidswas obtained by Goldman and his co-workers(104, 115) who showed that the cell-free ex-tracts of M. tuberculosis H37Ra possess bu-tyryl CoA dehydrogenase, enoyl hydrolase, (3-hydroxyacyl dehydrogenase, and fl-ketoacylthiolase. These enzymes have been partiallypurified; the characteristics of these enzymesare similar to those from the animal tissues.However, the prosthetic group (FAD) of thebutyryl dehydrogenase of M. tuberculosisH37Ra is easily dissociated, and the apoen-zyme does not bind the substrate. Addition ofFAD to the apoenzyme restores the enzymaticactivity (113).

Biosynthesis of Fatty AcidsSaturated straight-chain fatty acids. It is

now known that the steps involved in the bio-synthesis of straight-chain saturated fattyacids in mammalian tissues and in bacteria areessentially the same. However, unlike the fatty

70 BACTERIOL. REV.


acid synthetase from yeast and liver, the fattyacid synthetases from Escherichia coli andClostridium kluyveri have been resolved intoindividual components. One of these, the acylcarrier protein (ACP), has been purified andits role in fatty acid synthesis has been investi-gated (246). Kusunose et al. (218) demon-strated acetyl coenzyme A (CoA) carboxylaseactivity in the cell-free extracts of M. avium atabout the same time that the role of malonatein fatty acid biosynthesis was being resolvedby Wakil (392) in pigeon liver. The incorpora-tion of 14CO0 into malonate requires acetate(or acetyl phosphate), CoA, and a divalentmetal ion; adenosine triphosphate (ATP) in-hibits the reaction. The soluble enzyme, whichis partially purified, catalyzes the synthesis offatty acids from acetate-1-"4C in presence ofbicarbonate and ATP (CO2 has a stimulatoryeffect), and palmitic acid is the major product.However, the fatty acid synthetase from M.avium, unlike that of the avian and mam-malian tissues, forms from acetate, fatty acidsof longer chain length (up to C24) such asstearic, arachidic, behenic, and lignoceric acids(218, 219). In the presence of malonate, mostof the radioactivity from labeled acetate ap-pears in fatty acids higher than palmitic, andlignoceric acid is the major product. Synthesisof myristic and octanoic acids has also beenreported in the same organism (77).

Subsequently the demonstration of the syn-thesis of malonyl CoA from acetyl CoA and offatty acids from "4C-labeled acetate in M. tu-berculosis H37Ra, M. smegmatis, and BCGestablished the existence of malonyl pathwayof fatty acid synthesis in mycobacteria (284,287, 407). The fatty acids formed include C.,C89 C 16, C 18, C20, and at least two longer-chainacids in M. smegmatis and BCG and stilllonger-chain acids (C16 to C,20) in M. tubercu-losis H37Ra. The fatty acid-synthesizing sys-tems of M. tuberculosis H37Ra and M. smeg-matis 607 require ATP, CoA, Mn2+ or Mg2+,and NADPH (or NADPH generating system).The fatty acids synthesized are in the esteri-fied form. Surprisingly, in both the systems,NADH stimulates the incorporation of acetateinto fatty acids. Avidin inhibits the synthesisof fatty acids from acetate, and this inhibitioncould be reversed by biotin. Even high concen-trations (1.5 mg/ml) of avidin show only 80%inhibition of the synthesis of fatty acids fromacetate in M. smegmatis 607, and it was there-fore suggested that, in this organism, part ofthe fatty acid synthesis (about 20%) proceedsby the avidin-insensitive chain elongationprocess (284). The optimum pH for fatty acid

synthesis in M. smegmatis 607 is 8.6.The composition of the fatty acids synthe-

sized in M. tuberculosis H37Ra shows someinteresting features (287). No significant ra-dioactivity from labeled acetate is found instraight-chain, saturated, even-numbered fattyacids having less than 16 carbons or more than32 carbons, or in unsaturated and branched-chain fatty acids. In particular, neither tuber-culostearic acid nor oleic acid, present in theextracts, shows any radioactivity. Hexacosa-noic (C26) acid, which is considered to be in-volved in the synthesis of mycolic acids, is themajor product formed.

Recently, two fatty acid synthetases (systemI and system II) have been demonstrated in M.phlei (48, 251, 252). The first (system I) waspurified from the particle-free extracts of M.phlei grown in presence of 3H-f3-alanine as amarker for 4'-phosphopantotheine (48). Ra-dioactivity is found in the fatty acid synthe-tase, indicating that the M. phlei synthetase,like the fatty acid synthetases of liver andyeast, has a bound ACP-like component. It is amultienzyme complex and has a high molec-ular weight of 1.7 x 106. However, the M.phlei fatty acid synthetase differs from thefatty acid synthetases described so far in thefollowing properties. (i) It requires a heat-stable stimulating factor (SF) for its activity,and this stimulating factor does not appear tobe ACP or acetyl CoA. There is, however,some interdependence between acetyl CoAand SF concentrations required for optimalactivity. (ii) Its acyl transferase activity has amuch broader specificity. It uses not onlyacetyl CoA but longer-chain acyl CoA deriva-tives, such as octanoyl CoA and stearoyl CoA,as primers for malonyl CoA incorporation intolong-chain, even-numbered fatty acids (C,2 toC26). (iii) The fatty acids produced (C14 to C26)show a biphasic chain-length distribution, i.e.,C,, and C24 are the major acids produced, andthe others are in smaller quantities. (iv) It isunstable in solutions of low ionic strength(phosphate buffer 10 mM or less) and disso-ciates into smaller units. SF prevents suchdecay of activity. The ACP of M. phlei ispresent in a bound form (in the fatty acid syn-thetase multienzyme complex) and also in afree form (251, 252). The bound ACP can bereleased by alkaline hydrolysis. The ACP (M.phlei) has been purified, and its propertieshave been compared with that of ACP (E. colt)(251, 252). The ACP (M. phlei) is a low-molec-ular-weight, heat-stable compound, and itsamino acid composition is similar to that ofACP (E. colt). The former has no histidine and

71VOL. 36, 1972


has four proline residues compared to only onein ACP (E. coli).

In addition to the above synthetase, Blochand his coworkers (48, 252) observed the pres-ence in M. phlei of a second type (system II) offatty acid synthetase for chain elongation. Thisresembles the fatty acid synthetases of bac-teria in that it is totally dependent on the ad-dition of ACP from either M. phlei or E. coli.Its molecular weight is less than 250,000. Aninteresting property of this fatty acid synthe-tase is that it uses only palmitoyl-CoA or stea-royl-CoA, but not octanoyl-CoA or acetyl-CoA,for chain initiation.

As mentioned previously, the long-chainfatty acid synthesis may also be taking placeby an avidin-insensitive chain-elongationprocess in the crude extracts of M. smegmatis607 (284). The presence of two enzyme systemsfor the chain elongation of fatty acids in M.tuberculosis H37Ra has also been demon-strated (182, 393). In the first one, which is anavidin-insensitive mechanism, acetate-1- "4C isincorporated into a medium-chain (C,-C10)acid yielding a fatty acid, two carbons longerthan the starting material (Cn+2 acid from aCn acid and acetate). In this type of reaction,the stimulation is maximal with octanoate asthe substrate. Similar elongation was observedwith palmityl CoA also. For this reaction ATP,CoA, a metal ion, and NADH are necessary.ATP is not necessary when fatty acyl CoA andacetyl CoA are used. The elongation appears tooccur by the addition of a single C2 unit to thecarboxyl end of the acceptor fatty acid. Thesecond reaction is a direct condensation of twoor more molecules of fatty acid (Cn) to give alonger chain (C2n or C3n) acid. For this reac-tion, a metal ion, ATP, and NADH are neces-sary. From octanoyl-1- 4C CoA, radioactivityis found in palmitate and tetracosanoate. Sim-ilarly, eicosanoate is the product with decanoylCoA as the substrate. Matsumara, Brindley,and Bloch (252) reported, in M. phlei, a fattyacid synthetase which requires an externalsupply of ACP. Palmitoyl CoA and stearoylCoA, when used as primers, show incorpora-tion of malonyl CoA. Octanoyl CoA is found tobe less active.Unsaturated straight-chain fatty acids.

Two pathways-aerQbic and anaerobic-areknown to be involved in the biosynthesis ofmonounsaturated fatty acids (186, 222, 231,276, 305). Of these two, the aerobic pathwaywhich brings about dehydrogenation of the cor-responding saturated acids in presence of ox-ygen and NADPH was demonstrated in theparticulate fraction of M. phlei (94). Unlike

the other bacteria having the aerobic pathway,the M. phlei system requires Fe2+ and a fla-vine (FAD or FMN), in addition to NADPHand oxygen, for the synthesis of A9-unsatu-rated fatty acids. NADPH is required in sub-strate amounts and flavine in catalyticamounts. Stearoyl CoA and palmitoyl CoA aredesaturated to A9-octadecenoic and A9-hexa-decenoic acids, respectively. The formation ofthe cis-A9-unsaturated acids in the cell-freesystem is surprising because, when M. phleicells are grown on palmitic acid-l- "C, theproduct is primarily the A`-isomer of hexa-decenoic acid (319). The difference betweenthe products obtained with the intact cells andcell-free system in the same organism remainsto be explained. The synthesis of unsaturatedfatty acids by the particulate fraction is inhib-ited by sulfhydryl reagents and is irreversiblylost on dialysis or treatment with ammoniumsulfate. A recent report (29) that about 5% ofthe lipids of M. phlei contain polyunsaturatedfatty acids calls for an evaluation of the bio-synthetic process of this group of fatty acids.Branched-chain fatty acids. The

branched-chain fatty acids which occur inmycobacteria may be divided into threegroups: (i) acids with one methyl branch in themiddle of the chain (tuberculostearic acidgroup), (ii) acids with three or four methylbranches at the carboxyl end (phthienoic andmycocerosic acid group), and (iii) acids withlong branches and one or two oxygenated func-tions (mycolic acid group).Methyl branched acids. The biosynthesis of

tuberculostearic (10-methyl stearic) acid by M.phlei (Fig. 1) proceeds by dehydrogenation ofstearic acid to oleic acid and then, by additionof a C1 unit from methionine (in the form of S-adenosylmethionine). Similarly, when palmiticacid is the substrate, the product is methyl

CH3(CH2) ,,COOHStearic

I-2H

CH3(CH2)7CH = CH(CH2)7COOHoleic

C from S-adenosylmethionine

CH3(CH2)7CH-CH2(CH2)7COOHCH3

Tuberculostearic

FIG. 1. Biosynthesis of tuberculostearic acid.

72 BACTERIOL. REV.


palmitate (232, 319). The source of the C-methyl group in tuberculostearic acid is shownto be methionine in M. tuberculosis H37Raalso (226). However, using whole cells of M.smegmatis and cell-free extracts of M. phleiand with methyl-labeled methionine, Jaure'qui-berry et al. (169, 170) found that in the methylgroup of the branched fatty aqid only two ofthe three hydrogens are derived from the me-thionine methyl group. In support of this ob-servation, 10-methylene stearic acid is identi-fied to be an intermediate in the conversion ofoleic acid to tuberculostearic acid: Further,when M. phlei cells are incubated with labeled10-methylene stearic acid, there is radioac-tivity in tuberculostearic acid. By mass spec-trometric studies with deuterated compounds,Lenfant, Audier, and Lederer (229) showedthat a shift of H occurs from C10 of oleic acidto C9 during the methylation step. Similarresults were reported by Bristol andSchroepfer (49). The conversion of oleic acid to10-methylene stearic acid and the reduction ofthe latter to tuberculostearic acid takes placeonly on the endogenous phospholipid sub-strates (phosphatidylglycerol, phosphatidyl-inositol, and phosphatidylethanolamine), andexternally added phospholipids do not haveany effect (3). However, when partially puri-fied enzyme preparations are used, exogenousphosphatidylethanolamine stimulates the syn-thesis of alkylated fatty acid derivatives (4).The reduction step requires NADPH but noNADH as the cofactor, and the following reac-tions are suggested: (i) Oleyl phospholinid +S-adenosylmethionine - 10-methylene stearylphospholipid + S-adenosylhomocysteine; (ii)10-methylene stearyl phospholipid + NADPH+ H+ - 10-methyl-stearic acid + NADP. Thisenzymatic alkylenation occurs on the fattyacids at position 1 or 2 of the glyceride mole-cule (5). In addition, a soluble enzyme systemfrom M. phlei capable of esterifying fatty acids(carboxyl group alkylation) has also been de-scribed (6); oleic acid is the most effectivefatty acid acceptor and S-adenosyl methionineis the methyl donor.

Campbell and Naworal (58a) suggested thatleucine and isoleucine can serve as "starters"in the biosynthesis of branched-chain fattyacids, as in other bacteria (186, 222, 231, 276,305). They also postulated a general schemefor the synthesis of all methyl branched fattyacids involving the participation of a methyltransferase. This scheme, however, needs ex-perimental support.

Multiple methyl branched acids (phthienoicand mycocerosic acids). Yet another pathway

seems to be operating for the biosynthesis ofmycocerosic acids and phthienoic acids, whichare fatty acids with multiple (3 or 4)-branchedmethyl groups, of which 2,4,6, 8-tetra-methyl-octacosanoic acid in a typical example.

In the extracts of M. smegmatis, an avidin-sensitive carboxylation of propionyl-CoA tomethylmalonyl CoA has been observed, andthe propionyl CoA carboxylase therefore mayhave a role in the biosynthesis of branched-chain fatty acids (341). The whole cells of M.tuberculosis H37Ra synthesize 2,4,6, 8-tetra-methyl octacosanoic acid from a C20 straight-chain acid by the successive addition of fourmolecules of propionic acid (101). The conden-sation is on the a-position of the propionatemolecule (102, 186). In M. bovis also, propio-nate-i-1 4C is incorporated into mycocerosicacids (427).

Phthienoic acids, present only in virulent M.tuberculosis, are similar to the mycocerosicacids but have trans a-# unsaturation and arebelieved to be synthesized by similar mecha-nisms (102, 186, 226). Multiple-branched (7 to8 methyl branches) carboxylic acids from my-cobacterial sulfolipids have been recently char-acterized (124), but their biosynthesis is un-known.Mycolic acids. Biosynthesis of a-smegma-

mycolic acid (Fig. 2) in the cells of M. smeg-matis grown in presence of tetracosanoic acid-1- 14C and 14C-methionine seems to involveincorporation of tetracosanoic acid moleculesas such into the carboxyl terminal (24 carbons)and methyl group of methionine into themethyl branch (87). When palmitate-1-14C isused in the medium, radioactivity is incorpo-rated into the 60-carbon mycolic acids (211).The mycolic acids of M. tuberculosis and M.bovis have more branched chains (the branchchain at the a-position derived probably fromhexacosanoic acid) and additional oxygenfunctions such as hydroxyl, methoxy, or car-boxy group. The biosynthesis of these acids isnot yet fully understood (27, 221, 225). In thisconnection, it is interesting to recall that thefatty acid synthetases of different species ofmycobacteria are capable of synthesizing notonly C16 or C18 acids but also C24 and C26acids, which in turn may serve as buildingblocks for the biosynthesis of more complexmycolic acids. However, the biosynthesis ofmycolic acids using cell-free enzymes willthrow further light on the details of the mecha-nism. Phthiocerol, a methoxydiol present inM. tuberculosis, is synthesized starting fromtetracosanoic acid; methionine serves asmethyl donor (100, 186).

73VOL. 36, 1972


CH4(CH2) ,7CH = CH(CH2),8CH = CH-CH(CHJ),7-CHOH-CH-COOH

CH3FIG. 2. Structure of a-smegmamycolic acid.

Phospholipids

Interest in the phospholipids of mycobac-teria, which were originally described by An-derson (13), has in recent years been revived,and their immunological role is being activelystudied. The structure of the mycobacterialphosphatides has been recently reviewed (281).The phosphatides of mycobacteria show a

high turnover. In M. smegmatis 607 exposed to32p1 for a short time, lysophosphatidylethanol-amine and phosphatidylinositol undergo rapidturnover when compared with other phospha-tides (343). There is a sudden loss of radioac-tivity in lysophosphatidylethanolamine, butthe radioactivity in phosphatidylinositol in-creases for 1 hr. In growing M. phlei cells, phos-pholipids have an appreciable turnover (7) but,in contrast to M. smegmatis 607, cardiolipinhas the highest turnover rate, whereas phos-phatidylinositol oligomannoside and phospha-tidylethanolamine have low turnover rates. It ispossible that these variations are due either tothe difference in the species of mycobacteriainvestigated or technique used. Incorporationof 32P into the lipids of M. tuberculosis H37Rarequires at least 24 hr (165).The biosynthesis of phosphatidylinositol

mannosides in the cell-free extracts of M.phlei proceeds in a manner which is not yetcompletely understood (45, 46, 151). Guanosinediphosphate mannose (GDP-mannose) is themannose donor and phosphatidylmyoinositol isthe acceptor; the major products are threephosphatidylmyoinositoldiamannosides. Theseare designated as dimannophosphoinositidesA, B, and C, which differ from one another inthe number of acyl groups contained (4, 3, and2, respectively). It is assumed that two of thefatty acids are on the glycerol moiety and therest (in A and B forms) are attached to avail-able hydroxyls of mannose or myoinositolmoieties. The enzyme catalyzing the biosyn-thesis of the dimannosides is present both inthe particulate and supernatant fractions, butthe former has higher activity than the latter.The enzyme has been solubilized from the par-ticles by treatment with acetone; the solubi-lized enzyme shows a requirement for externalphosphatidylmyoinositol. The enzyme is in-hibited by Tween 80; Mg2+ stimulates the ac-

tivity at low concentrations but inhibits athigh concentrations. However, the particulateenzyme uses the endogenous phosphati-

dylmyoinositol itself as an acceptor of GDP-mannose-14C. The particulate fraction of M.phlei has another enzyme system which acyl-ates, in presence of ATP, CoA and the fattyacids or fatty acyl CoA, the dimannophos-phoinositides (46). The fatty acids incorpo-rated include not only palmitic acid, which isnaturally present in the phosphatides, but alsomyristic, stearic, and oleic acids. The incorpo-ration of tuberculostearic acid is only in traces.However, the recent demonstration that thebiosynthesis of tuberculostearic acid from oleicacid occurs in the presence of phospholipids(5) is of interest. In the interconversion ofdimannophosphoinositides, the formationof polyacyldimannophosphoinositides (havingmore than four fatty acids) is also implicated(46). Similar results were obtained by theabove authors with BCG. The pentamanno-phosphoinositide is expected to be formed byfurther addition of more mannose units to themannose already attached at position 6 of themyoinositol ring of the dimannophos-phoinositide.

In cell-free extracts of M. tuberculosisH37Ra, the particulate transmannosylase en-

zyme system has two independent reactions(356): one for the synthesis of phosphatidyl-inositomonomannoside (from phosphatidyl-myoinositol and GDP-mannose) and the otherfor the phosphatidylmyoinositoldimannoside(from phosphatidylmyoinositolmonomannosideand GDP-mannose). This conclusion is basedon the observation that, when the particulatefraction is incubated with GDP-mannose (i.e.,using endogenous acceptor): (i) radioactivityis observed both in the mono- and di-manno-sides; and (iii) radioactivity of the dimannosideis in the mannose moiety attached to the 6-position of the myoinositol. In the above reac-tion, the major product is a phosphorylatedisoprenylalcohol, the structure of which wasproposed as mannosyl-1-phosphoryl decapre-nol (357).Two phospholipases-phospholipase A and

lysophospholipase-are present in the mem-

brane fraction of M. phlei (277). Phosphatidyl-ethanolamine, phosphatidylcholine, and cardi-olipin serve as substrates, but the former ishydrolyzed more rapidly. The pH optima are

5.1 to 5.3 and 4.0 to 4.2, respectively. Ac-cording to Ono and Nojima (278), phospholi-pase A is also a regulatory enzyme with Fe3+as a negative effector. The purified phospholi-

C 22H45

74 BACTERIOL. REV

INTERMEDIARY METABOLISM OF MYCOBACTERIA7

pase A is inhibited by ferric ion (0.12 mM Ki).The substrate (phosphatidylethanolamine) sat-uration curve shows a sigmoid shape. The en-zyme undergoes reversible interconversions. Inpresence of Fe3 , it exists in an inactive pre-cipitate form (aggregate) but assumes an activesoluble form on the addition of ascorbic acid.The recent demonstration of the presence of

phosphonolipids and glyceryl ethers in myco-bacteria and the observation that the amountof these constituents varies in different speciesof mycobacteria make the study of their me-tabolism interesting (62, 296). There is someevidence that M. phlei can slowly utilize 2,3-dihydroxypropylphosphonate for growth (253).

Cord FactorCell-free extracts of M. tuberculosis H37Ra

synthesize trehalose-6-phosphate (a precursorfor cord factor) from glucose-6-phosphate anduridine diphosphate glucose (116). Mycoribnin,an oligoribonucleotide, containing adenine andguanine is a noncompetitive inhibitor of thistransglycosylase reaction.

Mycosides A and BThe biosynthesis of the aglycon (a phenol

glycol) of the mycosides A and B has been in-vestigated by Gastambide-Odier, Sarda, andLederer (103). The phenolic ring of the phenolglycol comes from tyrosine and the methylbranches from propionic acid.

LipopolysaccharideThe cell-free extracts of M. phlei catalyze

the transfer of methyl groups from S-adeno-sylmethionine methyl-1 4C to endogenous ac-ceptor yielding a labeled lipopolysaccharide(88). This polysaccharide methyl transferaseexhibits a pH optimum of 7.0 and an apparentKm of 0.1 mm for S-adenosylmethionine whenamylooligosaccharides are used as the accep-tors.

Effect of isoniazid on lipid metabolism.The mode of action of antitubercular drugs hasbeen discussed in another section of this re-view. However, the effect of isoniazid on lipidmetabolism is pertinent. The elegant work ofBrennan, Rooney, and Winder (47) shows thatthe methods used for extraction of lipids andtheir fractionation influence the results ob-tained. Isoniazid produces three distinct ef-fects on the lipid metabolism of BCG. (i) Itreduces the incorporation of radioactivity from"4C-glycerol into ether-soluble bound lipidslike mycolic acids and phospholipids, (ii) itdecreases the amount of longer-chain acids(above C,6) and increases the amount of

shorter chain acids, and (iii) it diminishes theamount of triglyceride extracted with ethanol-ether in the classical Anderson-type procedure,leaving an increased amount of the chloro-form-soluble and bound lipids. This redistribu-tion effect is masked when a more vigorouslipid extraction procedure of Brennan andBallou (45) is followed. This was, therefore,considered to be due to alteration in the prop-erties of the cell wall as a result of inhibition ofmycolic acid synthesis by isoniazid (408).Singhvi and Subrahmanyam (327) also ob-served that both isoniazid and dihydro-streptomycin inhibit the incorporation of 3P,into all the major phospholipids of M. avium.

Other LipidsAlthough cholesterol could not be detected

in mycobacteria, the organisms have theability to decompose cholesterol (157, 158,200). M. smegmatis is capable of esterifyingsome hydroxy sterols with fatty acids and suc-cinic acid (320, 321).

ELECTRON TRANSPORT ANDOXIDATIVE PHOSPHORYLATIONStudies on electron transport and oxida-

tive phosphorylation have been carried outextensively with M. phlei by Brodie and hiscolleagues (51, 52, 56), but not much is knownabout other species of mycobacteria.

Preparation, Properties, and Compositionof the Particulate (Membrane) FractionElectron transport activity in M. phlei is

localized in the membrane or particulate frac-tion (52, 55, 397, 398). Lysozyme treatment,which has yielded from Micrococcus lysodeik-ticus (164) a single fraction exhibiting bothoxidation and phosphorylation, has not beenquite successful in lysing mycobacteria unlesslysozyme is included in the growth medium forseveral hours before harvesting the cells (1,257, 367, 403). Relatively more harsh methodssuch as sonic treatment (see Table 1) em-ployed in the case of other microorganisms(154, 185, 288) have been used for mycobac-teria. Consequently in M. phlei, as in manyother bacteria, both particulate and solublesupernatant fractions are necessary for elec-tron transport and oxidative phosphorylation.Recently in the laboratory of one of us, it hasbeen possible to obtain a particulate fractionfrom M. smegmatis which oxidizes malate (bythe nicotinamide nucleotide pathway) andshows coupled phosphorylation (296). Themethod consists in treating the cells with lyso-zyme, subjecting them to osmotic shock, and

75VOL. 36, 1972


TABLE 1. Methods used in the preparation of cell-free extracts

Organism Methods in disintegration Activities demonstrated Reference no.

Mycobacterium phlei ... Sonic oscillation Enzymes of the electron transport 48, 54, 149, 397chain, oxidative phosphorylationand tricarboxylic acid cycle; fattyacid synthetase

M. smegmatis .......... Grinding with glass NADH-diaphorase, enzymes of the 61, 257, 326powder, sonic oscilla- tricarboxylic acid cycle and glyco-tion and treatment lytic pathway; NADH- and cyto-with lysozyme chromic-oxidases; malate oxida-

tion; phosphorylation

M. avium .. .. Grinding with sea sand Enzymes of fatty acid synthesis 219

M. tuberculosis H37Ra.. Grinding with alumina, NADH- and NADPH-oxidases, en- 114, 161, 420colloid mill and sonic zymes of glycolysis and the tricar-oscillation boxylic acid cycle

M. tuberculosis H37Rv.. Sonic oscillation Enzymes of the tricarboxylic acid 121, 162, 272, 350cycle, glycolytic, glyoxylate andhexose monophosphate pathways,NADH- and NADPH-oxidases,NADase and NADH-diaphorase

M. bovis BCG ......... Grinding with quartz Fatty acid synthetase and acetyl 217, 380, 408sand or alumina, and CoA carboxylase; isolation of ri-high pressure disrup- bosome and their proteosynthetiction ability

aAbbreviations: NADH, nicotinamide adenine dinucleotide (reduced form); NADPH, nicotinamide ade-nine dinucleotide phosphate (reduced form); NADase, nicotinamide adenine dinuclease; CoA, coenzyme A.

sonically treating them for a very brief period.The membranes isolated from M. smegmatisby Cesari et al. (61) and Mison et al. (257) con-tain NADH oxidase or diaphorase activity, butnothing is known about their phosphorylation.The M. phlei particles-70 to 180 nm long-

swell in hypotonic solutions and tend to clumpin hypertonic solutions (55). They containsome dehydrogenase (most of them are in thesupernatant fraction), NADH (exogenousNADH) oxidase, NAD, vitamin K9H, cyto-chromes b, c, a, a3, and o, nonheme iron, andlipids (22, 55, 76, 96, 216, 308, 398, 399). Thepresence of phospholipids also has been re-ported in M. tuberculosis H37Ra (114). Thecytochrome composition of different species ofmycobacteria reveals some interesting fea-tures. Cytochromes b, c, and a are common toall the species of mycobacteria grown in vitro,namely M. smegmatis, M. phlei, BCG, M.avium, M. tuberculosis H37Ra, and M. paratu-berculosis (52, 188, 217). In addition to thesecytochromes, M. smegmatis and M. phlei con-tain a carbon monoxide-binding pigment sim-ilar to cytochrome o (217, 308). However, cyto-chromes could not be detected in BCG grownin vivo and in M. leprasmurium isolated from

leprous nodules (217).

Respiratory ChainUsing techniques similar to those adopted

with mitochondria, Asano and Brodie (22) de-termined the sequence of the components ofthe respiratory chain. The respiratory chainshown in Fig. 3 is based on the scheme givenby Brodie and Adelson (52) incorporating theresults of subsequent studies of Brodie and hisco-workers (212, 216, 279, 308). Both oxidationand coupled phosphorylation, with malate andother NAD-linked substrates, require the par-ticulate as well as the supernatant fractions(21, 22). With succinate as the substrate, parti-cles alone show some oxidation and associatedphosphorylation; addition of the supernatantfraction, however, stimulates both oxidationand phosphorylation (21, 50). In the reconsti-tuted system containing both the particulateand supernatant fractions, cytochromes b, c, a,+ a3,, and o are reduced with succinate, malate,and fl-hydroxybutyrate as the substrates.Thus, both the succinate and NAD-linkedpathways converge at cytochrome b level, andthen the electrons flow cytochromes b to c to a+ a3 and to oxygen. The exact location of cy-

76 BACTERIOL. REV.


SUBSTRATE ALATEASCORBATE

SOLUBLE TMPDNADP LINKED F~ D

SUBSTRATE PqNAD INL AVO -K9 H - b-~-c c _ a 0a3 OXYGEN

LIGHT )SUCCINATE FLAVO- -a SENSITIVE - NON-HEML IRON

PROTEIN COMPONENT

SOLUBLESUCCINATEFACTOR

FIG. 3. Respiratory chain in Mycobacterium phlei. KII, Naphthoquinone vitamin K.H; b, cl, c, a, anda3, the cytochromes; TMPD, tetramethylparaphenylenediamine.

tochrome o in the chain is not yet determined(308). Before cytochrome b, the NAD and thesuccinate segments show some differences inaddition to specific flavines.The naphthoquinone, vitamin K9H, is con-

sidered to play an important role in the NAD-linked pathway and the malate-vitamin Kreductase pathway (shown in Fig. 3 as malateFAD pathway) for the following reasons (51,52, 56, 135). (i) Both oxidation and phosphoryl-ation are lost with all the substrates on expo-sure of the particulate and supernatant frac-tions to near ultraviolet light (360 nm), whichis believed to destroy the natural naphthoqui-none but does not apparently affect the othercofactors or the structural integrity of the par-ticulate fraction. (ii) If the natural naphthoqui-none or vitamin K, or some of its homologuessuspended in phospholipid are added to suchan irradiated system, both oxidation and phos-phorylation are restored with NAD-linked sub-strates and malate-vitamin K reductasepathway but not succinate. (iii) Under phos-phorylating conditions, incorporation oftritium from the medium occurs only into vi-tamin K1, but not into 2,3 dihydrophytyl vi-tamin K, or lapachol. In view of the fact thatFAD is required for quinone reduction in irra-diated particles, vitamin K appears to be lo-cated between the flavine and cytochrome b.However, studies by Di Mari et al. (72) andSnyder and Rapoport (337) have clearly indi-cated that, in the irradiated M. phlei system,tritium incorporation into vitamin K, is 0.1%or less.The succinate segment is more sensitive to

light (360 nm) than is the NAD segment. Nei-ther naphthoquinones nor benzoquinones re-store succinoxidase activity lost by irradiation(53), but addition of fresh supernatant fluid or

extract of boiled whole cells restores 50 to 60%of the original activity lost by irradiation (212).The protein-bound succinate factor is light-sensitive but, after partial purification, it isthermostable and resistant to irradiation. Itacts at a site between flavine and cytochrome(212). A similar factor is present in rat livermitochondria (212, 213). The cyanide-sensitivemetal in the succinate chain has been shown tobe nonheme iron; it is located between thelight-sensitive component and cytochrome b(215, 216). Krishna Murti et al. (213) have suc-ceeded in dissociating succinate dehydro-genase (SDH) from the particles by alkali ex-traction and recombining the fresh SDH (richin labile sulfur and nonheme iron) with alkali-treated particles.

In addition to the NAD and succinate path-ways, a third pathway has been shown to occurin M. phlei, which accounts for malate oxida-tion requiring naphthoquinone and FAD butnot NAD and entering the main respiratorychain at the naphthoquinone level (22). Theenzyme responsible for this reaction, malate-vitamin K reductase, is present both in theparticulate and in the supernatant fractions.The latter has been used as a convenient sol-uble system for studying the mechanism ofphosphorylation in M. phlei (26, 347, 349), un-like the NAD-independent malate dehydro-genases reported in other sources (66, 92, 235,370). In 2n-nonyl hydroxyquinoline N-oxide(NHQNO)-blocked particles, with ascorbate inpresence of tetramethyl paraphenylene-dia-mine (TMPD) as the substrate, the electronsenter at cytochrome c level and then flow tocytochrome (a + a,). However, with succinateas the substrate, the electrons bypass cyto-chrome b and reenter the respiratory chain atcytochrome c level (212, 279). The role of elec-

77VOL. 36, 1972

RAMAKRISHNAN, MIJRTHY, AND GOPINATHAN

tron-transport component with NADP-likeactivity isolated from M. phlei by Sutton (352)needs further study.

PhosphorylationAs in the case of other bacteria, the P/O

values obtained in M. phlei are lower thanthose reported with mitochondria. In the re-constituted system wherein particulate frac-tion and ammonium sulfate fractionated su-pernatant fluid are used, the P/O values withthe malate-vitamin K reductase and succinatechains are between 0.4 and 0.8, whereas, withthe NAD-linked substrates f3-hydroxybutyrateand ethanol, the values are 1.11 and 1.23, re-spectively. The low P/O values are attributedto loss of sites of phosphorylation and the pres-ence of nonphosphorylating electron-transportbypass reactions (23). The bypass mechanismsappear to play a role in the oxidation of exoge-nous NADH or NADPH with which the elec-trons can be transferred directly to oxygen (en-tirely bypassing the respiratory chain) orreenter the chain at a higher oxidation reduc-tion level, depending on the electron acceptorused (23, 24, 38, 39, 54, 56, 348). The phospho-rylation is sensitive to uncoupling agents likedinitrophenol, m-chlorocarbonyl cyanide phen-ylhydrazone, pentachlorophenol, dicumarol,and atebrin but not to oligomycin (23).

Sites of phosphorylation. Brodie and co-workers (reviewed by Brodie and Adelson, 52)demonstrated three sites of phosphorylationassociated with the respiratory chain in M.phlei. In cell-free systems of M. phlei, the P/Ovalues for the oxidation of succinate or malate(using FAD-mediated malate-vitamin K re-ductase) are similar and range from 0.4 to 0.8,whereas the same system gives P/O valuesgreater than 1.0 when NAD-linked substrateslike f,-hydroxybutyrate or ethanol are oxidized.This indicates the presence of one phosphoryl-ation site on the respiratory chain betweenNAD and the point of interaction of succinateor malate (FAD)-reduced vitamin K, and an-other on the oxygen side of this point. Furtherproof of the first site of phosphorylation comesfrom the observation that the phosphorylationassociated with the oxidation of an NAD-linked substrate like ,B-hydroxybutyrate inpresence of lapachol is sensitive to amytal,whereas the oxidation of exogenous NADH inpresence of lapachol is insensitive to amytal.By using the M. phlei system irradiated withUV light (360 nm), which destroys both oxida-tion and phosphorylation, and studying therestoration of these two activities with vitaminK1 and some of its analogues, it has been

shown that the second site of phosphorylationis in the quinone-cytochrome b region of thechain. In presence of NHQNO, which blocksthe respiratory chain at cytochrome b, the oxi-dation of ascorbate in presence of TMPD(which enters the chain at cytochrome c level)is accompanied by phosphorylation sensitiveto dinitrophenol. This indicates the presenceof the third site of phosphorylation betweencytochrome c and oxygen. Thus, in the case ofM. phlei, the number and location of the cou-pling sites appear to agree generally with thoseof mammalian mitochondria (23, 52).

Coupling factors. A factor which showsphosphorylation when added to purified non-phosphorylating malate-vitamin K reductaseand which stimulates phosphorylation coupledto malate oxidation when added to particleswashed in the absence of magnesium has beenpurified from the supernatant fraction of M.phlei (347, 349). Subsequently, four factorscalled bacterial coupling factors (BCF, toBCF4) were isolated from M. phlei (40, 149,150). Two of them (BCF, and BCF4) are fromthe particulate fraction. BCF4 separates as awhite turbid layer when the particles are sub-jected to sucrose density gradient centrifuga-tion in the absence of Mg2+ (149). When thebottom or "heavy" layer (containing most of theNADH oxidase activity and cytochromes) andBCF, are recombined in presence of Mg2+ andsoluble coupling factors, phosphorylationcoupled to NADH oxidation occurs. BCF, isessential for phosphorylation with all the sub-strates and therefore common to all the sitesof phosphorylation (149). BCF, is released fromthe particles by urea treatment (150) and ap-pears to participate in reactions common toboth succinate and NAD-linked pathways.This, however, is not effective with exogenousNADH or ascorbate-TMPD and is differentfrom the soluble coupling factors (150). Boginet al. (40) purified (from the supernatant frac-tion) BCF2 and BCF, which operate at differentphosphorylating sites. The coupling factors,BCF,, BCF2, and BCF3, seem to be site-spe-cific because they are required in addition toBCF, for different sites (40, 149, 150). The puri-fied coupling factors, BCF,, BCF2, and BCF3,are proteins and contain some adenosine tri-phosphates, adenylate kinase, 14C-ADP-ATP-,and 32Pi-ATP exchange activities. They have nosignificant effect on the level of oxidation. Thecoupling factors from M. phlei and beef heartmitochondria are interchangeable (42), andthere is interaction between the electron trans-port particles of M. phlei and of mammaliansystems after cytochrome b and before cyto-

78 BACTERIOL. REV.


chrome c (39). Treatment of particles with ei-ther heat or trypsin or a combination of bothstimulates phosphorylation (41, 43). The signif-icance of these treatments vis a vis the role ofcoupling factors in phosphorylation is not yetfully known.Intermediates of oxidative phosphoryla-

tion at the naphthoquinone level of the res-piratory chain. The mechanisms involved inthe formation of ATP from reduced naphtho-quinone have been reviewed (51). Watanabeand Brodie (394) reported the formation of aquinol phosphate similar to, but not identicalwith, the 6-chromanyl derivative, of vitamin K,(25, 199, 315). Since there is no significant in-corporation of tritium from the medium into thequinone, the quinone methide mechanismsmay not be operating, although chromanolformation could still be possible (72, 337).

Electron Transport in Other MycobacteriaSonic extracts (10,000 x g supernatant) of

M. tuberculosis H37Rv have NADH oxidaseand NADPH oxidase activities, NADH andNADPH cytochrome c reductases and NADHdiaphorase and NADPH diaphorases (350).These extracts, however, oxidize citrate, isocit-rate, and cis-aconitate only in the presence ofan artificial electron acceptor like phenazinemethosulfate or menadione. This may meanthat some component(s) of the electron trans-port chain might have been destroyed duringthe sonic treatment (350). It is also possiblethat part of the respiratory chain might havebeen bypassed in the electron transport asso-ciated with these substrates.

In M. tuberculosis H37Ra, NADH oxidaseand NADPH oxidase activities have beendemonstrated in the sonic extracts (161, 272).Goldman and his co-workers have studied ingreater detail the NADH oxidase activity ofthe particulate fraction of M. tuberculosisH37Ra (118, 188, 273, 325, 418). NADH oxi-dase activity is not coupled to phosphoryla-tion. On addition of NADH to the NADH oxi-dase under anaerobic conditions, cytochromesc and a + a3, but not cytochrome b, of theparticulate fraction are reduced. The NADHoxidase activity is inhibited by cyanide,NHQNO, PCMB, N-ethylmaleimide and di-cumarol. This indicates that, under the experi-mental conditions of Kearney and Goldman(188), at least part of the electron transportchain around cytochrome b seems to have beenbypassed.Heinen et al. (145) have compared the prop-

erties of NADH diaphorases of M. tuberculosisH37Ra solubilized from the particulate frac-tion by various procedures, such as ultrasonic

treatment, freezing and thawing, digitonin oralkali extraction, with those in the solublesupernatant fraction. The two types of diaph-orase have the same electron acceptor speci-ficity, but they differ from each other in theirphysical properties, such as response to heatinactivation, etc. The authors have thereforeconcluded that the soluble NADH diaphoraseis derived from the comminution of theNADH oxidase of the cytoplasmic membrane.The variations between the different solubi-lized diaphorases are attributed to differencesin the size of the fragments.

In M. smegmatis, NADH oxidase and cyto-chrome c oxidase activities have been demon-strated in a membrane fraction sedimentingbetween 5,000 and 25,000 x g (257), and diaph-orase activity has been demonstrated in themembrane mesosome fraction (61).

Regulation of Electron Transport andCoupled Phosphorylation

The particulate NADH oxidase of M. tuber-culosis H37Ra is an allosteric enzyme (418,419, 420). NADH oxidase is activated specifi-cally by adenosine monophosphate (AMP)(half maximal at 0.2 mM) but not by adenosinediphosphate (ADP), ATP, 3'5'-cyclic AMP orcytidine monophosphate; deoxyadenosinemonophosphate is only 30% as effective asAMP; AMP protects NADH oxidase from al-kali (pH 8.5) inactivation. According to Wor-cel, Goldman, and Cleland (420) AMP bringsabout a conformational change of proteinstructure, because the enzyme remains in theactive state even after the AMP is dissociatedfrom the enzyme. NAD catalyzes the reversionof the AMP-activated enzyme to the nativestate. Based on the kinetic data, it was sug-gested that there is one site for AMP but twosites for NADH-one allosteric and anothercatalytic on the native protein. Neither inor-ganic phosphate nor Mg2+ is required for thestimulation of NADH oxidase by AMP.The phosphate acceptor system has a regula-

tory role in M. phlei when Sephadex (G-25)-treated particles (called depleted particles)are used (307). Inorganic phosphate (Pi) andAMP stimulate (the former more than the lat-ter) the oxygen uptake with NADH (generatedwith alcohol dehydrogenase system). ADP,which inhibits the oxidation in the absence ofPi, stimulates the oxygen uptake in presence ofPi, and ATP shows inhibition of the latter. Thestimulation is on the reduction of cytochromesa + a3 but not of cytochromes b and c.The oxidation of exogenous NADH in M.

phlei and its regulation have also been studied

79VOL. 36, 1972


(38, 39). As in the case of M. tuberculosisH37Ra (420), the particulate NADH oxidase(both cyanide-sensitive and cyanide-insensi-tive) are inhibited by NAD (allosteric), andthis inhibition is relieved by AMP. However,the M. phlei NADH-oxidases show some dif-ferences. Both the cyanide-sensitive and thecyanide-insensitive particulate NADH oxi-dases are stimulated by Pi and fumarate. Fur-thermore, the NAD inhibition also is reversedby P, or fumarate; P., fumarate, and AMP donot stimulate the soluble NADH oxidasewhereas NAD shows some inhibition. SinceNADH can be oxidized by many pathways,Bogin et al. (38, 39) suggest that the levels ofNADH and NAD, as well as the level of AMPand P1, may play an important role in the reg-ulation of the pathway utilized for NADH oxi-dation. Thus the basic mechanism is similar tothat suggested by Worcel et al. (420), althoughthe role of inorganic phosphate in M. tubercu-losis H37Ra in this context needs a closerstudy.

OTHER ASPECTS OF CARBONMETABOLISM

Aromatic CompoundsMycobacteria can utilize a number of aro-

matic compounds. Gale (95) showed as early as1952 that mycobacteria oxidize benzoate di-rectly to catechol without the intermediateformation of salicylate. Whereas M. fortuitumis capable of converting salicylate to catechol(385), M. smegmatis cannot use salicylate forgrowth or metabolize it to any great extent(303). When M. smegmatis is grown on shi-kimic acid as sole source of carbon, salicylicacid, anthranilic acid, and 3, 4-dihydroxy-benzoic acid are excreted into the medium.Since there is no evidence that aromatic com-pounds such as anthranilic acid or tryptophangive rise to salicylic acid, it was concludedthat in this organism salicylic acid arises bythe shikimic acid pathway via chorismic acid(303). The salicylate accumulated is postulatedto condense with serine to form N-salicyloyl-serine, followed by ring closure to form the 2-(O-hydroxyphenyl)-2 oxazoline-4-carboxylicacid moiety of mycobactin. Under conditionsof iron deficiency there is an increase in theamount of salicylic acid excreted-an observa-tion which cannot be explained at the presentmoment.

Guerin et al. (133) have studied the metabo-lism of benzoic, phenylacetic, and phenylbu-tyric acids by whole cells and cell-free extractsof M. phlei. After incubation of "4C-labeled

acids with the cell-free extracts, a strongly ra-dioactive neutral fraction consisting of fattyacid esters of the corresponding aromatic alco-hols has been isolated.

Hydrocarbon UtilizationHydrocarbon utilization seems to be an in-

trinsic capability of mycobacteria, but practi-cally nothing is known about the mechanismby which hydrocarbons are utilized by them.An attempt to understand this has been madeby Lukins and Foster (240) who found that M.smegmatis 422 produces the homologousmethyl ketones during the oxidation of pro-pane, n-butane, n-pentane, or n-hexane. Ali-phatic alkane-utilizing mycobacteria are ableto grow at the expense of several aliphaticmethyl ketones as sole sources of carbon. Anoxygenase reaction is postulated for the attackon methyl ketones.

Oxidation of Phenolic CompoundsThe oxidation of phenolic compounds by M.

leprae has been studied by Prabhakaran andco-workers (290, 295). M. leprae isolated frominfected human tissues oxidizes D- and L-3,4-dihydroxyphenylalanine, a property not pos-sessed by other mycobacteria (290, 291, 294).This oxidation occurs only under aerobic con-ditions, and the intermediate product is in-dole-5, 6-quinone. In the range of substratesutilized and in the nature of the productsformed, the phenoloxidase in M. leprae ap-pears to be different from that obtained frommammalian and plant sources. Compoundswhich bind copper, for instance, diethyl dithio-carbamate, inhibit the enzyme activity mark-edly (293, 295). Therefore, it has been sug-gested that nontoxic inhibitors of phenol oxi-dase in leprosy bacilli may be of value in de-veloping a rational approach to the chemo-therapy of the disease, especially since dihy-droxyphenylalanine is present in tissues of thehuman body (like skin and nerve) where theorganisms proliferate.Beaman and Barkodale (33) have isolated, in

pure culture, anaerobic corynebacteria (pro-pionibacteria) from the plasma of tuberculoidleprosy and from lepromata of cases of lep-romatous leprosy and found that these orga-nisms, like the M. leprae shown earlier, exhibitphenoloxidase activity. The enzyme was alsoinhibited strongly by diethyl dithiocarbamate.

NITROGEN METABOLISM

Nitrate MetabolismDe Turk and Bernheim (71) were the first to

80 BACTERIOL. IREV.


report that nitrate as well as nitrite and hy-droxylamine are assimilated by washed sus-pensions of BCG. In an extensive study of ni-trate reduction by mycobacteria, Virtanen(390) has reported that all of the species inves-tigated reduce nitrate to nitrite, though quan-titative differences are observed; there was noreduction of nitrite when cell suspensions wereexamined in buffer solutions. Nitrate nitrogenis readily utilized by M. butyricum, M. smeg-matis, and M. tuberculosis H37Ra, while bothnitrite and nitrate are utilized as sole sourcesof nitrogen by M. tuberculosis H37Rv (144).Nitrate reductase of M. tuberculosis is inhib-ited by tungstate, but this inhibition is re-versed by molybdate. It is a particulate en-zyme present constitutively in the organism(32). The enzyme is specific for NADH, andNADPH cannot replace NADH; 2,heptyl-4, hydroxyquinoline-N-oxide, a strong inhibitorof the oxidation of cytochrome b, inhibits theenzyme by 70% at a concentration of 1 um.Reduction does not go far beyond nitrite,which is toxic to the cell, and therefore theauthors have concluded that "its physiologicalproperties fit the pattern of the so-called dis-similatory reduction, a process in which ni-trate only interferes in the normal energy-yielding reactions of the cell by acting as anon-essential electron acceptor." If this is true,it is necessary to look for other enzyme sys-tems which metabolize nitrate, since it is usedas a sole source of nitrogen for growing myco-bacteria.

Ellfolk and Katunuma (84) have detectedthe presence of an ammonia-activating enzymein M. avium: ATP + NH3 = AMP - NH2 +pyrophosphate.

Amino Acids and AmidesGeneral. Our knowledge of amino acid me-

tabolism in mycobacteria has not changed inprinciple in the period covered by this review,though many details of the metabolism have,of course, been studied. When "4C-labeled ace-tate is incorporated into the growth medium,the highest specific activities are found in glu-tamic acid, aspartic acid, proline, isoleucine,and arginine (377). The distribution of activityin different amino acids is dependent on theposition of the labeled atom in acetate.The utilization of amino acids during growth

of M. tuberculosis H37Ra in rotary cultureshas been studied by Lyon et al. (243). The effi-ciency of amino acids in promoting growth isin the order: alanine >> glutamate > aspara-gine > aspartate. Utilization of asparagineduring early growth is greater than of alanine

and glutamate, and during asparagine metabo-lism extracellular amino acids are accumulatedin the medium. The authors suggested thatduring the metabolism of asparagine by theH37Ra strain in rotary cultures, metaboliccontrols may be exerted which impede proteinsynthesis. In contrast to M. tuberculosisH37Ra, M. smegmatis 607 grows well with allthe nitrogen sources. A comparison with theH37Rv strain of M. tuberculosis is also desir-able.The uptake of L-glutamate shows Michaelis-

Menten kinetics in M. avium whereas that ofD-glutamate in both M. avium and M. smeg-matis is accomplished by a passive processshowing diffusion kinetics (422, 423). The up-take of D-alanine in M. smegmatis is an activeprocess displaying saturation kinetics charac-teristic of enzyme function, while the uptakeof L-alanine, L-glutamine, and D- and L-valinetakes place by both the active and passiveprocesses described above. The passive processis, however, sensitive to sulfhydryl blockingagents and shows competition among structur-ally related amino acids, suggesting that thepassive process is a facilitated diffusion.The cell-free extracts of M. tuberculosis

H37Rv and BCG catalyze the transfer ofamino groups from L-asparagine, DL-asparticacid, L-isoleucine, L-valine, L-leucine, L-phen-ylalanine, L-tryptophan, DL-norleucine, and DL-norvaline to a-ketoglutarate (318). M. aviumalso possesses similar transaminase activitybut cannot utilize L-phenylalanine and L-tryp-tophan as substrates.Asparagine and aspartic acid. Asparagine

is the preferred source of nitrogen for thegrowth of mycobacteria. From the uptake anddistribution of labeled carbon from '4C-aspar-agine by M. tuberculosis H37Ra it was shownthat asparagine supplies not only nitrogen butalso carbon to growing cells (245). The carbonof asparagine is rapidly taken up and exten-sively utilized by this organism. Washed cellsas well as cell-free preparations of M. tubercu-losis H37Rv, H37Ra, and BCG deamidate as-paragine to aspartic acid and ammonia (137,197). Ott (280) has described asparaginasefrom M. tuberculosis H37Ra and M. smeg-matis and has determined the kinetics for theformation of L-aspartic acid both with wholecells and cell-free preparations. A single peakof maximal enzyme activity is found at pH8.5, and the two strains of mycobacteria showonly quantitative differences in asparaginaseactivity. In contrast, Jayaram, Ramakrishnan,and Vaidyanathan (172) have demonstratedtwo L-asparaginases with pH optima 9.0 and

VOL. 36, 1972 81


9.6, respectively, in M. tuberculosis H37Raand a single enzyme exhibiting maximal ac-tivity at pH 9.0 in M. tuberculosis H37Rv. Thetwo enzymes (pH 9.0 and 9.6 enzymes) differalso in kinetic properties, inhibition by L-as-partic acid, and sensitivity to general enzymeinhibitors. The pH 9.6 enzyme, characteristicof the H37Ra strain is inhibitory to the growthof Yoshida ascites tumor in rats (342). Thisenzyme is inducible in the presence of L-as-paragine in the growth media (171). Besides,an aspartotransferase, which specifically cata-lyzes the transfer of the amido group of L-as-paragine to hydroxylamine, forming aspartohy-droxamic acid, is shown to be present in M.tuberculosis H37Ra but absent from H37Rv(173). The enzyme has been separated andfreed of L-asparaginase and glutamotrans-ferase; the purified enzyme exhibits maximumactivity at pH 9.0. The observations on aspara-ginase and aspartotransferase represent thefirst examples of qualitative enzymatic differ-ences between the virulent and avirulentstrains of M. tuberculosis, but what relevancethese enzymes have to the virulence of theorganism is not known. In addition to the spe-cific aspartotransferase, another nonspecifictransferase which acts on both asparagine andglutamine is present in both strains. The en-zyme has a pH optimum of 9.6.The presence of aspartokinase (which cata-

lyzes the conversion of L-aspartic acid to $-aspartylphosphate) and homoserine dehydro-genase (which catalyzes the reduction of L-as-partic acid f3-semialdehyde to homoserine) inM. album has been reported (184). The syn-thesis of aspartokinase is repressed by DL-threonine and L-methionine, and the activity isinhibited by DL-threonine and DL-allothreonine.The synthesis of homoserine dehydrogenase isrepressed by DL-threonine and DL-valine, andits activity is inhibited by DL-threonine, DL-iSo-leucine, L-valine, L-methionine, or L-phenylala-nine. The activity of this enzyme is stimulatedby Na+ and K+ in the reverse reaction and in-hibited slightly in the forward reaction.Glutamine and glutamic acid. Glutamine

can substitute for asparagine as nitrogensource for the growth of mycobacteria, thoughthe yield is slightly lower. The extracts of M.phlei possess glutamotransferase activity andhydrolyze aspartohydroxamic acid, but do nothave aspartotransferase and glutamo- and as-parto-synthetases. The transfer and hydrolyticreactions, therefore, may be catalyzed by dif-ferent enzymes (130). M. tuberculosis also pos-sesses aspartotransferase and glutamo-transferase activities which are distinct from

each other, but not aspartosynthetase (172).Washed cell preparations of M. tuberculosisH37Ra and M. smegmatis 607 grown in Sau-ton's medium oxidize glutamate after a lagperiod, but when the cells are grown in a mod-ified medium containing glutamate there is nolag (244). The lag is due to the induction of aglutamate transport system.

Prabhakaran and Braganca (292) have re-ported that M. leprae separated from humanlepromatous nodules possesses glutamic aciddecarboxylase activity, and the product of theenzyme reaction, y-aminobutyric acid occursin skin lesions of leprosy as a product of thebacillary metabolism. They could detect theenzyme in M. phlei and M. kedrowsky but notin M. smegmatis and M. tuberculosis H37Rv.

Alanine. Lyon and Hall (242) have recentlyreported that L-alanine can substitute for L-asparagine as nitrogen source for growing M.tuberculosis, though the growth rate with theformer is lower. The purification and proper-ties of dehydrogenase from M. tuberculosisH37Ra and H37Rv have been described (112,299). The enzyme is specific for L-alanine andrequires NAD for activity. The enzyme is alsopresent in M. phlei, M. smegmatis, and M.lacticola; the properties of the enzymes fromthe two strains of M. tuberculosis and the sap-rophytic strains are similar (302).Lysine. The production of lysine from dia-

minopimelic acid by cell-free extracts of M.tuberculosis H37Rv and H37Ra strains hasbeen demonstrated by Willett (402). Diamino-pimelic acid is decarboxylated to give lysine,and there is no difference in the activities ofthe virulent and avirulent strains.

Isoleucine and L-valine. In addition to theknown enzymes of the isoleucine-valine bio-synthetic pathway, cell-free extracts of M.tuberculosis possess a new enzyme, acetohy-droxy acid isomerase which isomerizes a-ace-tolacetate to a-keto ,B-dihydroxyisovalerate,which is, in turn reduced to a-fl-dihydroxyiso-valerate in the presence of NADPH (9). Thisis an alternate pathway to convert a-acetol-acetate to a-f3-dihydroxyisovalerate, mediatedby an isomerase, followed by a reductase, inaddition to the usual isomeroreductasepathway. The isomerase has been purifiedabout 100-fold and separated from reductaseand isomeroreductase (10). The enzyme re-quires specifically L-ascorbic acid and sulfatefor its activity. Since the requirement of thisenzyme for L-ascorbic acid is specific, it shouldbe possible to use the enzyme in a test systemfor L-ascorbic acid and its antagonists. Theisomeroreductase from M. tuberculosis H37Rv

82 BACTERIOL. REV.


has also been purified about 40-fold (11). L-Ascorbic acid has no effect on the purifiedisomeroreductase, unlike the isomerase. Thus,the three enzyme activities reside in three sep-arate proteins. Since ascorbic acid is presentin extracts of M. tuberculosis (12), this vitaminmay be a natural cofactor in the isomerizationreaction.

L-Valine inhibits acetohydroxy acid synthe-tase, the first common step in the biosynthesisof isoleucine and valine and, when added tothe growth medium, inhibits the growth of M.tuberculosis H37Rv (9). On the other hand, inM. pellegrino valine coordinately increases thelevels of acetohydroxy acid synthetase, dihy-droxy acid dehydratase, and threonine deami-nase, three of the enzymes participating in thebiosynthesis of isoleucine and valine in theorganism, and L-isoleucine inhibits the effectof valine (153). The inductive effect of L-valineappears to be due to its ability to inhibit theactivity of acetohydroxy acid synthetase, thuscausing isoleucine deficiency, which in turnleads to the derepression of the three enzymes.Acetylated amino acids. Fowler, Camien,

and Dunn (91) have identified acetyl L-isolei-cine and acetyl L-leucine as extracellular prod-ucts of M. ranae. The nature of the biosyn-thesis of these compounds would be most in-teresting, since acetylated amino acids haveonly rarely been encountered in natural mate-rials, but when they do occur, as in mam-malian systems, have been shown to possessprofound physiological significance. Examplesof the latter are acetyl-L-glutamic acid andacetyl-L-aspartic acid, intermediates in aminoacid metabolism.

Paraaminobenzoic AcidSloane and co-workers (329-334) have

studied in detail the metabolism of p-amino-benzoic acid (PABA). The washed cells of M.smegmatis form p-amino phenol from PABAas well as aniline. The first step in the se-quence of metabolism of PABA is a directenzymatic reduction of the carboxyl group togive p-aminobenzyl alcohol, which is followedby the transformation of p-aminobenzyl al-cohol to p-hydroxy aniline. The authors havesuggested that this unique transformation maybe a specific case of a more general mechanisminvolved in enzymatic hydroxylation of arylcompounds. In the metabolism of aniline bymycobacteria also, aniline hydroxylation is pre-ceded by hydroxymethylation. The sequence ofreactions is:

PABA

p-aminobenzyl alcohol ,

p-hydroxyanilineaniline

However, p-aminobenzyl alcohol was not de-tected when "4C-aniline was incubated withwhole cells of M. tuberculosis in the presenceor absence of 14C-methyl methionine (230).When aniline is incubated with cell-free ex-tracts, in the presence of labeled methionine,N-methyl aniline and small amounts of dimeth-ylaniline can be isolated; methionine acts asthe C-1 donor.

CarboligaseYamasaki and Moriyama (426) have de-

tected a-ketoglutarate carboligase activity inM. phlei. The reaction product, 6-(OH) levu-linic acid, inhibits porphyrin synthesis from 6-amino levulinic acid in cell-free extracts of M.phlei, and therefore the carboligase may playan important part in the regulation of por-phyrin synthesis (by inhibiting the 6-amino-levulinate dehydratase).

PROTEIN SYNTHESISThe synthesis of proteins and nucleic acids

by mycobacteria has attracted attention par-ticularly from the view point of drug action(383, 384, 386, 387). While isoniazid inhibitsthe incorporation of a-ketoglutarate and DL-glutamate into proteins in M. tuberculosisH37Rv as well as M. jucho (an avirulent strainof M. avium), it does not inhibit the incorpora-tion of glycine or leucine (386, 387).The activation of various L-amino acids by

cell-free extracts of M. phlei and M. tubercu-losis H37Rv has been analyzed by the hydroxa-mate formation method (97, 98, 147). Cell-freeextracts of M. phlei also activate D-aminoacids such as D-glutamic acid or D-alanine, butthese are not incorporated into transfer ribonu-cleic acid (tRNA). The activation of aminoacids in nine different strains of mycobacteriais independent of the sensitivity or resistanceof these organisms to streptomycin, isoniazid,p-amino salicylic acid, and ethionamide (146).The incorporation of amino acids into pro-

teins and the effect of antibiotics known toinhibit protein synthesis have been studied inmycobacteria also. The effect of streptomycinon the incorporation of 14C-DL-tyrosine intoproteins by cell-free extracts of M. friburgensis

83VOL. 36, 1972


was examined by Erdos and Ullmann (85) whoclaimed that streptomycin (100 Ag/ml) inhibitsthe incorporation of tyrosine into proteins instreptomycin-sensitive cells but not in thestreptomycin-resistant cells. However, theauthors have employed the pH 4.5 precipitateobtained from the cell-free system prepared bycentrifugation of extracts at 105,000 x g for 60min and thus devoid of ribosomes. Later, theseauthors (86) noticed much lower inhibition oftyrosine incorporation in whole cells of M. fri-burgensis by streptomycin. Streptomycincauses almost complete inhibition of proteinsynthesis in whole cells of streptomycin-sensi-tive M. tuberculosis H37Rv but not in a strep-tomycin-resistant mutant (327a).The amino acid incorporation by M. smeg-

matis 607 and BCG extracts is inhibited bychloramphenicol, tetracycline, erythromycin,and streptomycin (80). The steroidal anti-biotics fusidic acid and helvolinic acid inhibitthe incorporation of 14C-leucine and 14C-valineas well as polyuracil (poly U)-directed incorpo-ration of phenylalanine in cell-free systems ofM. smegmatis 607 (424).

Although Tomcsanyi (376) has described asystem from M. tuberculosis H37Rv capable ofincorporating labeled glycine, tyrosine, leu-cine, and valine in the absence of any addedmessenger, a systematic description of anamino acid incorporating system from myco-bacteria was given for the first time by Rieberand Imaeda (309). They determined the op-timal conditions for poly U-directed phenylal-anine incorporation in the extracts of M.smegmatis 607 and demonstrated a slightstimulation of polyphenylalanine formationcaused by isoniazid, presumably by enhancingthe rate of activation of phenylalanine. An effi-cient system from M. tuberculosis H37Rv ca-pable of incorporating phenylalanine directedby poly U has also been described recently(327a).

RibosomesSince mycobacteria are classified as Actino-

mycetes and are considered to be advancedprokaryotes, a look at the ribosomal structureto see whether they possess 70S ribosomes asthe prokaryotes, or 80S ribosomes as the eu-karyotes, should be fascinating. The isolationand characterization of ribosomes from M.smegmatis 607 and BCG have been reportedby Eda (78, 79). The protein synthesizing ac-tivity of membrane-bound ribosomes from M.smegmatis 607 is higher than that of free ribo-somes (93). Trnka, Wiegeshaus, and Smith

(381) have characterized the ribosomes fromBCG in greater detail. The BCG ribosomessediment as a main peak of 70S and one peakeach of 50S and 30S at an Mg2+ concentrationof 10 mm. When the Mg2+ concentration islowered to 0.1 mm, only 30S and 50S particlesare observed, and the 70S particles completelydisappeared. Under their experimental condi-tions, these authors could not detect any poly-somes.

While analyzing the various cellular frac-tions of human tubercle bacilli for antigenicactivity, Youmans and Youmans (431) de-scribed the preparation of ribosomes from M.tuberculosis H37Ra and demonstrated that theantigenicity was associated with the RNAcomponents of these ribosomes. However, adetailed and systematic analysis of ribosomesfrom M. tuberculosis H37Ra was carried outby Worcel, Goldman, and Sachs (421). TheH37Ra ribosomes also sediment as a 70S peakon sucrose gradients at 10 mm Mg2+, whereasthey dissociate into 50S and 30S particleswhen the Mg2+ concentration is lowered to 0.1mM. Further, the RNA isolated from these ri-bosomes exhibits a sedimentation profile sim-ilar to the Escherichia coli ribosomal RNA,showing two major peaks of sedimentation,23S and 16S, respectively, and a minor peak of4 to 5S.The structural integrity and functional ca-

pacity of the ribosomes, however, depend onthe age of the culture and the method of prep-aration of bacterial extracts (378, 421). Theribosomal structures seem to disappear totallyfrom old cells (4 weeks or older), and the pro-tein synthesizing ability is also considerablyless. In fact, Trnka and Smith (378) observedthat poly U-dependent 14C-phenylalanine in-corporation by BCG ribosomes and superna-tant fluids from 18-day-old cells is only 1.5 to10% of the activity of 11-day-old cells, and 60to 75% of the activity was lost by storing theextracts for 24 hr at 4 C. The system describedby Trnka and Smith (378) is very efficient andgives an almost 100-fold stimulation of aminoacid incorporation over background in re-sponse to the added messenger. Since severalof the mycobacteria are slow-growing, a slowerrate of protein synthesis was expected; how-ever, the amount of RNA (about 26% of thedry weight) and proteins at the logarithmicphase of growth was approximately the sameas found with bacteria having a faster rate ofgrowth, although the amount of DNA (0.66% ofdry weight) is lower than that found with mostmicroorganisms (432).

84 BACTrERIOL. REV.


NUCLEIC METABOLISM

GeneralThe nucleic acids of tubercle bacilli attracted

attention as early as 1898, and a product called"Tuberculinsaure" was isolated from "crushedtubercle bacilli" by Ruppel (314) which he de-scribed as possessing all the properties of a nu-cleic acid and also the "poisonous" and "im-munizing" properties. The isolation and char-acterization of nucleic acids from M. avium(65), M. phlei, and M. tuberculosis (175, 220),and BCG (388) were described quite early. It iswell known that mycobacterial DNA is charac-terized by an extremely high guanine-cytosine(GC) content, even though the significance ofthis is not known (64). The occurrence of mod-ified bases such as 5-methyl cytosine and 6-methyl amino purine in mycobacterial nucleicacids was also established early (75, 174). Inspite of all the early information available on

the nucleic acids of mycobacteria, very littleattention has been devoted to intermediarynucleic acid metabolism.

In the 1960's again several papers have ap-

peared on the isolation of DNA from variousspecies of mycobacteria and its GC contents.Among these, the only report of significance isfrom Wayne and Gross (396), who have de-scribed a technique for the isolation of DNAafter autolysis of cells and have successfullyapplied this technique to eight species of My-cobacteria. Subsequently, these authors (128)using immobilized DNA (from various myco-

bacteria), labeled reference DNA (from M.tuberculosis and M. kanasasii), and annealingtechniques compared the degrees of related-ness among various mycobacteria. Relative percent binding and thermal stability of boundDNA are used as parameters for determiningthe homology; their results agree well with theestablished taxonomic data.A gentle, but fast, method for isolating DNA

from mycobacteria has been reported by Mi-zuguchi and Tokunaga (258) who lysed thecells with ethylenediaminetetraacetate and gly-cine or cycloserine before phenol extraction.

Recently Michalska and Lorenc (254) havedescribed the isolation of nucleic acids fromM. smegmatis 607 and BCG and their resolu-tion on methylated albumin Kieselghur(MAK) columns into tRNA, DNA, and high-molecular-weight ribosomal RNA by salt gra-dient elution. However, this is only an exten-sion of earlier work of Odrzywolska (274), whohad examined the synthesis of RNA by la-beling 32pi in normal cells of M. smegmatis607 and in cells infected with phage D29 and

had observed a rapidly labeled fraction, dis-playing a rapid turnover, presumably corre-sponding to the messenger RNA.

DNA and RNAA report on the partial purification (430-

fold) of DNA polymerase from M. smegmatisrepresents the only available information re-garding the biosynthesis of DNA (415).On growth under conditions of iron and zinc

deficiency, an overall decrease has been ob-served in the synthesis of nucleotides or nu-cleic acid in M. smegmatis (137, 138, 139, 409,416, 417). In a series of papers, Winder and co-workers (410-413) reported the presence andpartial purification of the nucleoside triphos-phate-dependent deoxyribonuclease in M.smegmatis and the characterization of theproducts of the enzyme reaction. There is amarked increase per unit of protein in theDNA polymerase and the ATP-dependentdeoxyribonuclease activities, in iron-limitedcultures, reaching the maximum between 48and 72 hr of growth (414). The changes in ac-tivities of both enzymes can be prevented orreversed by addition of Fe2+ to normal values,within 24 hr. The two activities are apparentlynot the functions of one enzyme, since theyhave been separated [unpublished observa-tions of F. G. Winder, M. Levin, and M. S.McNulty, cited by Winder and McNulty (415) ].The only enzyme pertaining to RNA metab-

olism in mycobacteria studied so far is polynu-cleotide phosphorylase. The enzyme has beenreported to be present in M. phlei (57), M.smegmatis (59, 163), M. avium (14), and M.tuberculosis (250). Polynucleotide phosphoryl-ase, partially purified from M. tuberculosisH37Rv, catalyzes the synthesis of polymersfrom nucleotide diphosphates, the phosphory-lysis of polynucleotides, and the exchange re-action between nucleotide diphosphates and32Pi. Further, the enzyme purified from H37Rvand H37Ra as well as from streptomycin-re-sistant (100 Aig/ml) H37Rv cells exhibits verysimilar properties. The sensitivity to strepto-mycin of the enzymes derived from strepto-mycin-sensitive or streptomycin-resistant cellsis the same; thus, the polynucleotide phos-phorylase cannot be implicated as the locus ofaction of streptomycin.

Purines and PyrimidinesThe de novo biosynthesis of nucleic acid

purines in M. tuberculosis H37Rv has beenstudied by using "4C-labeled precursors, withspecial reference to the precursors of C2 andC8 of the purine ring by Malathi and Ramak-

85VOL. 36, 1972


rishnan (248). Both the ureide carbon atomsare derived most effectively from the ,8-carbonof serine and a-carbon of glycine, while for-mate, the "classical" precursor of the purinering, is not an efficient Cl donor in this orga-nism. Cell-free extracts of the organism arealso able to incorporate serine into the nucleicacid purines. Although l4C-methyl-labeledmethionine is not a precursor for the purines,the high specific radioactivity of the nucleicacids isolated from the organism grown in itspresence suggests the methylation of bases innucleic acid. Shaila and Ramakrishnan (328)have shown the presence of active tRNA-methylating enzymes in cell-free extracts ofM. tuberculosis by using S-adenosyl methio-nine as methyl donor and methyl-deficienttRNA from E. coli as substrate.Whole cells as well as cell-free extracts of M.

tuberculosis H37Rv are capable of utilizationand interconversion of preformed purines,adenine and guanine (247, 248). The conver-sion of guanine to adenine may be through theintermediate formation of guanosine mono-phosphate, inosine monophosphate, and AMP,and the deamination of guanine is ruled out.Mycobacteria can, however, deaminate adeno-sine (60), and some properties of adenosinedeaminase from M. tuberculosis H37Ra havebeen described (134).The ability of M. butyricum and M. smeg-

matis to oyidize uric acid to CO2 and NH3with the intermediate formation of allantoin,allantoic acid, glyoxyl urea, and glyoxylic acidis reported by Klemperer, Scott, and Bagchi(198).Very little information is available on the

pyrimidine metabolism of mycobacteria. Re-cently, Vitol, Shaposhnikov, and Shvachkin(391) have reported that two strains of myco-bacteria catabolize orotic acid to uracil, tobarbituric acid, and to urea.

VITAMINS AND COENZYMES

Nicotinic Acid and NicotinamideCoenzymes

The production of various B-complex vita-mins by M. tuberculosis was analyzed by Popeand Smith (286) and Bird (36) and the syn-thesis of markedly high levels of nicotinic acidwas observed. Later, Konno and his co-workers(205-207) made use of this excessive nicotinicacid production by M. tuberculosis as amethod for differentiating the human strain ofM. tuberculosis from other mycobacteria byemploying a simple color reaction undergoneby nicotinic acid with cyanogen bromide and

aniline. Konno's experiments were repeated byseveral laboratories, and presently this methodof differentiation has formed the basis of asimple biochemical test; however, each phaseof Konno's original technique has undergonemodification. In addition, these studies led tothe search of other biochemical properties forthe differentiation of tubercle bacilli.A large number of workers have attempted

to elucidate the mechanism of biosynthesis ofnicotinic acid in mycobacteria (8, 125-127, 261,264, 311). From both radioactive incorporationand nutritional supplementation studies, it hasbeen shown that the biosynthetic mechanismin M. tuberculosis may be the following:

3-carbonunit(from

glycerol) ( +NN

Aspartic Quinolinic Nicotinicacid acid acid

There is, however, no direct conversion ofquinolinic acid to nicotinic acid.A decrease in the production of nicotinic

acid by BCG as a result of zinc deficiency oran increased supply of phosphate has also beenreported (263, 435).The biochemically functional forms of nico-

tinic acid are the nicotinamide nucleotidecoenzymes (NAD and NADP), which are syn-thesized by way of nicotinic acid mononucleo-tide (NaMN) and nicotinic acid adenine dinu-cleotide (NaAD). The direct conversion ofquinolinate to NaMN without the interme-diary formation of free nicotinic acid by theenzyme quinolinate transphosphoribosylasehas been reported for various strains of myco-bacteria, including M. tuberculosis H37Rv andH37Ra (209, 210). This is in contrast with theresults of Dudley and Willet (73, 74) who re-ported the conversion of nicotinic acid toNaMN in M. bovis and M. tuberculosis. How-ever, earlier studies of Gopinathan (119) haddemonstrated the inability of H37Rv extractsto incorporate 'IC-labeled nicotinic acid ornicotinamide into NaMN and NaAD, in agree-ment with the results of Konno et al. (210).Thus the excretion of nicotinic acid may repre-sent the inability of the organism to utilizethis compound for the biosynthesis of thecoenzymes. Under conditions of ATP limita-tion, quinolinate transphosphoribosylase candegrade the NaMN formed into free nicotinicacid (258), and the enzyme may thus possessregulatory properties.

86 BACTERIOL. REV.


The degradation of NAD (or NADP) is ac-complished by the enzyme NADase (NAD gly-cohydrolase) which cleaves the nicotinamide-riboside bond of the oxidized form of the coen-zymes. This enzyme dan, in addition to thehydrolytic cleavage, catalyze an exchange reac-tion between NAD and free nicotinamide orcompounds structurally related to it, resultingin the formation of an analogue of NAD (433).The potent antitubercular drug isoniazid canparticipate in the NADase-catalyzed exchangereaction, and the isoniazid analogue of NADformed may not be effective as the coenzyme.This was suggested as the mechanism of actionof isoniazid, and therefore the enzyme is ofgreat significance in mycobacteria.NADases have been reported in M. butyr-

icum (189, 371) and M. tuberculosis H37Rv(121, 122), and in both the cases the enzymesare associated with a heat-labile protein inhib-itor. The partially purified enzymes from bothof these strains do not catalyze the NAD-iso-niazid exchange reaction. The proteinaceousinhibitor of NADase from M. tuberculosisH37Rv has also been purified and its speci-ficity as well as certain aspects of enzyme-in-hibitor complex formation were evaluated(120). The inhibitor is specific for mycobac-terial NADase and the enzyme-inhibitor com-plex formed is undissociable. The complexformation between highly purified NADase(molecular weight, 39,000) and its proteina-ceous inhibitor (molecular weight, 26,000) fromM. butyricum has recently been studied byOgasawara et al. (275) by use of Sephadex G-100 chromatography, sucrose gradient centrifu-gations, and cellulose acetate electrophoresis.The NADase inhibitor may play a role in the

regulation of NAD levels and seems to be sen-sitive to the action of isoniazid. The latterproperty will be discussed in the section ofmechanism of drug action.The presence of active NADase in lung-

grown tubercle bacilli was shown by Artmanand Bekierkunst (20); it is presumably derivedfrom the infected tissues. The tremendous in-crease in NADase activity in tuberculous tis-sues has also been shown to be of host tissueorigin and not derived by activation of bac-terial enzyme (123).The nicotinamide, rising out of NADase ac-

tion, can be deamidated to nicotinic acid byan enzyme nicotinamidase, which is alsopresent in several strains of mycobacteria,with the exception of M. bovis (208). Moredetails about the enzyme are given in anothersection of this review.

Other B VitaminsCompared to the volume of literature avail-

able on nicotinic acid and its derivatives, theinformation available on other vitamins andcoenzymes in mycobacteria is scanty.

Distribution of thiamine and its mono-, di-,and triphospho derivatives was quantitativelydetermined in M. lacticola and M. fortuiitumby Rossi-Fanelli et al. (313). All of the thia-mine esters can be dephosphorylated to thia-mine in cell-free extracts of M. lacticola.The effects of medium composition (C, N, P,

Fe, K, Mn, and surface active agents) andgrowth conditions (the intensity of aeration,temperature, and pH of the medium) on theyield of flavine from M. smegmatis strain 104were studied by Mil'ko and Ivanova (256). Fla-vine synthesis increases with increasing con-centrations of Na and K, but increasing con-centration of PO, decreases it; low initial pHof the medium favors flavine biosynthesis.The synthesis of dihydrofolate and dihy-

dropteroate from dihydroneopterin by extractsof M. smegmatis has been reported by Jaen-icke et al. (168). The isomers of the pteridineprecursors are neither substrates nor inhibitorsof folate biosynthesis.The synthesis of vitamin B12 by various

strains of mycobacteria was examined byAithal and Sirsi (2), who found some differ-ences in the nature and contents of B,2 inisoniazid-sensitive and -resistant strains ofmycobacteria, assayed microbiologically withLactobacillus leichmannii 313. The signifi-cance of these results, however, is not estab-lished.

Vitamin KAmong the fat-soluble vitamins, only the K-

group of vitamins from mycobacteria has re-ceived attention; their role in electron trans-port has been well documented and has al-ready been considered under the section ofelectron transport. The natural naphthoqui-none (dihydromenaquinone-9) has been re-solved into two geometric isomers, and the cisisomer is active in oxidative phosphorylation(76). The report that M. phlei also containsdihydromenaquinone-8, and probably dihydro-menaquinone-10, shows the necessity for de-tailed studies on the role of these quinones, ifany, in oxidative phosphorylation (58).Attempts have been made to establish the

biosynthesis of vitamin K in mycobacteria. [2-14C]Mevalonic acid and acetate-2-14C are notincorporated into vitamin K2 in M. tubercu-

VOL. 36, 1972 87


losis or M. phlei (301). The incorporation ofradioactivity from methionine-methvl-14C intothe 2-methyl group of vitamin K2-(45)-H bywhole cells as well as cell-free extracts of M.phlei has been reported by Azerad, Bleiler-Hill, and Lederer (30) and Guerin, Azerad, andLederer (131). Under their conditions, labeledtyrosine-U-'4C is not incorporated into vi-tamin K2. Recently Azerad and co-workers(132, 228) have presented some evidence forthe biosynthesis of the ring of naphthoquinonein M. phlei and M. avium derived from studiesof the incorporation of labeled substrates intothe vitamin. While p-hydroxybenzaldehydeand 3,4-dihydroxybenzaldehyde are not incor-porated, [U-14C]shikimic acid served as goodprecursor of the napthoquinone rings. Tenta-tive schemes for naphthoquinone ring biosyn-thesis involving 0-carboxyphenylpyruvic acidas an intermediate have been suggested buthave yet to be confirmed. According to theauthors, menadione may not be a true inter-mediate in menaquinone biosynthesis. Incor-poration of a-naphthol-1-'4C into naphthoqui-nones has also been examined; while one strainof M. phlei (Legroux) shows a fair amount ofincorporation of radioactivity, only a smallamount is incorporated by another strain, M.phlei (ATCC 354).

MISCELLANEOUS ENZYME SYSTEMSA number of unrelated enzymes in mycobac-

teria have been reported, and for the most partthese have been used to differentiate mycobac-terial species and strains from one another.The role of most of these enzymes in theoverall metabolism of mycobacteria is notclear. The enzymes include phosphatases (15,269, 317, 363, 389), nicotinamidase and formi-dase (129, 136, 189-194, 208, 266), urease (18,286), arylsulfatase (285, 360-362), reductase(359, 366, 382), esterases (28, 395), and deami-nase (434); special mention should be madeabout catalase and peroxidase in mycobacteria.Mycobacteria are catalase-positive organismsand also show peroxidative activity towardscertain phenols like pyrogallol. Both activitiesare lacking in isoniazid-resistant strains of M.tuberculosis (255). There has been a contro-versy whether catalatic and peroxidatic activi-ties of these organisms are due to one and thesame enzyme or to two distinct enzymes. Insupport of the second hypothesis it has beenreported (368, 369) that some species of myco-bacteria and some strains of M. tuberculosisare negative to a qualitative test for peroxi-datic activity but give a positive catalase test.

Further, the two activities from certain strainsof M. avium are separated by zone electropho-resis at pH 8.6 on cellulose acetate (16). On theother hand, Winder (404) has presented evi-dence that peroxidatic activity in M. smeg-matis is due to catalase since, during 60-foldpurification of catalase, all fractions showedthe same ratio of catalase to peroxidase ac-tivity as the crude extracts. Similar experi-ments carried out with M. tuberculosis andother mycobacteria may clear up the contro-versy.

MECHANISM OF ACTION OFANTITUBERCULAR DRUGS

IsoniazidThe drugs currently in use against tubercu-

losis are isoniazid, streptomycin, p-aminosali-cylic acid (PAS), cycloserine, ethambutol, rifa-mycin, pyrazinamide, ethionamide, kanamy-cin, capreomycin, viomycin, and thioaceta-zone. An exhaustive account of all the antitu-bercular drugs known so far is given by Robsonand Sullivan (312). Isoniazid is the most po-tent antitubercular drug available and is spe-cific in its action against mycobacteria. A vo-luminous amount of literature has accumu-lated on the mode of action of this drug andthe subject has been reviewed periodically(113, 405, 428). Some of the earlier hypothesesput forth to explain the action of isoniazidare: (i) the drug inhibits the synthesis or ac-tivity of hemoproteins like catalases or peroxi-dase; (ii) isoniazid is active because of its che-lating property; (iii) hydrogen peroxide andfree radicals formed during the metabolism ofthe drug are toxic to M. tuberculosis; (iv) thedrug acts through its metabolic products; (v)the drug acts as a respiratory inhibitor; (vi) thedrug interferes with pyridoxal and nicotinicacid function; (vii) the drug inhibits the as-partic-glutamic transaminase of the organism;and (viii) the drug interferes with the carbohy-drate, lipid, protein, and nucleic acid metabo-lism of the organism. We shall deal in this ar-ticle mostly with literature which has appearedafter the last review by Youatt (428) was pub-lished.Any hypothesis proposed to explain the ac-

tion of isoniazid should account for the fol-lowing facts: (i) the drug is specific in its ac-tion against mycobacteria, and (ii) the growth-inhibitory concentration of the drug is verylow. Two hypotheses which apparently satis-fied these conditions at the time the last re-view appeared are the following. (i) Isoniazid

88 BACTERIOL. REV.


combines with an enzyme that is specific tothe drug-susceptible strains of M. tuberculosis,modifying the enzymes and at the same timedisplacing a molecule which gives rise to a

pigment precursor. Further reactions of eitherthe pigment precursor or the modified enzymein the presence of appropriate substrates leadto derangement of the various metabolic activ-ities of the organism. Youatt and Tham (429)have shown that this enzyme reaction hasmany features in common with the growth-in-hibiting action of isoniazid. Further, the reac-

tion is not observed in isoniazid-resistant cells.The biochemical characteristics of this enzymesystem from M. tuberculosis H37Rv have beendescribed recently from the authors' laboratory(103a). (ii) Isoniazid combines with the heat-labile inhibitor of NADase present in M. tu-berculosis and this causes a decrease in thecontent of NAD. The lowering of NAD contentaffects the metabolic activities of the orga-nism. In support of this hypothesis, Bekier-kunst and Bricker (5) reported that in an isoni-azid-resistant mutant of M. tuberculosisH37Ra the inhibitor of NADase is altered insuch a way that it is not inactivated by isoni-azid. The resistant organism they used is a

multiple-step mutant, and it is not clear there-fore that the observed reaction is a result ofthe primary action of the drug or one of theseveral changes which has occurred. To clarifythe situation, Sriprakash and Ramakrishnan(340) isolated a number of independent mu-

tants of M. tuberculosis H37Rv which were

exposed to the lowest inhibitory concentrationof isoniazid and hence would reasonably bealtered in only one biochemical step. Theseindependent mutants can be divided into twogroups-one in which the inhibitor has lost itssensitivity to the drug, and the other where theinhibitor is unchanged. In both groups, theuptake of "4C-labeled isoniazid was very muchdecreased as compared to the sensitive orga-nism, but when the permeability barrier to thedrug was removed by the use of surface-activeagents, the drug proved to be lethal to the or-

ganism. Even though the NADase inhibitorhas lost its sensitivity to isoniazid in one groupof isoniazid-resistant mutants, not only doesthe drug exert its lethal activity against thesemutants (in the presence of surface-activeagents), but it also decreases their NAD con-

centration, as in the case of the drug-sensitiveparents (338). There seems to be, therefore, a

direct correlation between the lethality of iso-niazid and the lowering of NAD content; sinceisoniazid cannot bind to the NADase inhibitorin this case, nor does it directly activate the

NADase of M. tuberculosis (122), the mecha-nism by which the drug brings about the de-crease of NAD in the organism is unknown.However, isoniazid at a concentration of 0.5mm inhibits the synthesis of NAD by cell-freeextracts of M. tuberculosis H37Rv and theisoniazid-resistant mutants to the same extent(339).One has to conclude that up to the present

time no satisfactory hypothesis about themode of action of isoniazid is available. Re-cently, Winder and Collins (408) have sug-gested that the primary site for the inhibitoryaction of isoniazid lies in the biosyntheticpathways to the mycolic acids. The inhibitionof mycolic acid synthesis may lead to the for-mation of defective envelope material low inmycolic acid, resulting in loss of materialsfrom the cell leading ultimately to loss of acidfastness and death. The model is yet to beproved experimentally.

Other Antitubercular AgentsAlthough the mechanism of action of strep-

tomycin is now well established in E. coli, veryfew reports on its mode of action on mycobac-teria are available. In view of the reports thatthe lipid content is doubled in a streptomycin-resistant strain of M. tuberculosis H37Rv ascompared to a sensitive strain (63), that thedrug inhibits arginase in whole cells of M.smegmatis but not in cell-free extracts (270),and that it induces premature lysis of phage-infected mycobacteria (374), a systematicexamination of the mode of action of strepto-mycin in mycobacteria seems to be desirable.Hedgecock (140-143) demonstrated that

strains of M. kansasii have more resistance toPAS in vitro than M. tuberculosis and thatpartial reversal of PAS inhibition occurs in thepresence of methionine only for the formerorganisms. The fact that M. kansasii can uti-lize the free methionine present in humanserum, while M. tuberculosis cannot, probablyexplains why PAS is ineffective as a chemo-therapeutic agent against M. kansasii.Among rifamycins, rifamycin SV and ri-

fampin inhibit at low concentrations thegrowth of M. tuberculosis and other mycobac-teria. White, Lancini, and Silvestri have dem-onstrated (401) that the primary event in theinhibition of M. smegmatis by rifampin is theblock of DNA-dependent RNA polymerase,just as in E. coli. Since, however, the effect ofrifampin on mycobacteria is slower than on E.coli the authors conjecture that there might bea difference in the stability of the rifampin-RNA polymerase complex between those bac-

89VOL. 36, 1972


teria which are killed very rapidly and thosewhich are killed more slowly. The other re-ported effects of rifampin on the incorporationof 32P into the nucleic acids of M. tuberculosis(384) and on the poly U-dependent phenylala-nine incorporation by isolated ribosomes ofmycobacteria (379) are probably only sec-ondary.

D-Cycloserine is employed as a second-linechemotherapeutic agent against M. tubercu-losis. D-Alanine antagonizes the inhibition ofgrowth of M., tuberculosis and M. phlei by cy-closerine, while L-alanine competes in the pro-tection given by D-alanine (262, 436). D-Ala-nine is a constituent of the mycobacterial cellwall, and in the presence of D-cycloserine amucopeptide accumulates in the drug-sensi-tive strain of M. acapulcensis.

David, Takayama, and Goldman (69, 70)have isolated D-alanyl-D-alanine synthetasefrom M. tuberculosis H37Ra and shown thatthe kinetics of the inhibition of this enzyme byD-cycloserine are similar to that from Staphy-lococcus aureus and Streptococcus faecalis,which are less sensitive to D-cycloserine. Onthe other hand, these authors have shown thatthe synthesis of Wax D peptidoglycolipid of theH37Ra strain is inhibited by D-cycloserine atlow concentration and that this inhibition iscompletely reversed by D-alanine. An arabino-galactan-galactosamine-(diaminopimelic acid)-mycolate accumulates in the organism exposedto the antibiotic, and the authors suggestedthat this lipid is a possible precursor to thesynthesis of Wax D. Further work on thissystem may reveal the particular enzymewhich is inhibited by the drug and the roleplayed by D-alanine in the reaction.D-Carbamyl serine is another antibiotic

which has antitubercular action. Its mode ofaction in S. faecalis has been reported to bethrough its inhibition of alanine racemase,thus depriving the organism of D-alanine nec-essary for cell wall biosynthesis (241). Thebasis of its antitubercular action, however,appears to be different. The inhibitory effect ofD-carbamyl serine in M. tuberculosis H37Rv isnot reversed by L-alanine or D-alanine unlessthe bacilli are previously grown in media sup-plemented by D- or L-alanine, suggesting thatthere is an inducible alanine permease systemin the organism (68). The drug potentiates theinhibitory action of D-cycloserine, and the po-tentiation is partially reversed by D-alanine.The author has concluded without any addi-tional direct evidence that D-carbamyl serineinhibits the induction of an alanine permeasesystem.

Ethambutol has specific antimycobacterialactivity and has been shown to be therapeuti-cally effective in tuberculosis in experimentalanimals and in man. Even though the drug istaken up rapidly by both proliferating andnonproliferating M. smegmatis, it has no effecton the viability and metabolism of nonprolifer-ating cells (89, 90, 214). The synthesis of DNAand protein and, to a lesser extent, RNA, isinhibited in the presence of the drug. Poly-amines and magnesium added to the culturereverse the inhibitory action of the drug. Sincespermidine and magnesium are necessary formaximal synthesis of RNA, ethambutol maybe exerting its inhibitory effect by interferingwith a function of polyamines and cations innucleic acid metabolism. Whether these effectsare due to the primary action of the drug orare secondary effects is not clear. A more ex-tensive investigation of the mode of action ofethambutol in mycobacteria is therefore calledfor.

Bacitracin at low concentrations inhibits thegrowth of M. smegmatis and, at higher concen-trations, the growth of BCG (310). Ultrastruc-ture studies indicated that the main target ofbacitracin on mycobacteria may be the mem-brane.From the above account it may be seen that

the mode of action of practically none of thecurrently available drugs on mycobacteria hasbeen satisfactorily explained and remains eventoday a challenge to intending investigators inthe area.

GENETICS OF MYCOBACTERIA

GeneralA beginning has been made in recent years

to study the genetics of mycobacteria. Theresults of such studies would be of valuableassistance in relating the data on metabolismto the genetic constitution of mycobacteriaand to understand the problem of drug resist-ance and virulence. Of the genetic processesreported to be operative in mycobacteria, onlylysogenic conversion and transduction are rea-sonably well documented. A large number ofbacteriophages of mycobacteria has been iso-lated; the work carried out on these has beenreviewed by Redmond (304). These phageshave been mostly used for typing mycobac-teria.

Lysogenic ConversionThe mycobacteriophage B2 lysogenizes the

host strain M. phlei F89 and on reisolation itshows a genotype which differs in several re-

90 BACTERIOL. REV.


spects from B2; previously restricted to M.phlei F89, its host range now extends to M.smegmatis SN2 (179). This phage was calledB2h. Since no absorption studies of the phagesB2 and B2h with the two bacterial strains havebeen reported, it is difficult to judge whetherthe ability of B2h to plaque on M. smegmatisSN2 is due to an extension of the host range ofthe phage or due to the ability of B2h to ad-sorb on to SN2. Concurrently with the changesin phage type, the respective lysogenic hostsshow an altered colony morphology and a de-creased growth rate, and the authors refer tothis as lysogenic conversion. When phage B2his grown on M. smegmatis SN2, a fraction ofthe infected cells gives rise to typical M. phleicolonies (177). In similar experiments with B2hgrown on M. phlei F89, conversion of M. phleito M. smegmatis was found. The conversion ismediated by the hybrid phage B2h, which isable to lysogenize both M. phlei and M. smeg-matis. One interpretation from these resultsfor the difference between M. phlei and M.smegmatis chromosomes is that an addition ofa specific region to M. smegmatis chromo-somes results in the M. phlei chromosome, anda deletion of this region from the M. phleichromosome gives rise to M. smegmatis chro-mosome. B2h could mediate this process (di-rectly or indirectly).

In another set of experiments, Juhasz (178)studied the changes in M. phlei due to B2hlysogeny by using the replica plating method.The changes could be divided into three cate-gories: conversion to slow growth and smallcolony size, dependence on related but com-plex nutritional requirements, and conversionto thiamine auxotrophy.The infection of M. smegmatis 607 by

phages D29 and B4 results in colony changefrom rough to smooth and increased nitratereductase activity accompanying lysogeniza-tion (176). Loss of the lysogenic state reversesthese changes. It has been suggested that theobserved rapid loss and gain of such charactersare due to genetic changes brought by aplasmid. Since lysogens of E. coli which con-tain defective prophages have been reported toform wrinkled colonies (233, 234), this explana-tion for the colony change is probably the mostlogical one. Juhasz, Gelbart, and Harize (180)have reported decrease in the amidase, nitratereductase, and phenolphthalein sulfatase activ-ities of M. smegmatis SN2 following lysogeni-zation by phage B1, showing that a wide rangeof changes may follow lysogenization and alterpotentially important taxonomic characters.An extreme case of such changes has been

found in Mycobacterium strain 80 which isobtained following lysogenization of M. phleiF89 with phage B2h (105). The per cent GCcontent (68.9) of the DNA of strain 80 is inter-mediate between that of M. phlei (69.9) andM. smegmatis (67.9). The authors admit that a1% difference may not be considered signifi-cant but add that another F89 (B2h) lysogenwhich does not show any of the changes asstrain 80 has the same GC content (69.9) asthe parental M. phlei.Some of the earlier investigators had re-

ported the isolation of smooth colonies of my-cobacteria from previously rough strains afterexposure to mycobacteriophages (316, 400).Russell, Jann, and Froman (316) have alsodemonstrated that some smooth colonies werelysogenized with the infecting phage. Becauseof the low frequency with which smooth colo-nies are detected and the inability to demon-strate lysogeny in many of the smooth colo-nies, they believed that selection of sponta-neous mutants is the preferable explanationfor their findings. White, Foster, and Lyon(400), however, isolated smooth colonies of M.smegmatis 607 after exposure to certain phagesin a higher frequency than would be explainedby selection of mutants alone. The high fre-quency with which smooth colonies are demon-strated to be lysogenic suggests that lysogenyis responsible for the altered colony mor-phology. Thus, reports of lysogenic conversionin mycobacterium have appeared from dif-ferent laboratories, but the spectrum of geneticcharacters altered in the lysogenic conversionsstudied by Bonicke and Juhasz is muchbroader than those studied by others and re-quires careful analysis for any possible artifactbrought about by the problem of clumping inmycobacteria. Interconversion of species medi-ated by phage, if confirmed, would have far-reaching genetic and taxonomic implications,especially in light of the hypothesis that theatypical mycobacteria have originated fromthe tubercle bacilli "through genetic mecha-nisms" (152).

TransductionA mycobacteriophage capable of mediating

transduction in M. smegmatis SN2 has beenisolated recently by Sundar Raj and Ramak-rishnan (345). Transduction has been shownfor histidine, glycine, arginine, alanine, andadenine markers; the transduction of resist-ance to the drug isoniazid (to which only my-cobacteria are susceptible) can now be at-tempted. The authors have also describedmethods of isolating auxotrophs of M. smeg-

91VOL. 36, 1972


matis by using ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (NG) asmutagens (346). The use of NG, hydroxyl-amine, ultraviolet irradiation, and ethyl-methane sulfonate for isolating mutants of M.phlei (auxotrophic, drug-resistant, as well asmutants with changed pigmentation) havebeen described by Konickova-Radochova andco-workers (201, 204). An interesting observa-tion they have reported is that among the aux-otrophic mutants, 50% required either glycineor serine for growth. Transduction in M. phleiby another mycobacteriophage, Bo2, has beenrecently reported (105a).

TransfectionTokunaga and Nakamura (372, 373) have

successfully infected M. tuberculosis and M.smegmatis in the late log phase of growth withDNA extracted from mycobacteriophage B1.One microgram of DNA (the equivalent of 1.5x 108 phage) produced 84 infective centers.The activity was destroyed by deoxyribonu-clease but not by phage antiserum. Transfecti-bility was increased by presensitization of bac-teria to cycloserine and amino acids such as D-serine, D-alanine, D-lysine, D-threonine, andglycine, apparently by alteration of the cellsurface (375). M. smegmatis 607 becomeshighly competent for transfection when incu-bated in the standard acid medium, which is amodification of the minimal medium for my-cobacteria (267).

ConjugationMizuguchi and Tokunaga (259) recently re-

ported genetic recombination between auxo-trophs of M. jucho and M. lacticola in solidmedia at a frequency of about 10-3 to 10-5.Crosses between mutants of homologousstrains were not fertile, nor were recombinantsproduced in liquid broth. Pretreatment of ei-ther of the parental types with acridines didnot abolish the ability to produce recombi-nants. This observation eliminated the possi-bility that the donor in the recombination cor-responded to an F+ mating type, but the use ofvery few auxotrophic markers in the recombi-nation experiments makes it difficult to judgewhether the donor corresponded to the Hfrtype. It is hoped that with the isolation of agreater variety of auxotrophic mutants notonly will this problem be solved but also thatattempts will be made to map the mycobac-terial chromosomes. Mycobacterial conjugationhas sexual polarity and in a study of the ki-netics of gene transfer, the glycine marker en-

tered earlier than the leucine marker (To-kunaga and Mizuguchi, personal communica-tion). This observation supports the idea thatan Hfr mediates the conjugation.

MappingA partial map of the genome of mycobacter-

iophage D29 by the isolation of temperature-sensitive mutants and the determination ofrecombinational sequences has been con-structed by Mizuguchi and Sellers (260). Bygrowing the mutants at the permissive temper-ature for varying intervals and then subjectingthem to the restrictive temperature they wereable to find out the physiological role of thevarious genes, as has been done in T4 phages.

CONCLUDING REMARKSThe information presented here shows that

a significant amount of work on the metabo-lism of mycobacteria has been carried outsince the last review was published. However,some metabolic problems have received littleattention, while others seem to have been over-emphasized.The metabolism of carbohydrates and lipids

by mycobacteria has been extensively studied,and the operation of classical pathways such asthe glycolytic pathway, hexose monophosphateshunt, citric acid cycle, and ,B-oxidation hasbeen well documented. Several enzymes ofthese pathways have been partially purified,but the lactate oxidative decarboxylase of M.phlei and M. smegmatis is the only mycobac-terial enzyme that has been crystallized. Againa comparison of oxidative and glycolytic path-ways in virulent and avirulent strains of myco-bacteria has been attempted. This attemptwas made in order to discover whether thecapacity of the virulent strains to multiply inthe host tissues is due to differences in oxida-tive metabolism or not; it is known that theoxygen tension in the host tissues is low. Be-cause the differences between virulent andavirulent strains seem to be quantitative, nodefinitive explanation has been possible.Some of the metabolic properties of tuberclE

bacilli grown in vivo (in susceptible laborator3animals) and in vitro (in chemically definedmedia) have been compared, and some inter-esting differences have been found. More de-tailed studies on such differences would bedesirable.

Electron transport and oxidative phos-phorylation in mycobacteria, particularly in M.phlei, have been studied thoroughly by Brodieand his colleagues. The phosphorylation sites

92 BACTERIOL. REV.


and the coupling factors of the respiratorychain have been characterized. Limited infor-mation is available on M. tuberculosis also, butthe pathogenic strains of M. tuberculosis needsystematic investigation.Some headway has been made on problems

of the biosynthesis of simple fatty acids inmycobacteria. Our knowledge of the biosyn-thesis of more complex fatty acids and lipids isstill based on data obtained from whole cellsby using some labeled precursors, or from cell-free systems in which only a few steps havebeen worked out. Detailed studies with puri-fied enzymes or enzyme complexes of lipidbiosynthesis and the determination of cofactorrequirements of the enzymes should be under-taken.

There are certain qualitative differences inthe amino acid metabolism of the virulent(H37Rv) and the avirulent (H37Ra) strains ofM. tuberculosis. The enzyme asparaginase,responsible for the breakdown of asparagine(which is the preferred nitrogen source fortheir growth in synthetic media), is distributeddifferently in the virulent and avirulentstrains. While the H37Rv strain possesses onlyone type of asparaginase, the H37Ra strainpossesses two asparaginases. Notably, the en-zyme specific to H37Ra is inducible; it inhibitsthe growth of Yoshida ascites sarcoma in rats,and its inhibitory activity is superior to that ofother bacterial asparaginases studied hitherto.Yet another difference between the two strainsis that the avirulent strain possesses a specificaspartotransferase, which transfers the as-

partyl moiety of asparagine to hydroxylamine,and the avirulent strain does not. The possiblerole of these enzymes in the phenomenon ofvirulence is an open question.

Studies on nucleic acids and protein syn-thesis of mycobacteria are still at the prelimi-nary stage; such attempts have been largelyrestricted to studies on the incorporation ofsimple labeled precursors into these macro-

molecules and the effects of antituberculardrugs on precursor incorporation. A systematicapproach to this problem is urgently needed.The study of the genetics of mycobacteria is

still in its infancy. Successful use of variouschemical mutagens for the isolation of auxo-

trophic and other mutants of mycobacteria hasbeen reported, but here again peculiar prob-lems are posed because of the tendency ofmycobacteria to extreme clumping and theirrelatively long generation time. The formerproblem, however, may be solved by isolatingmutants which do not clump. Various bacterio-phages specific for mycobacteria have been iso-

lated, including some capable of transducinggenetic markers. The existence of differentmating types in mycobacteria has also beenreported recently. These observations raisehopes for unveiling the genetic make-up of themycobacterial chromosome. Although suc-cessful transfection has been carried out withisolated phage DNA, all attempts to demon-strate transformation in mycobacteria havefailed. The fragmentary genetic information onmycobacteria available at present is restrictedto the nonpathogenic, relatively fast-growingspecies.Knowledge on the mode of action of various

antitubercular drugs also is still scanty. Mostof the research on the action of drugs has beendone at the level of substrate utilization, or bytesting them on isolated enzymes. Hence,these studies fail to explain the specificity ofthe drug or the development of drug resist-ance. Earlier work showed that the develop-ment of resistance to the drug isoniazid is ac-companied by a loss of catalase or peroxidaseactivity of mycobacteria; this finding suggeststhat the study of metabolic differences, as aconsequence of the development of drug resist-ance, could lead to fruitful results.The information currently available on the

metabolic pathways in mycobacteria is not suf-ficient for devising new chemotherapeuticdrugs against mycobacteria. However, a poten-tial method for developing such drugs couldprobably be based on the biosynthetic path-ways unique to these organisms, such as thosefor vitamin K and mycobactins. A drug whichinhibits any of the enzymatic steps in thesepathways is likely to prevent the growth of anorganism like M. tuberculosis without in anyway harming the human host.Except for a few reports on the phenolases

and glutamate decarboxylase, nothing isknown about the metabolic activities of theimportant species, M. leprae. The develop-ment of suitable culture media for the in vitrogrowth of such organisms would greatly stimu-late research in this important area.

ACKNOWLEDGMENTS

The work described in this paper from the authors' labo-ratory was supported by grants from the Indian Institute ofScience, the Indian Council of Medical Research, and theBhabha Atomic Research Centre. Most of the equipmentused in the work has been provided by grants from theRockefeller Foundation, New York, and from the WellcomeTrust, London. The preparation of this article is supportedby the Indian Institute of Science.

The authors are also grateful to several of their colleaguesand in particular to Barry Bean, C. K. R. Kurup, and P. S.Sastry for reading, criticizing, and discussing the manu-script.

93VOL. 36, 1972


LITERATURE CITED

1. Adamek, L., P. Mison, H., Mohelska, and L.Trnka. 1969. Ultrastructural organization ofspheroplasts induced in Mycobacteriumsmegmatis by lysozyme or glycine. AMch.Mikrobiol. 69:227-236.

2. Aithal, H. N., and M. Sirsi. 1964. Vitamin B12synthesis by mycobacteria, Part II/ Isoniazidresistance and vitamin B,2 activity of Myco-bacterium tuberculosis H,7R,. Indian J.Biochem. 1:166-167.

3. Akamatsu, Y., and J. H. Law. 1968. Enzymaticsynthesis of 10-methylene stearic acid andtuberculostearic acid. Biochem. Biophys. Res.Commun. 33:172-176.

4. Akamatsu, Y., and J. H. Law. 1969. Enzymaticsynthesis of alkylated fatty acids in Mycobac-terium phlei extracts. Fed. Proc. 28:538 (Ab-stract).

5. Akamatsu, Y., and J. H. Law. 1970. Enzymaticalkylenation of phospholipid fatty acid chainsby extracts of Mycobacterium phlei. J. Biol.Chem. 245:701-708.

6. Akamatsu, Y., and J. H. Law. 1970. The enzy-matic synthesis of fatty acid methyl esters bycarboxyl group alkylation. J. Biol. Chem.245:709-713.

7. Akamatsu, Y., Y. Ono, and S. Nojima. 1967.Studies on the metabolism of phospholipidsin Mycobacterium phlei. 1. Difference inturnover rates of individual phospholipids. J.Biochem. (Tokyo) 61:96-102.

8. Albertson, J. W., and A. G. Moat. 1965. Biosyn-thesis of nicotinic acid by Mycobacteriumtuberculosis. J. Bacteriol. 89:540-541.

9. Allaudeen, H. S., and T. Ramakrishnan. 1968.Biosynthesis of isoleucine and valine inMycobacterium tuberculosis H37Rv. Arch.Biochem. Biophys. 125:199-209.

10. Allaudeen, H. S., and T. Ramakrishnan. 1970.Biosynthesis of isoleucine and valine in My-cobacterium tuberculosis H37Rv. II. Purifica-tion and properties of acetohydroxy acidisomerase. Arch. Biochem. Biophys. 140:245-256.

11. Allaudeen, H. S., and T. Ramakrishnan. 1971.Biosynthesis of isoleucine and valine in My-cobacterium tuberculosis H37Rv. III. Purifi-cation and properties of reductase and reduc-toisomerase. Indian J. Biochem. Biophys. 8:23-27.

12. Allaudeen, H. S., and T. Ramakrishnan. 1971.Occurrence of ascorbic acid in Mycobac-terium tuberculosis H37Rv. Indian J. Exp.Biol. 9:278-279.

13. Anderson, R. J. 1930. The chemistry of the li-poids of tubercle bacilli. XIV. The occurrence

of inosite in the phosphatide from humantubercle bacilli. J. Amer. Chem. Soc. 52:1607-1608.

14. Ando, K. 1960. Effect of streptomycin on po-lynucleotide phosphorylase from avian tu-bercle bacilli. Nagoya J. Med. Sci. 23:39-51.

15. Andrejew, A. 1968. Differentiation des Phos-

phatases acides des mycobacteries. Ann. Inst.Pasteur 115:343-349.

16. Andrejew, A., and A. Renard. 1968. Tentativeseparation of the catalase and peroxidase ac-tivities in mycobacteria. Ann. Inst. Pasteur115:3.

17. Ariji, F., J. Yamaguchi, K. Fukushi, and S.Oka. 1968. Electron microscopic localizationof enzymes in Mycobacterium tuberculosis.Sci. Rep. Res. Inst. Tohoku Univ. Ser. C.(Med). 15:88-97.

18. Arora, K. L., and S. R. Guha. 1961. Studies inMycobacterium tuberculosis-urease activity.J. Sci. Indust. Res. 20C:227-231.

19. Arora, K. L., T. N. Srikantan, and C. R.Krishna Murti. 1959. Studies in Mycobac-terium tuberculosis-aldolase activity. J. Sci.Industr. Res. 18C:191-195.

20. Artman, M., and A. Bekierkurst. 1961. Myco-bacterium tuberculosis H37Rv grown in vivo:nature of the inhibitor of lactic dehydro-genase of Mycobacterium phlei. Proc. Soc.Exp. Biol. Med. 106:610-614.

21. Asano, A., and A. F. Brodie. 1963. Oxidativephosphorylation in fractionated bacterial sys-tems. XI. Separation of soluble factors neces-sary for oxidative phosphorylation. Biochem.Biophys. Res. Commun. 13:416-421.

22. Asano, A., and A. F. Brodie. 1964. Oxidativephosphorylation in fractionated bacterial sys-tems. XIV. Respiratory chains of Mycobac-terium phlei. J. Biol. Chem. 239:4280-4291.

23. Asano, A., and A. F. Brodie. 1965. Oxidativephosphorylation in fractionated bacterial sys-tems. XVIII. Phosphorylation coupled to dif-ferent segments of the respiratory chains ofMycobacterium phlei. J. Biol. Chem. 240:4002-4010.

24. Asano, A., and A. F. Brodie. 1965. The proper-ties of the non-phosphorylative electrontransport by-pass enzymes of Mycobacteriumphlei. Biochem. Biophys. Res. Commun. 19:121-126.

25. Asano, A., A. F. Brodie, A. F. Wagner, P. E.Wittreichs, and K. Folkers, 1962. The newsynthetic 6-chromanyl phosphate of vitaminK1 (20) and its J. Biol. Chem. 237:2411-2412.

26. Asano, A., T. Kaneshiro, and A. F. Brodie.1965. Malate-vitamin K reductase, a phos-pholipid requiring enzyme. J. Biol. Chem.240:895-905.

27. Asselineau, J., and P. Bennet. 1964. Biosyn-thesis of branched-chain fatty acids of myco-bacteria, p. 111-123. Metab. Physiol. Signifi-cance Lipids, Proc. 1963. Cambridge, Eng-land.

28. Asselineau, J., and E. Lederer. 1964. Constitu-ents of mycobacteria, p. 1-37. In V. C. Barry(ed.), Chemotherapy of tuberculosis. Butter-worths, London.

29. Asselineau, C., H. Montrozier, and J. C. Prome.1969. Presence d'acides polyinsature's dansune bacterie; isolement, a partir des lipidesde Mycobacterium phlei, d'acide hexatriacon-tapentaens-4,8,12,16,20 oique et d'acides

94 BACTERIOL. IREV.


analogues. Eur. J. Biochem. 10:580-584.

30. Azerad, R., R. Bleiler-Hill, and E. Lederer.1965. Biosynthesis of vitamin K2 by cell-freeextracts of Mycobacterium phlei. Biochem.Biophys. Res. Commun. 19:194-197.

31. Bastarrachea, F., D. G. Anderson, and D. S.Goldman. 1961. Enzyme systems in the my-

cobacteria. XI. Evidence for a functional gly-colytic system. J. Bacteriol. 82:94-100.

32. Bastarrachea, F., and D. S. Goldman. 1961.Diphosphopyridine nucleotide-linked nitratereductase from Mycobacterium tuberculosis.Biochim. Biophys. Acta 50:174-176.

33. Beaman, L., and L. Barksdale. 1970. Phenoloxi-dase activity in organisms isolated from lep-romatous and tuberculoid leprosy. J. Bac-teriol. 104:1406-1408.

34. Bekierkunst, A., and M. Artman. 1959. Effectof cell-free extracts from Mycobacteriumtuberculosis H37Rv on lung succinoxidase.Nature (London) 184:458.

35. Bekierkunst, A., and A. Bricker. 1967. Studieson the mode of action of isoniazid on myco-

bacteria. Arch. Biochem. Biophys. 122:385-392.

36. Bird, 0. D. 1947. Vitamin content of tuberclebacilli. Nature (London) 159:33.

37. Bloch, H. 1960. Biochemical properties of viru-lent and avirulent strains of Mycobacteriumtuberculosis. Ann. N.Y. Acad. Sci. 88:1075-1086.

38. Bogin, E., T. Higashi, and A. F. Brodie. 1969.Exogenous NADH oxidation and particulatefumerate reductase in Mycobacterium phlei.Arch. Biochem. Biophys. 129:211-220.

39. Bogin, E., T. Higashi, and A. F. Brodie. 1969.Extra particulate chain interaction betweendifferent electron transport particles. Science165:1364-1367.

40. Bogin, E., T. Higashi, and A. F. Brodie. 1970.Oxidative phosphorylation in fractionatedbacterial systems. XLIII. Coupling factorsassociated with the NAD+-linked electrontransport pathway. Arch. Biochem. Biophys.136:337-351.

41. Bogin, E., T. Higashi, and A. F. Brodie. 1970.The effect of trypsin and heat treatment on

oxidative phosphorylation in Mycobacteriumphlei. Biochem. Biophys. Res. Commun. 41:995-1001.

42. Bogin, E., T. Higashi, and A. F. Brodie. 1970.Interchangeability of coupling factors frombacterial and mammalian origin. Biochem.Biophys. Res. Commun. 38:478-483.

43. Bogin, E., T. Higashi, and A. F. Brodie. 1970.Effects of heat treatment of electron-trans-port particles on bacterial oxidative phos-phorylation. Proc. Nat. Acad. Sci. U.S.A. 67:1-6.

44. Bowles, J. A., and W. Segal. 1965. Kinetics ofutilization of organic compounds on thegrowth of Mycobacterium tuberculosis. J.Bacteriol. 90:157-163.

45. Brennan, P., and C. E. Ballou. 1967. Biosyn-thesis of mannophosphoinositides by Myco-

bacterium phlei. The family of dimmanno-phosphoinositides. J. Biol. Chem. 242:3046-3056.

46. Brennan, P., and C. E. Ballou. 1968. Biosyn-thesis of mannophosphoinositides by Myco-bacterium phlei. Enzymic acylation of thedimannophosphoinositides. J. Biol. Chem.243:2975-2984.

47. Brennan, P. J., S. A. Rooney, and F. G.Winder. 1970. The lipids of Mycobacteriumtuberculosis B.C.G.: fractionation, composi-tion, turnover and the effects of isoniazid.Irish J. Med. Sci. 3:371-390.

48. Brindley, D. N., S. Matsumara, and K. Bloch.1969. Mycobacterium phlei fatty acid synthe-tase-a bacterial multienzyme complex. Na-ture (London) 224:666-669.

49. Bristol, G., and G. J. Schroepfer, Jr. 1966. Bio-synthesis of tuberculostearic acid. Biochim.Biophys. Acta 125:389-391.

50. Brodie, A. F. 1959. Oxidative phosphorylationin fractionated bacterial systems. J. Biol.Chem. 234:398-404.

51. Brodie, A. F. 1965. The role of naphthoquinonesin oxidative metabolism, p. 355-404. In R. A.Morton (ed.), Biochemistry of quinones. Aca-demic Press Inc., London.

52. Brodie, A. F., and J. W. Adelson. 1965. Respi-ratory chains and sites of coupled phospho-rylation. Science 149:265-269.

53. Brodie, A. F., and J. Ballantine. 1960. Oxida-tive phosphorylation in fractionated bacterialsystems. II. The role of Vitamin K. J. Biol.Chem. 235:226-231.

54. Brodie, A. F., and C. T. Gray. 1956. Phospho-rylation coupled to oxidation in bacterial ex-tracts. J. Biol. Chem. 219:853-862.

55. Brodie, A. F., and C. T. Gray. 1957. Bacterialparticles in oxidative phosphorylation. Sci-ence 125:534-537.

56. Brodie, A. F., and T. Watanabe. 1966. Mode ofaction of vitamin K in microorganisms, p.447-463. In International symposium on vita-mins K and related quinones, Elsinore, Den-mark; Vitamins and hormones, vol. 24. Aca-demic Press Inc., New York.

57. Brummond, D. O., M. Stachclin, and S. Ochoa.1957. Enzymatic synthesis of polynucleotides.II. Distribution of polynucleotide phospho-rylase. J. Biol. Chem. 225:835-849.

58. Campbell, I. M., and R. Bentley. 1968. Inhomo-geneity of vitamin K2 in Mycobacteriumphlei. Biochemistry 7:3323-3327.

58a. Campbell, I. M., and J. Naworal. 1969. Com-position of the saturated and monounsatur-ated fatty acids of Mycobacterium phlei. J.Lipid Res. 10:593-598.

59. Cattaneo, C., P. L. Ipata, and M. C. C. Gabrielli.1960. Polynucleotide phosphorylase of myco-bacteria. 1. Exchange reactions between di-nucleotides and 32P in Mycobacterium smeg-matis strain Milch. Ann. Inst. Carlo Forlanini20:325-337.

60. Cattaneo, C., M. Morallini, L. Felicetti, and L.Ferri. 1962. Deaminases of purine nucleosides

95VOL. 36, 1972

96 RAMAKRISHNAN, MURTHY, AND GOPINATHAN

in mycobacteria. Ann. Inst. Carlo Forlanini22:290-302.

61. Cesari, I. M., M. Rieber, and T. Imaeda. 1969.Localization and properties of enzyme in-volved with electron transport activity inmycobacteria. J. Bacteriol. 98:767-773.

62. Chandramouli, V., G. Raghupathi Sarma, andT. A. Venkitasubramanian. 1971. Phospho-lipids of mycobacteria. Possible presence ofglyceryl ethers. Indian J. Biochem. Biophys.8:55-56.

63. Chandrasekhar, S., A. J. H. de Monte, and T.A. Venkitasubramanian. 1958. Fattyacidsfrom the lipids of streptomycin-resistant tu-bercle bacilli. Indian J. Med. Res. 46:643-647.

64. Chargaff, E. 1955. Isolation and composition ofthe deoxypentose nucleic acids and of thecorresponding nucleoproteins, p. 307-371, InE. Chargaff and J. N. Davidson (ed.), Thenucleic acids, vol. 1. Academic Press Inc.,New York.

65. Chargaff, E., and H. F. Saidel, 1949. On thenucleoproteins of avian tubercle bacilli. J.Biol. Chem. 177:417-428.

66. Cohn, D. V. 1958. The enzymatic formation ofoxaloacetic acid by non-pyridine nucleotidemalic dehydrogenase of Micrococcus lyso-deikticus. J. Biol. Chem. 233:299-304.

67. Cousins, F. B. 1956. The lactic acid oxidase ofthe mycobacteria. Biochem. J. 64:297-304.

68. David, H. L. 1970. Effect of O-carbamyl-D-serine on the growth of Mycobacterium tu-berculosis. Amer. Rev. Resp. Dis. 102:68-74.

69. David, H. L., D. S. Goldman, and K. Tak-ayama. 1970. Inhibition of the synthesis ofwax D peptidoglycolipid of Mycobacteriumtuberculosis by D-cyloserine. Infect. Immunity1:74-77.

70. David, H. L., K. Takayama, and D. S.Goldman. 1969. Susceptibility of mycobac-terial D-alanyl-D-alanine synthetase to D-cy-closerine. Amer. Rev. Resp. Dis. 100:579-581.

71. De Turk, W. E., and F. Bernheim, 1958. Effectsof ammonia, methylamine, and hydroxyl-amine on the adaptive assimilation of nitriteand nitrate by a mycobacterium. J. Bacteriol.75:691-696.

72. Di Mari, S. J., C. D. Synder, and H. Rapport.1968. The role of the quinone in oxidativephosphorylation: evidence against carbon-hydrogen bond cleavage. Biochemistry 7:2301-2317.

73. Dudley, M. A., and H. P. Willett. 1965. Nico-tinamide adenine dinucleotide synthesis byMycobacterium tuberculosis var. bovis. Proc.Soc. Exp. Biol. Med. 119:807-812.

74. Dudley, M. A., and H. P. Willett. 1966. Com-parison of nicotinamide adenine dinucleotidesynthesis by Mycobacterium tuberculosis andMycobacterium bovis. Can. J. Microbiol. 12:1225-1233.

75. Dunn, D. B., and J. D. Smith. 1958. The occur-rence of 6-methylaminopurine in deoxyribo-nucleic acids. Biochem. J. 68:627-636.

BACTERIOL. REV.

76. Dunphy, P. J., D. L. Gutnick, P. G. Phillips,and A. F. Brodie. 1968. A new naturalnaphthoquinone in Mycobacterium phlei cis-dihydro-menaquinone-9, structure and func-tion. J. Biol. Chem. 243:398-407.

77. Ebina, T., M. Motomiya, T. Munakata, and G.Kobuga. 1961. Effect de l'isoniazide sur lesacides gras des mycobacteries. C. R. Soc.Biol. 155:1176-1178.

78. Eda, T. 1964. Studies on the protein biosyn-thesis in mycobacteria. I. Preparation andcharacterization of the ribosomes of Myco-bacterium 607. Jap. J. Bacteriol. 19:201-206.

79. Eda, T. 1964. Studies on the protein biosyn-thesis in mycobacteria. II. Isolation and char-acterization of the ribosomes of BacillusCalmette Guerin. Jap. J. Bacteriol. 19:411-417.

80. Eda, T. 1964. Studies on the protein biosyn-thesis in mycobacteria. III. Influence of var-ious antibiotics on the protein biosynthesis inmycobacteria. Jap. J. Bacteriol. 19:437-442.

81. Edson, N. L. 1951. The intermediary metabo-lism of mycobacteria. Bacteriol. Rev. 15:147-182.

82. Edson, N. L., G. J. E. Hunter, R. G. Kulka,and D. E. Wright. 1959. The metabolism ofglycerol by Mycobacterium butyricum.Biochem. J. 72:249-261.

83. Ellard, G. A., and P. H. Clarke. 1959. Acetateand fumarate permeases of Mycobacteriumsmegmatis. J. Gen. Microbiol. 21:338-343.

84. Ellfolk, N., and N. Katunuma. 1959. The occur-rence of ammonia activating enzyme in var-ious organisms. Arch. Biochem. Biophys. 81:521-522.

85. Erdos, T., and A. Ullmann. 1959. Effect ofstreptomycin on the incorporation of aminoacids labelled with '4C into RNA and proteinin a cell-free system of mycobacterium. Na-ture (London) 183:618-619.

86. Erdos, T., and A. Ullmann. 1960. Effect ofstreptomycin on the incorporation of tyrosinelabelled with 14C into protein of mycobac-terium cell fractions in vivo. Nature (London)185:100-101.

87. Etemadi, A. H., and E. Lederer. 1965. Bio-synthese de l'acide a-smegmamycolique.Biochim. Biophys. Acta 98:160-167.

88. Ferguson, J. A., and C. E. Ballou. 1970. Biosyn-thesis of a mycobacterial lipopolysaccharide.Properties of the polysaccharide methyltransferase. J. Biol. Chem. 245:4213-4223.

89. Forbes, M., N. A. Kuck, and E. A. Peets. 1962.Mode of action of ethambutol. J. Bacteriol.84:1099-1103.

90. Forbes, M., N. A. Kuck, and E. A. Peets. 1965.Effect of ethambutol on nucleic acid metabo-lism in Mycobacterium smegmatis and itsreversal by polyamines and divalent cations.J. Bacteriol. 89:1299-1305.

91. Fowler, A. V., M. Camien, and S. Dunn. 1961.Acetyl L-isoleucine and acetyl L-leucine asextracellular products of Mycobacteriumranae. J. Bacteriol. 81:163-164.


92. Francis, M. J. O., D. E. Hughes, H. L. Korn-berg, and P. J. R. Phizackesby. 1963. Theoxidation of L-malate by Pseudomonas sp.Biochem. J. 89:430-438.

93. Fukuyama, K., and J. Tani. 1968. Protein syn-thetic activities of mycobacterial ribosomesin vivo. Biochim. Biophys. Acta 155:611-613.

94. Fulco, A. J., and K. Bloch. 1964. Cofactor re-quirements for the formation of A9-unsatu-rated fatty acids in Mycobacterium phlei. J.Biol. Chem. 239:993-997.

95. Gale, G. R. 1952. The oxidation of benzoic acidby mycobacteria. I. Metabolic pathways inMycobacterium tuberculosis, Mycobacteriumbutyricum, and Mycobacterium phlei. J. Bac-teriol. 63:273-278.

96. Gale, P. H., B. H. Arison, N. R., Trenner, A. C.Page, Jr., K. Folkers, and A. F. Brodie. 1963.Characterisation of vitamin K9 (H) fromMycobacterium phlei. Biochemistry 2:200-203.

97. Gasior, E. 1961. Aktywacja aminokwaso'w uMycobacterium phlei. Ann. Univ. MariaeCuris-Sklodowska, Lublin-Polonia Sec. D 16:163-181.

98. Gasior, E. 1966. Enzymatic activation of D-glutamic acid and D-alanine in Mycobac-terium phlei. Acta Microbiol. Polon. 15:3-11.

99. Gastambide-Odier, M. 1959. Determination ofdehydrogenases in atypical strains and inother mycobacteria. J. Bacteriol. 77:748-752.

100. Gastambide-Odier, M. 1966. Biosynthese duphtiocerol: incorporation d'acide propioniquedans le groupement methyle lateral en posi-tion 4 du phtiocerol. C. R. Acad. Sci. Ser. C.262:1401-1404.

101. Gastambide-Odier, M., J. M. Delaumeny, andE. Lederer. 1963. Biosynthesis de l'acids C82mycoserosique: incorporation d'acide pro-pionique. Biochim. Biophys. Acta 70:670-678.

102. Gastambide-Odier, M., H. Grisebach, H. Ach-enbach, and E. Lederer. 1964. Biosynthesis ofbranched-chain fatty acids of mycobacteria,p. 111-123. In J. Asselineau and P. Bennet(ed.), Metab. Physiol. Significance Lipids,Proc. 1963. Cambridge, Endland.

103. Gastembide-Odier, M., P. Sarda, and E. Led-erer. 1967. Biosynthese des aglycones desmycosides A at B. Bull. Soc. Chim. Biol. 49:849-864.

103a. Gayathri Devi, B., T. Ramakrishnan and K. P.Gopinathan. 1971. Enzymatic interactionbetween isoniazid and nicotinamide-adeninedinucleotide, p. 83. In Proceedings of theBiochemical Society, Agenda Papers, of theGolden Jubilee Meetings of Department ofBiochemistry, Indian Institute of Science,Bangalore, under the auspices of The Bio-chemical Society (U.K.) and The Society ofBiological Chemists (India).

104. Gelbard. A., and D. S. Goldman. 1961. Enzymesystems in the mycobacteria. X. The butyryldehydrogenase system. Arch. Biochem. Bio-phys. 94:228-235.

105. Gelbart, S. M., and S. E. Juhasz. 1969. Myco-

bacterium sp. strain 80, Mycobacterium phleior Mycobacterium smegmatis? J. Gen. Micro-biol. 59:141-143.

105a. Gelbart, S. M., and S. E. Juhasz. 1970. Ge-netic transfer in Mycobacterium phlei. J.Gen. Microbiol. 64: 253-254.

106. Goldman, D. S. 1956. Enzyme systems in my-cobacteria. I. The isocitric dehydrogenase. J.Bacteriol. 71:732-736.

107. Goldman, D. S. 1956. Enzyme systems in themycobacteria. II. The malic dehydrogenase.J. Bacteriol. 72:401-405.

108. Goldman, D. S. 1957. Enzyme systems in themycobacteria. III. The condensing enzyme. J.Bacteriol. 73:602-607.

109. Goldman, D. S. 1958. Enzyme systems in themycobacteria. IV. The pyruvic oxidase.Biochim. Biophys. Acta 27:506-512.

110. Goldman, D. S. 1958. Enzyme systems in themycobacteria. V. The pyruvic dehydrogenasesystem. Biochim. Biophys. Acta 27:513-518.

111. Goldman, D. S. 1959. Enzyme systems in themycobacteria. VI. Further studies on the py-ruvic dehydrogenase system. Biochim. Bio-phys. Acta 32:80-95.

112. Goldman, D. S. 1959. Enzyme systems in themycobacteria. VII. Purification, propertiesand mechanism of action of the alanine dehy-drogenase. Biochim. Biophys. Acta 34:527-539.

113. Goldman, D. S. 1961. Enzyme systems in my-cobacteria. A review. Adv. Tuberc. Res. 11:1-44.

114. Goldman, D. S. 1970. Subcellular localization ofindividual mannose containing phospholipidsin Mycobacterium tuberculosis. Amer. Rev.Resp. Dis. 102:543-555.

115. Goldman, D. S., and A. Gelbard. 1959. Enzymesystems in the mycobacteria. VIII. The fattyacid oxidizing system. Arch. Biochem. Bio-phys. 83:360-370.

116. Goldman, D. S., and F. A. Lornitzo. 1962. En-zyme systems in the mycobacteria. XII. Theinhibition of the transglycosidase-catalyzedformation of trehalose 6-phosphate. J. Biol.Chem. 237:3332-3338.

117. Goldman, D. S., and M. J. Wagner. 1962. En-zyme systems in mycobacteria. XIII. Glycinedehydrogenase and the glyoxylic acid cycle.Biochim. Biophys. Acta 65:297-306.

118. Goldman, D. S., M. J. Wagner, T. Oda, and A.L. Shug. 1963. Oxidation of reduced nicotin-amide adenine dinucleotide by subcellularparticles from Mycobacterium tuberculosis.Biochim. Biophys. Acta 73:367-379.

119. Gopinathan, K. P. 1965. Studies on the metab-olism of nicotinamide adenine nucleotides inMycobacterium tuberculosis H37Rv. Ph.D.thesis, Indian Institute of Science, Bangalore,India.

120. Gopinathan, K. P., T. Ramakrishnan, and C. S.Vaidyanathan. 1966. Purification and proper-ties of an inhibitor for nicotinamideadeninedinucleotidase from Mycobacterium tubercu-losis H37Rv. Arch. Biochem. Biophys. 113:376-382.

97VOL. 36, 1972

98 RAMAKRISHNAN, MUR

121. Gopinathan, K. P., M. Sirsi, and T. Ramak-rishnan. 1963. Nicotinamide-adenine nucleo-tides of Mycobacterium tuberculosis H37Rv.Biochem. J. 87:444-448.

122. Gopinathan, K. P., M. Sirsi, and C. S. Vai-dyanathan. 1964. Nicotinamide-adenine dinu-cleotide glycohydrolase of Mycobacteriumtuberculosis H37Rv. Biochem. J. 91:277-282.

123. Gopinathan, K. P., M. Sirsi, and C. S. Vai-dyanathan. 1965. Nicotinamdie-adenine dinu-cleotide glycohydrolase activity in experi-mental tuberculosis. Biochem. J. 94:446-451.

123a. Goren, M. B. 1972. Mycobacterial lipids: se-lected topics. Bacteriol. Rev. 36:32-63.

124. Goren, M. B., 0. Brokl, B. C. Das, and E. Led-erer. 1971. Sulfolipid I of Mycobacteriumtuberulosis strain H37Rv. Nature of acyl sub-stituents. Biochemistry 10:72-81.

125. Gross, D., P. Banditt, A. Zureck, and H. R.Schutte. 1968. Cholinsaure als Zwischenstufein der Nicotinsaure-Biosynthes bei Mycobac-terium bovis stamm BCG. Z. Naturforsch.23b:390-391.

126. Gross, D., A. Feige, R. Stiecher, A. Zureck, andH. R. Schutte. 1965. Untersuchungen zurBiosynthese dur Nikotinsaure bei Mycobac-terium tuberculosis. Z. Naturforsch. 20b:11 16-1118.

127. Gross, D., H. R. Schutte, G. Hubner, and K.Mothes. 1963. The biosynthesis of nicotinicacid by Mycobacterium tuberculosis. Tetra-hedron Lett. 9:541-544.

128. Gross, W. M., and L. G. Wayne. 1970. Nucleicacid homology in the genus Mycobacterium.J. Bacteriol. 104:630-634.

129. Grossowicz, N., and Y. S. Halpern. 1956. Inhi-bition of nicotinamidase activity in cell-freeextracts of Mycobacterium phlei by 3-acetyl-pyridine. Biochim. Biophys. Acta 20:576-577.

130. Grosswicz, N., and Y. S Halpern. 1957. Enzy-matic transfer and hydrolysis involving glu-tamine and asparagine. J. Biol. Chem. 228:643-653.

131. Guerin, M., R. Azerad, and E. Lederer. 1965.Sur l'origine biogenetique du methyle en po-sition 2 de la vitamine K2 de Mycobacteriumphlei. Bull. Soc. Chim. Biol. 47:2105-2114.

132. Guerin, M., M. M. Leduc, and R. G. Azerard.1970. Biosynthese du noyau naphtoqui-nonique des menaquinones bacteriennes. Eur.J. Biochem. 15:421-427.

133. Guerin, M., R. G. Azerad, and E. Lederer. 1960.Metabolism des acides benzoique, phenylacetique et phenyl butyrique per des cellulusentieres et des extraites acellulaires de Myco-bacterium phlei. Bull. Soc. Chim. Biol. 50:187-193.

134. Guha, S. R., R. P. Saxena, and K. L. Arora.1962. Studies in Mycobacterium tuberculosisadenosine deaminase activity. J. Sci. Industr.Res. 21c:66-69.

135. Gutnick, D. L., and A. F. Brodie. 1965. Phos-phate dependent incorporation of tritium intoa naphthoquinone during oxidative phosphor-ylation. J. Biol. Chem. 240:3698-3699.

CT'HY, AND GOPINATHAN BACTERIOL. REV.

136. Halpern, Y. S., and N. Grossowicz. 1957. Hy-drolysis of amides by extracts from mycobac-teria. Biochem. J. 65:716-720.

137. Harris, A. B. 1967. Inhibition of growth andnucleic acid synthesis in iron-deficient Myco-bacterium smegmatis. J. Gen. Microbiol. 47:111-119.

138. Harris, A. B. 1969. Inhibition of growth andnucleic acid synthesis in zinc-deficient Myco-bacterium smegmatis. J. Gen. Microbiol. 56:27-33.

139. Harris, A. B. 1969. Effect of iron deficiency onnucleotide levels in Mycobacterium smeg-matis. Biochim. Biophys. Acta 190:554-556.

140. Hedgecock, L. W. 1956. Antagonism of the in-hibitory action of aminosalicylic acid onMycobacterium tuberculosis by methionine,biotin, and certain fatty acids, amino acids,and purines. J. Bacteriol. 72:839-846.

141. Hedgecock. L. W. 1958. Mechanisms involvedin the resistance of Mycobacterium tubercu-losis to para-aminosalicylic acid. J. Bacteriol.75:345-350.

142. Hedgecock, L. W. 1958. Antagonism of methio-nine in aminosalicylate inhibition of Myco-bacterium tuberculosis. J. Bacteriol. 75:417-421.

143. Hedgecock, L. W. 1965. Comparative study ofthe mode of action of para-aminosalicylicacid on Mycobacterium kansasii and Myco-bacterium tuberculosis. Amer. Rev. Resp.Dis. 91:719-727.

144. Hedgecock, L. W., and R. L. Costello. 1962.Utilization of nitrate by pathogenic and sap-rophytic mycobacteria. J. Bacteriol. 84:195-205.

145. Heinen, W., M. Kusunose, E. Kusunose, D. S.Goldman, and M. J. Wagner, 1964. Propertiesand origin of DPNH diaphorases from Myco-bacterium tuberculosis. Arch. Biochem. Bio-phys. 104:448-457.

146. Heirowski, M., and I. Westfal. 1962. Aminoacid activation in tuberculosis bacilli ofvarying sensitivity to antibacillary drugs.Bull. Soc. Amis. Sci. Lettr. Poznan Ser. C.12:17-23.

147. Heirowski, M., and I. Westfal. 1963. Activationof amino acids and the influence of strepto-mycin on this process in Mycobacterium tu-berculosis H37Rv. Acta Microbiol. Polon. 12:125-129.

148. Hey-Ferguson, A., and A. D. Elbein. 1970. Puri-fication of a D-mannose isomerase from My-cobacterium smegmatis. J. Bacteriol. 101:777-780.

149. Higashi, T., E. Bogin, and A. F. Brodie. 1969.Separation of a factor indispensable for cou-pled phosphorylation from the particulatefraction of Mycobacterium phlei. J. Biol.Chem. 244:500-502.

150. Higashi, T., E. Bogin, and A. F. Brodie, 1970.Oxidative phosphorylation in fractionatedbacterial systems. XLIII. The effect of cou-pling factors on urea treated particles. Arch.Biochem. Biophys. 136:331-336.

151. Hill, D. L., and C. E. Ballou. 1966. Biosynthesis


of mannophospholipids by Mycobacteriumphlei. J. Biol. Chem. 241:895-902.

152. Hobby, G. L. 1967. A look at the atypicals.Amer. Rev. Resp. Dis. 96:357-360.

153. Horvath, I., A. Szentirmai, and J. Zsadenyi,1967. Induced phenotypic resistance to valinein Mycobacterium pellegrino. J. Bacteriol.94:850-854.

154. Hovenkamp. H. G. 1959. Fractionation of thesystem bringing about oxidative phosphoryla-tion in Azotobacter vinelandii. Nature (Lon-don) 184:471.

155. Hudson, A. R., and R. Bentley. 1970. Impurityin a common growth medium component:presence of an isoflavanoid in samples of L-asparagine. J. Bacteriol. 104:599-600.

156. Hunter, G. J. E. 1953. The oxidation of glycerolby mycobacteria. Biochem. J. 55:320-328.

157. Imshenetskii, A. A., E. F. Efimochkina, V. A.Zomin, and L. E. Nikitin. 1968. Cholesteroldecomposition by mycobacteria. Mikrobiolo-giya 37:31-37.

158. Imshenetskii, A. A., and L. A. Marssina. 1968.Cholesterol decomposition by mycobacteria.Mikrobiologiya 37:620-627.

159. Indira, M. 1964. Studies on the glucose metabo-lism of the virulent and avirulent strains ofMycobacterium tuberculosis H37 Rv andH37Ra. Ph.D. thesis, Indian Institute of Sci-ence, Bangalore, India.

160. Indira, M., and T. Ramakrishnan. 1959. Biologyand biochemistry of microorganisms, p. 26.Abstracts, Golden Jubilee Symp. Indian In-stitute of Science, Bangalore, India.

161. Indira, M., and T. Ramakrishnan. 1962. Glu-cose dissimilation by Mycobacterium tuber-culosis H37Ra J. Sci. Industr. Res. 21:1-6.

162. Indira, M., and T. Ramakrishnan. 1963. Glu-cose dissimilation by Mycobacterium tuber-culosis H37Ra. J. Sci. Industr. Res. 21c:1-6.516.

163. Ipata, P. L., and C. Cattaneo. 1960. Polynucleo-tide phosphorylase of mycobacteria: 2) Par-tial purification of the enzyme. Ann. Inst.Carlo Forlanini 20:338-346.

164. Ishikawa, S., and A. L. Lehninger. 1962. Recon-stitution of oxidative phosphorylation inpreparations from Micrococcus lysodeikticus.J. Biol. Chem. 237:2401-2408.

165. Iwainsky, H. 1963. Analysis of the metabolismof mycobacteria with the aid of "P and 14C,p. 119-127. In J. F. Pasquier, L. Trnka, andK. R. Urbanci (ed.), The use of radioactiveisotopes in tuberculosis research: Proc. Inter-national Symp. Prague. Pergamon Press,Oxford, England.

166. Jacobs, N. J., and P. J. Van Demark. 1960.Comparison of the mechanism of glyceroloxidation in aerobically and anaerobicallygrown Streptococcus faecalis. J. Bacteriol. 79:532-538.

167. Jacquet, A., F. Tison, B. Polspod, P. Ross, andB. Devulder. 1966. Etude de la ,B-D-galactosi-dase des mycobacteries. Ann. Inst. Pasteur111:86-89.

168. Jaenicke, L., Ch. Molberger, I. Schile, R.Tschesche, K. Brandau, and A. Otto. 1969.Neopterin und Neopterin-3'-phosphate alsVerlasufer der Fobsaure bei Mycobacteriumsmegmatis. Z. Naturforsch. 24:569-574.

169. Jaure'guiberry, G., J. H. Law, J. M. Mc-Closkey, and E. Lederer. 1966. Studies on themechanism of biological carbon alkylationreactions. Biochemistry 4:347-353.

170. Jaure'guiberry, G., M. Lenfant, R. Toubiana, R.Azerad, and E. Lederer. 1966. Biosynthesis oftuberculostearic acid in a cell-free extract:Identification of 10-methylenestearic acid asan intermediate. Chem. Commun. (Chem.Soc. London) 23:855-857.

171. Jayaram, H. N., T. Ramakrishnan, M. Sirsi,and C. S. Vaidyanathan. 1970. Regulation ofL-asparagine amidohydrolases (EC 3.5.1.1)(L-asparaginase) from Mycobacterium tuber-culosis H37Ra and H37Rv. Indian J. Bio-chem. 7:237-240.

172. Jayaram, H. N., T. Ramakrishnan, and C. S.Vaidyanathan, 1968. L-asparaginases fromMycobacterium tuberculosis strains H37Rvand H37Rv. Arch. Biochem. Biophys. 126:165-174.

173. Jayaram, H. N., T. Ramakrishnan and C. S.Vaidyanathan, 1969. Aspartotransferase fromMycobacterium tuberculosis H37Ra. IndianJ. Biochem. 6:106-110.

174. Johnson, T. B., and R. D. Coghill, 1925. Re-searches on pyrimidines. C.111. The dis-covery of 5-methyl cytosine in tuberculinicacid, the nucleic acid of tubercle bacillus. J.Amer. Chem. Soc. 47:2838-2844.

175. Jones, A. S., M. Stacey and B. E. Watson,1957. The nucleotide sequence in deoxypen-tosenucleic acids. Part IV. The deoxyribonu-cleic acid of Mycobacterium phlei. J. Chem.Soc. Part II: 2454-2459.

176. Jones, W., and A. White. 1968. Lysogeny inmycobacteria. I. Conversion of colony mor-phology, nitrate reductase activity andTween 80 hydrolysis of Mycobacterium Sp.ATCC 607. Can. J. Microbiol. 14:551.

177. Juhasz, S. E. 1967. Reciprocal in toto conversionof Mycobacterium phlei - Mycobacteriumsmegmatis by mediation of an intermediatehybrid genome: B2h. Nature (London) 214:518-520.

178. Juhasz, S. E. 1968. Growth retardation, colonialchanges and nutritional deficiency in Myco-bacterium smegmatis due to lysogeny. J. Gen.Microbiol. 52:237-241.

179. Juhasz, S.E., and R. Bonicke. 1966. Reciprocalgenetic changes in mycobacterial host-virussystems; effect of lysogeny on the phage.Nature (London) 210:1185-1186.

180. Juhasz, S. E., S. Gelbast, and M. Harize. 1969.Phage induced alteration of enzymic activityin lysogenic Mycobacterium smegmatisstrains. J. Gen. Microbiol. 56:251-255.

181. Juhasz, S. E., and G. Plummer (ed). 1970.Host-virus relationships in Mycobacterium,Nocardia and Actinomycetes. Proceedings of

99VOL. 36, 1972


a symposium. Charles C Thomas, Publisher,Springfield, Ill.

182. Kanemasa, Y., and D. S. Goldman. 1965. Directincorporation of octanoate into long chainfatty acids by soluble enzymes of Mycobac-terium tuberculosis. Biochim. Biophys. Acta98:476-485.

183. Kaplan, N. 0. 1955. Animal tissue DPNasepyridine transglycosidase), p. 660-663. In S.P. Colowick and N. 0. Kaplan (ed.), Methodsin enzymology, vol. 2. Academic Press Inc.,New York.

184. Karasevich, Yu. N., and S. A. Butenko. 1966.O-nekotorykh Svvistakh gomoserin degidro-genazy Mycobacterium album shtamm 13.Mikrobiologiya 35:212-219.

185. Kahket. E. R., and A. F. Brodie. 1963. Oxida-tive phosphorylation in fractionated bacterialsystems. VI. Role of particulate and solublefractions from Escherichia coli. Biochim.Biophys. Acta 78:52-65.

186. Kates, M. 1966. Biosynthesis of lipids in micro-organisms. Annu. Rev. Microbiol. 20:13-44.

187. Katz, N., and H. G. Wood. 1963. The use of"4CO2 yields from glucose-l-and-6- '4C for theevaluation of the pathway of glucose metabo-lism. J. Biol. Chem. 238:517-523.

188. Kearney, E. B., and D. S. Goldman. 1970. Fur-ther studies on the NADH oxidase of the cy-toplasmic membrane of Mycobacterium tu-berculosis. Biochim. Biophys. Acta 197:197-205.

189. Kern. M., and R. Natale. 1958. A diphospho-pyridine nucleotidase and its protein inhib-itor from Mycobacterium butyricum. J. Biol.Chem. 231:41-51.

190. Kimura, T. 1959. Studies on the metabolism ofamides in Mycobacteriaceae. I. Purificationand properties of nicotinamidase from Myco-bacterium avium. J. Biochem. (Tokyo) 46:973-978.

191. Kimura, T. 1959. Studies on metabolism ofamides in Mycobacteriaceae. II. Enzymatictransfer of nicotinyl group of nicotinamide tohydroxylamine in Mycobacterium avium. J.Biochem. (Tokyo) 46:1133-1139.

192. Kimura, T. 1959. Studies on metabolism ofamides in Mycobacteriacease. III. Amidaseand transferases in the extracts from Myco-bacteriaceae. J. Biochem. (Tokyo) 46:1271-1279.

193. Kimura, T. 1959. Studies on the metabolism ofamides in Mycobacteriaceae. IV. formationand hydrolysis of hydroxamate. J. Biochem.(Tokyo) 46:1399-1410.

194. Kimura, T., and Sasakura, T. 1956. Lipid me-tabolism of Mycobacterium avium. I. En-zymic synthesis of hydroxamic acid from freefatty acid and its specificity. J. Biochem.(Tokyo) 43:175-185.

195. Kimura, T., and T. Sasakawa. 1957. Studies onpolyol dehydrogenase from Mycobacteriumavium. J. Biochem. (Tokyo) 44:859-862.

196. Kimura, T., and J. Tobari. 1963. Participation

of flavin adenine dinucleotide in the activityof malate dehydrogenase from Mycobac-terium avium. Biochim. Biophys. Acta 73:399-405.

197. Kirchheimer, W. F., and C. K. Whittaker. 1954.Asparaginase of mycobacteria. Amer. Rev.Tuberc. 70:920-921.

198. Klemperer, F., C. Scott, and S. Bagechi. 1967.Uric acid oxidation by mycobacteria Amer.Rev. Resp. Dis. 95:833-837.

199. Klubes, P., and A. F. Brodie. 1966. Oxidativephosphorylation in fractionated bacterial sys-tems. XXIII. Enzymatically reduced deriva-tives of vitamin K1 (20) obtained by acetyla-tion. Biochemistry 5:4171-4176.

200. Kogan, L. M., I. I. Zoretskaya, Zh. Y., Sys., 0.B. Tikhomirova, G. K. Skryabin, and I. V.Torgov. 1968. Transformation and utilizationof chloresterol by microorganisms. Prik.Biochim. Mikrobiol. 4:82-87.

201. Konickova-Radochova, M., and I. Malek. 1969.Mutagenic effect of nitrosoguanidine onMycobacterium phlei. Folia Microbiol. (Pra-gue) 14:201-207.

202. Konickova-Radochova, M., and I. Malek. 1969.The induction of auxotrophic mutants ofMycobacterium phlei PA by ultraviolet irra-diation. Folia Microbiol. (Prague) 14:251-255.

203. Konickova-Radochova, M., and I. Malek. 1969.The use of ethyl methanesulfonate (EMS) forthe induction of mutants in Mycobacteriumphlei strain PA. Folia Microbiol. (Prague) 14:470-474.

204. Konickova-Radochova, M., and I. Malek. 1970.Study of mutagenesis in Mycobacteriumphlei. Folia Microbiol. (Prague) 15:88-102.

205. Konno, K. 1956. New chemical method to dif-ferentiate human type tubercle bacilli fromother mycobacteria. Science 124:985.

206. Konno, K., R. Kurzmann, and K. T. Bird. 1957.The metabolism of nicotinic acid in mycobac-teria: a method for differntiating tuberclebacilli of human origin from other mycobac-teria. Amer. Rev. Tuberc. 75:529-537.

207. Konno, K., R. Kurzmann, K. T. Bird, and A.Sbarra. 1958. Differentiation of human tu-bercle bacilli from atypical acid-fast bacilli.1. Niacin production of human tubercle ba-cilli and atypical acid-fast bacilli. Amer. Rev.Tuberc. 77:669-674.

208. Konno, K., H. Nagayama, and S. Oka. 1959.Nicotinamidase in mycobacteria: a methodfor distinguishing bovine type tubercle bacillifrom other mycobacteria. Nature (London)184:1743-1744.

209. Konno, K., K. Oizumi, and S. Oka. 1965. Bio-synthesis of niacin ribonucleotide from quino-linic acid by mycobacteria. Nature (London)205:874-875.

210. Konno, K., K. Oizumi, Y. Shimizu, and S. Oka.1965. Niacin metabolism in mycobacteria:quinolinic acid as a precursor of niacin ribonu-cleotide and the difference in niacin biosyn-thesis among various species of mycobacteria.

100 BACTERIOL. REV.


Amer. Rev. Resp. Dis. 91:383-390.211. Krembel, J., and A. H. Etemadi. 1966. Sur la

biogenese acides mycoliques en C.. isoles deMycobacterium smegmatis. Bull. Soc. Chem.Biol. 48:67-75.

212. Krishna Murti, C. R., and A. F. Brodie. 1969.New light-sensitive cofactor required for oxi-dation of succinate by Mycobacterium phlei.Science 164:302-304.

213. Krishna Murti, C. R., V. K. Kalra, and A. F.Brodie. 1970. Oxidative phosphorylation onsuccinate by membranes of Mycobacteriumphlei, p. 505-513. Symposium on Macromole-cules in storage and transfer of Biological in-formation. Department of Atomic Energy,Government of India, Bombay.

214. Kuck, N. A., E. A. Peets, and M. Forbes. 1963.Mode of action of ethambutol on Mycobac-terium tuberculosis strain H37Rv. Amer. Rev.Resp. Dis. 87:905-906.

215. Kurup, C. K. R., and A. F. Brodie. 1967. Oxida-tive phosphorylation in fractionated bacterialsystems. XXV. Studies on the involvement ofmetal in Mycobacterium phlei. J. Biol.Chem. 242:197-203.

216. Kurup, C. K. R., and A. F. Brodie. 1967. Oxida-tive phosphorylation in fractionated bacterialsystems. XXIX. The involvement of non-heme iron in the respiratory pathways ofMycobacterium phlei. J. Biol. Chem. 242:5830-5837.

217. Kusaka, T., R. Sato, and K. Shoji. 1964. Com-parison of cytochromes in mycobacteriagrown in vitro and in vivo. J. Bacteriol. 87:1383-1388.

218. Kusunose, M., E. Kusunose, Y. Kowa, and Y.Yamamura. 1959. Carbon dioxide fixationinto malonate in Mycobacterium avium. J.Biochem. (Tokyo) 46:525-527.

219. Kusunose, E., M. Kusunose, Y. Kowa, and Y.Yamamura. 1960. The synthesis of fatty acidsin the cell-free extracts of Mycobacteriumavium. J. Biochem. (Tokyo) 47:689-693.

220. Laland, S. G., W. G. Overend, and M. Webb.1952. Deoxypentosenucleic acids. Part IV.The properties and composition of deoxypen-tosenucleic acids from certain animal, plantand bacterial sources. J. Chem. Soc. p. 3224-3231.

221. Lamonica, G., and A. H. Etemadi. 1967. Sur lapresence simaltance d'acides mycoliquescomportant deux cycles propaniques on deuxinsaturations dans les lipides de Myco-bacterium phlei. Compt. Rend. Acad. Sci.(Paris). Ser. C. 264:1711-1714.

222. Law, J. J. 1967. Bacterial lipids, p. 87-105. InB. D. Davis and L. Warren (ed.), The spe-cificity of cell surface. Prentice-Hall Inc.,Englewood Cliffs, N.J.

223. LeCam, M., Y. Madec, and S. Bernard. 1969.Le metabolisme intermediare des mycobac-teries. I. Comparison de deux "Instantanesmetaboliques" realises respective-mant surMycobacterium phlei et Mycobacterium tu-

berculosis souche H37Ra. Ann. Inst. Pasteur.Paris. 118:158-167.

224. Lederer, E. 1963. Biosynthesis of lipids. 5thIntern. Congr. Biochem. Ed. Popjak, Per-gamon Press. pp. 90-103.

225. Lederer, E. 1964. Excursion dans le mondeMysterieux des Mycobacteries. Proceedingsof the plenary sessions of the sixth Intern.Congr. Biochem. New York, p. 63-78.

226. Lederer, E. 1964. The origin and function ofsome methyl groups in branched chain fattyacids, plant sterols and quinones. Biochem. J.93:449-468.

227. Lederer, E. 1967. Glycolipids of mycobacteriaand related microorganisms. Chemistry andPhysics of Lipids, 1:294-315.

228. Leduc, M. M., P. M. Dansette, and R. G.Azerad. 1970. Incorporation de l'acide shiki-mique dans de noyau des napthoquinones d'origine bacterienne et vegetal. Eur. J.Biochem. 15:428-435.

229. Lenfant, M., H. Audier, and E. Lederer. 1966.Migration of hydrogen during tuberculo-stearic acid biosynthesis. Bull. Soc. Chem.Fr. (a). 2775-2777.

230. Lenfant, M., P. F. Hunt, L. M. Therier, F.Printe, and E. Lederer. 1970. N-methylationsde 1' aniline per Mycobacterium tuberculosis.Biochem. Biophys. Acta 201:82.

231. Lennarz, W. J. 1966. Lipid metabolism in thebacteria. Adv. Lipid. Res. 4:175-226.

232. Lennarz, W. J., G. Scheurbrandt, and K.Bloch. 1962. The biosynthesis of oleic and 10-methylstearic acids in Mycobacterium phlei.J. Biol. Chem. 237:664-671.

233. Lieb, M. 1967. Studies of heat-inducible Xphage. IV. Conversion of host phenotype by adefective prophage. Virology 31:643-656.

234. Lieb, M. 1969. Repressor-less A: plasmids andprophages, p. 15. Abstracts of papers pre-sented at the Bacteriophage Meetings, ColdSpring Harbor, New York, 24 to 30 August1969.

235. Linnane, A. W., and J. L. Still. 1955. The Intra-cellular distribution of enzymes in Serratiamarcescens. Biochim. Biophys. Acta 16:305-306.

236. Lornitzo, F. A., and D. S. Goldman. 1964. Puri-fication and properties of the transglucosylaseinhibitor of Mycobacterium tuberculosis. J.Biol. Chem. 239:2730-2734.

237. Lornitzo, F. A., and D. S. Goldman. 1965. Re-versible effect of Bicarbonate on the inhibi-tion of mycobacterial and yeast transgluco-sylase by mycoribnin. J. Bacteriol. 89:1086-1091.

238. Lornitzo, F. A., and D. S. Clpldman. 1968. In-tracellular localization of a 6-O-methyl-D-glucose containing soluble polysaccharidefrom Mycobacterium tuberculosis. Biochim.Biophys. Acta 158:329-335.

239. Lornitzo, F. A., and D. S. Goldman. 1969.Transmethylation to a-glycerol phosphate: apossible precursor in the formation of 6-0-

VOL. 36, 1972 101


methyl-a-glucose in Mycobacterium tubercu-losis. Biochem. Biophys. Res. Commun. 35:215.

240. Lukins, H. B., and J. W. Foster. 1963. Methyl-ketone metabolism in hydrocarbon utilizingmycobacteria. J. Bacteriol. 85:1074-1087.

241. Lynch, J. L., and F. C. Neuhaus. 1966. On themechanism of action of the antibiotic D-car-bamyl-D-serine in Streptococcus faecalis. J.Bacteriol. 91:449-460.

242. Lyon, R. H., and W. H. Hall. 1969. Influence ofasparagine on growth and amino acid utiliza-tion by Mycobacterium tuberculosis (H37Ra).Amer. Rev. Resp. Dis. 99:981.

243. Lyon, R. H., W. H. Hall, and C. Costas-Mar-tinaz. 1969. Uptake and distribution of la-belled carbon from "C-asparagine by Myco-bacterium tuberculosis. J. Bacteriol. 98:317-318.

244. Lyon, R. H., P. Rogers, W. H. Hall, and H. C.Lichstein. 1967. Inducible glutamate trans-port in mycobacteria and its relation to gluta-mate oxidation. J. Bacteriol. 94:92-100.

245. Lyon, R. H., W. H. Hall, and C. Costes-Mar-tinez. 1970. Utilization of amino acids duringgrowth of Mycobacterium tuberculosis in ro-tary cultures. Infect. Immunity 1:513-520.

246. Majerus, P. W., and P. R. Vagelose. 1967. Fattyacid biosynthesis and the role of the acyl car-rier proteins Adv. Lipid Res. 5:1-33.

247. Malathi, V. G. 1966. Studies on the metabolismof nucleic acids in Mycobacterium tubercu-losis H37Rv. Ph.D. thesis, Indian Institute ofScience, Bangalore, India.

248. Malathi, V. G., and T. Ramakrishnan. 1966.Biosynthesis of nucleic acid purines in Myco-bacterium tuberculosis H37Rv. Biochem. J.98:594-597.

249. Malathi, V. G. T. Ramakrishnan, and M. Sirsi.1959. The immunological effects of coconutwater in experimental tuberculosis of mice. J.Indian Inst. Sci. 41:52-57.

250. Malathi, V. G., M. Sirsi, T. Ramakrishnan, andR. K. Maller. 1964. Polynucleotide phospho-rylase of Mycobacterium tuberculosis H37Rv.Indian J. Biochem. 1:71-76.

251. Matsumara, S. 1970. Conformation of acyl car-rier protein from Mycobacterium phlei.Biochem. Biophys. Res. Commun. 38:238-243.

252. Matsumara, S., D. N. Brindley, and K. Bloch.1970. Acyl carrier protein from Mycobac-terium phlei. Biochem. Biophys. Res.Commun. 38:369-377.

253. Mastalerz, P., Z. Wicczorek, and M. Kochman.1965. Utilization of carbon-bound phosphorusby microorganisms. Acta Biochim. Polon. 12:151-156.

254. Michalska, K., and R. Lorenc. 1970. Distribu-tion of MAK (methylated albumin adsorbedon kieselguhr) columns of nucleic acids iso-lated from different strains of mycobacteria.Acta Microbiol. Polon. Ser. A. 2(19):31-38.

255. Middlebrook, G. 1954. Isoniazid resistance and

catalase activity of tubercle bacilli. Amer.Rev. Tuberc. 69:471-472.

256. Mil'ko, E. S., and N. P. Ivanova. 1970. Vliyaniesostava sredy i uslovii kul'tivirovania nasintez flavinov Mycobacterium lacticolumShtamm 104. Microbiologiya 39:71-76.

257. Mison, P., L. Trnka, H. Mohelska, and L.Adamek. 1969. Isolation problems and struc-tural organization of membrane units inMycobacterium smegmatis. Arch. Mikrobiol.69:216-226.

258. Mizuguchi, Y., and T. Tokunaga. 1970. Methodfor isolation of deoxyribonucleic acid frommycobacteria. J. Bacteriol. 104:1020-1021.

259. Mizuguchi, Y., and T. Tokunaga. 1971. Recom-bination between Mycobacterium smegmatisstrains Jucho and Lacticola. Jap. J. Micro-biol. 15:359-366.

260. Mizuguchi, Y., and M. I. Sellers. 1970. Isolationand characterization of temperature-sensitivephage D 29, p. 65-79. In S. E. Juhasz and G.Plummer (ed.), Host-virus relationships inMycobacterium, Nocardia, and Actinomyces.Proceedings of a Symposium, Charles CThomas, Publisher, Springfield, Ill.

261. Moat, A. G., and J. N. Albertson. 1964. Niacinbiosynthesis by Mycobacterium tuberculosis.Fed. Proc. 23:528.

262. Mora, J., and L. F. Bojalil. 1965. Antagonism ofthe D-alanine reversal of D-cycloserine actionby L-alanine in Mycobacterium acapulcensis.Proc. Soc. Exp. Biol. Med. 119:49-52.

263. Mothes, D. 1964. Zinikmangel und Nikotin-saure-Biosynthe bei Mycobacterium tubercu-losis (stamm BCG) Z. Allgem. Mikrobiol. 4:42-58.

264. Mothes, E., D. Gross, H. R. Schutte, and K.Mothes. 1961. Zur Biosynthese der Nicotin-saure bei Mycobacterium tuberculosis(stamm BCG). Naturwissenchaft 48:623.

265. Murthy, P. S., M. Sirsi, and T. Ramakrishnan.1969. Preliminary studies on isocitrate lyaseof Mycobacterium tuberculosis H37Rv, p. 42.Abstracts, Symposium on the chemistry andmetabolism of lipids and related subjects,Delhi, India.

266. Nagayama, H., K. Konno, and S. Oka. 1961.Formamidase in mycobacteria and its use indifferentiating saprophytic mycobacteriafrom other mycobacteria. Nature (London)190:1219-1220.

267. Nakamura, R. M. Transfection of Mycobac-terium smegmatis in an acid medium, p. 166-178. In S. E. Juhasz and G. Plummer (ed.),Host-virus relationships in Mycobacterium,Nocardia, and Actinomyces. Proceedings of aSymposium. Charles C Thomas Publisher,Springfield, Ill.

268. Nakamura, S., Y. Nishizuka, and 0. Hayaishi.1964. Regulation of nicotinamide adeninedinucleotide biosynthesis by adenosine tri-phosphate. J. Biol. Chem. 239:2717-2718.

269. Nielsen, H. M., and J. Bennedsen. 1968. Locali-zation of acid phosphatase activity in myco-

102 BACTrERIOL. REV.


bacterial cells with electron microscope. ActaPathol. Microbiol. Scand. 74:51-60.

270. Nolte, H. 1967. Untersuchungen uber dieHemmung der Mykobakteriollan Arginasedurch Streptomycin und verwandte Tuberku-lostatika. (Ein Beitrag zum Wirkungsme-chanismus des Streptomycin) Zentalbl. Bak-teriol. Parasintenk. Infektionskr. Hyg. ErsteAbt. Orig. 202:503-520.

271. O'Barr, T. P., and M. V. Rothlauf. 1970. Me-tabolism of D-glucose by Mycobacteriumtuberculosis. Amer. Rev. Resp. Dis. 101:964-966.

272. O'Barr, T. P., and M. A. Smith. 1969. Compar-ative NADH-diaphorase content of isoniazidresistant and isoniazid susceptible Mycobac-terium tuberculosis. Amer. Rev. Resp. Dis.99:116-118.

273. Oda, T., and D. S. Goldman. 1962. Terminalelectron transport mechanisms in the tu-bercle bacillus. A reduced diphosphopyridinenucleotide nitro-blue tetrazolium Diaphorase.Biochim. Biophys. Acta 59:604-613.

274. Odrzywolska, A. 1966. Synthesis of nucleicacids in normal cells of Mycobacterium spe-cies 607 and in cells infected with mycophageD 29. Bull. Acad. Pol. Sci. Ser. Sci. Biol. 14:741-745.

275. Ogasawara, N., N. Suzuki, M. Yoshino, and Y.Kotake. 1970. Complex formation betweennicotinamide adenine dinucleotidase and pro-tein inhibitor from Mycobacterium butyr-icum. Fed. Eur. Biochem. Soc. Lett. 6:337-338.

276. O'Leary, W. M. 1970. Bacterial lipid metabo-lism, p. 229-264. In M. Florkin and E. H.Stotz (ed.), Lipid metabolism. Comprehen-sive biochemistry, vol. 18. American ElsevierPublishing Co., New York.

277. Ono, Y., and S. Nojima. 1969. Phospholipasesof the membrane fraction of Mycobacteriumphlei. Biochim. Biophys. Acta 176:111-119.

278. Ono, Y., and S. Nojima. 1969. Phospholipase Aof Mycobacterium phlei: a regulatory mem-brane enzyme with Ferric iron as effector. J.Biochem. (Tokyo) 65:979-981.

279. Orme, T. W., B. Revsin, and A. F. Brodie. 1969.Phosphorylation linked to ascorbate oxidationin Mycobacterium phlei. Arch. Biochem.Biophys. 134:172-179.

280. Ott, J. L. 1960. Asparaginase from mycobac-teria. J. Bacteriol. 80:355-361.

281. Pangborn, M. C. 1968. Structure of mycobac-terial phosphatides. Ann. N.Y. Acad. Sci.154:133-139.

282. Parvin Khan, R., and S. V. Pande, and T. A.Venkitasubramanian. 1963. Malate metabo-lism in Mycobacteria. Indian J. Exp. Biol. 1:225-226.

283. Parvin Khan, R., S. V. Pande, and T. A. Venki-tasubramanian 1964. Purification and prop-erties of malate dehydrogenase (decarboxyl-ating) from Mycobacterium tuberculosis 607.Biochim. Biophys. Acta 92:260-277.

284. Parvin Khan, R., and T. A. Venkitasubram-anian. 1964. Biosynthesis of long chain fattyacids by cell free preparations of Mycobac-terium 607. Indian J. Biochem. 1:16-18.

285. Pattyn, S. R., and M. T. Hermans Boveroulle.1965. A rapid method for the demonstrationof arylsulphatase in mycobacteria. Its pres-ence among members of the genus Mycobac-terium. Amer. Rev. Resp. Dis. 92:297-298.

286. Pawlicki, S. A., C. S. Hertz, and R. A. Green.1963. Urease activity of mycobacteria. Amer.Rev. Resp. Dis. 87:284-285.

287. Pierard, A., and D. S. Goldman. 1963. Enzymesystems in the Mycobacteria. 14. Fatty acidsynthesis in cell-free extracts of Mycobac-terium tuberculosis. Arch. Biochem. Biophys.100:56-65.

288. Pinchot, G. B. 1953. Phosphorylation coupledto electron transport in cell-free extracts ofAlcaligens faecalis. J. Biol. Chem. 205:65-74.

289. Pope, H., and D. T. Smith. 1946. Synthesis ofB-complex vitamins by tubercle bacilli whengrown on synthetic media. Amer. Rev.Tuberc. 54:559-563.

290. Prabhakaran, K. 1967. Phenoloxidase of Myco-bacterium leprae. Nature (London) 215:436-437.

291. Prabhakaran, K. 1968. Properties of phenoloxi-dase in Mycobacterium leprae. Nature (Lon-don) 218:973-974.

292. Prabhakaran, K., and B. M. Braganca. 1962.Glutamic acid decarboxylase activity of-My-cobacterium leprae and occurrence of GABAin skin lesions of leprosy. Nature (London)196:589-590.

293. Prabhakaran, K., E. B. Harris, and W. F. Kirch-heimer. 1969. Effect of inhibition on phenol-oxidase of Mycobacterium leprae. J. Bac-teriol. 100:935-938.

294. Prabhakaran, K., and W. F. Kirchheimer. 1966.Use of 3,4-dihydroxyphenylalanine oxidationin the identification of Mycobacterium le-preae. J. Bacteriol. 92:1267-1268.

295. Prabhakaran, K., W. F. Kirchheimer, and E. B.Harris. 1968. Oxidation of phenolic com-pounds by Mycobacterium leprae and inhibi-tion of phenolase by substrate analogues andmetal chelators. J. Bacteriol. 95:2051-2053.

296. Prasada Reddy, T. L., P. Suryanarayana Mur-thy, and T. A. Venkitasubramanian. 1971.Isolation of a particulate (membrane) fractionfrom Mycobacterium smegmatis showingphosphorylation coupled to malate oxidation.In Proceedings of Biochemical Society,Agenda Papers, p. 82, Golden Jubilee meet-ings of the Department of Biochemistry, In-dian Institute of Science, Bangalore, underthe auspices of The Biochemical Society(U.K.) and The Society of Biological Chem-ists (India).

297. Raghupathi Sarma, G., V. Chandramouli, andT. A. Venkitasubramanian. 1970. Occurrenceof phosphonolipids in Mycobacteria. Bio-chim. Biophys. Acta 218:561.

103VOL. 36, 1972


298. Ramakrishnan, T., M. Indira, and M. Sirsi.1957. A new growth-promoting factor forMycobacterium tuberculosis. Nature (Lon-don) 179:1356.

299. Ramakrishnan, T., M. Indira, and M. Sirsi.1958. A new growth factor for Mycobacteriumtuberculosis. J. Indian Inst. Sci. 40:15-22.

300. Ramakrishnan, T., M. Indira, and R. K. Maller.1962. Evaluation of the route of glucose utili-zation in virulent and avirulent strains ofMycobacterium tuberculosis. Biochim. Bio-phys. Acta 59:529-532.

301. Ramasarma, T., and T. Ramakrishnan. 1961.Incorporation of 2- 4C mevalonic acid and 2-"C acetic acid into lipids of mycobacteria.Biochem. J. 81:303-308.

302. Rao, N. A. N., T. Ramakrishnan, and M. Sirsi.1961. Alanine dehydrogenase in Mycobac-teria, p. 1-7. Proceeding of a symposium onproteins, August 1960. Published by theCFTRI, Mysore, India.

303. Ratledge, C., and F. G. Winder, 1966. Biosyn-thesis and utilization of aromatic compoundsby Mycobacterium smegmatis with particularreference to the origin of salicylic acid.Biochem. J. 101:274-283.

304. Redmond, W. B. 1963. Bacteriophages of themycobacteria-a review. Adv. Tuberc. Res.12:191-229.

305. Reeves, H. C., R. Rabin, W. S. Wegener, and S.J. Ajl. 1967. Fatty acid synthesis and metabo-lism in microorganisms. Annu. Rev. Micro-biol. 21:225-256.

306. Reutgen, H., and H. Iwainsky. 1967. Untersu-chungen zum endogenon Stoff-Wechsol vonMykobacterien: I Mitt. zur Trennung phos-phorylierter Metabolite der sanorlslichenFrankton. Z. Naturforsch. 22:1043-1050.

307. Revsin, B., and A. F. Brodie. 1967. An effect ofinorganic phosphate and AMP at the thirdphosphorylative site of the respiratory chainof Mycobacterium phlei. Biochem. Biophys.Res. Commun. 28:635-640.

308. Revsin, B., and A. F. Brodie. 1969. Carbon-monoxide binding pigments of Mycobac-terium phlei and Escherichia coli. J. Biol.Chem. 244:3101-3104.

309. Rieber, M., and T. Imaeda. 1969. Effect of isoni-azid on mycobacterial polyphenylalaninesynthesis. Biochim. Biophys. Acta 186:173-177.

310. Rieber, M., T. Imaeda, and I. M. Cesari. 1969.Bacitracin action on membranes in mycobac-teria. J. Gen. Microbiol. 55:155-159.

311. del Rio-Estrada, C., and H. Patino. 1962. Nico-tinic acid biosynthesis by Mycobacteriumtuberculosis. J. Bacteriol. 84:871-872.

312. Robson, J. M., and F. M. Sullivan. 1963. Anti-tuberculosis drugs. Pharmacol. Rev. 15:169-223.

313. Rossi-Fanelli, A., P. L. Ipata, and P. Fasella.1961. On the distribution and transformationof thiamine and its phosphoric esters in bio-logical material. Biochem. Biophys. Res.

Commun. 4:23-27.314. Ruppel, W. G. 1898. Zur Chemie der Tuberkel-

bacillen. 1. Mittheilung. Z. Physiol. Chem.26:218-232.

315. Russel, P. J., and A. F. Brodie. 1961. Chromanformation and its role in oxidative phosphor-ylation, p. 205-210. In G. E. W. Wolsten-holme and Cealia M. O.'Conor (ed.), CibaFoundation symposium on quinones in elec-tron transport.

316. Russell, R. L., G. J. Jann, and S. Froman. 1960.Lysogeny in the mycobacteria. Amer. Rev.Resp. Dis. 82:384-393.

317. Saito, S., H. Hosokawa, and H. Tasaka. 1967.Heat stable acid phosphatase activity ofmycobacteria. Jap. J. Bacteriol. 22:519.

318. Saxena, K. C., and K. L. Arora. 1959. Studiesin Mycobacterium tuberculosis-transaminaseactivity. J. Sci. Industr. Res. 18:237-241.

319. Scheuerbrandt, G., and K. Bloch. 1962. Unsatu-rated fatty acids in microorganisms. J. Biol.Chem. 237:2064-2068.

320. Schubert, K., G. Kaufmann, and H. Bodzi-kiewitz. 1969. Chelest-4-en-26-ol-3-on undCholesta-1, 4-dien-26-ol-3-on, als Kompo-nenten eines neuen Mikrobioll gebildetenEstertyps. Biochim. Biophys. Acta 176:170-177.

321. Schubert, K., G. Kaufmann, and C. Hoezhold.1969. Veresterung von Sterinen mit Fett-sauren und mit Bernsteinsaure in mycobak-terien. Biochim. Biophys. Acta 176:163-169.

322. Segal, W. 1964. A comparative study of Myco-bacterium tuberculosis grown in vivo and invitro. J. Gen. Microbiol. 35:VII-VIII.

323. Segal, W., and H. Bloch. 1956. Biochemical dif-ferentiation of Mycobacterium tuberculosisgrown in vivo and in vitro. J. Bacteripl. 72:132-141.

324. Segal, W., and H. Bloch. 1957. Pathogenic andimmunogenic differentiation of Mycobac-terium tuberculosis grown in vitro and invivo. Amer. Rev. Tuberc. 75:495-500.

325. Segel, W. P., and D. S. Goldman, 1963. Therequirement for a naphthoquinone in the re-duced nicotinamide adenine dinucleotideoxidase system of Mycobacterium tubercu-losis. Biochim. Biophys. Acta 73:380-390.

326. Seshadri, R., P. S. Murthy, and T. A. Venkita-subramanian. 1970. Studies on the carbohy-drate metabolism of Mycobacterium smeg-matis, p. 30-31. Abstracts, Second Interna-tional Convention of Biochemists, Baroda,India.

327. Senghvi, D. R., and D. Subrahmanyam. 1967.Effect of certain antitubercular drugs on themetabolism of phospholipids of mycobacteria,p. 18-22. Symposium on Chemotherapy oftuberculosis, Indian J. Chest. Dis.

327a. Sheila, M. S., K. P. Gopinathan, and T. Ra-makrishnan. 1971. Protein synthesis in Myco-bacterium tuberculosis H37Rv and the effectof streptomycin. In Proceectings of Biochem-ical Society, Agenda papers, p. 59, Golden

104 BACTERIOL. REV.


Jubilee meetings of the Department of Bio-chemistry, Indian Institute of Science, Ban-galore, under the auspices of the BiochemicalSociety (U.K.) and the Society of BiologicalChemists (India).

328. Shaila, M. S., and T. Ramakrishnan. 1970.Methylation of transfer RNA by Mycobac-terium tuberculosis H37Rv, p. 181-188. InSymposium on macromolecules in storageand transfer of biological information, Dept.of Atomic Energy, Government of India,Bombay.

329. Sloane, N. H. 1961. Metabolites of aminoben-zoic acid. I. Isolation, biological and chemicalproperties of two crystalline compounds met-abolically derived from p-amino benzoic acid.J. Biol. Chem. 236:448-454.

330. Sloane, N. H., C. Crane, and R. L. Mayer, 1951.Studies on the metabolism of p-aminobenzoicacid by Mycobacterium smegmatis. J. Biol.Chem. 193:453-458.

331. Sloane, N. H., M. Samuels, and R. L. Mayer.1954. Studies on the metabolism of p-ami-nobenzoic acid by Mycobacterium smeg-matis. J. Biol. Chem. 206:751-755.

332. Sloane, N. H., and K. G. Untch. 1962. Metabo-lites of p-aminobenzoic acid. II. p-aminobenzyl alcohol: a metabolite of PABA.Biochim. Biophys. Acta 59:511-512.

333. Sloane, N. H., and K. G. Untch. 1964. Metabo-lites of p-amino benzoic acid IV. Structure of"metabolite I" and aryl hydroxylation priorto hydroxy methylation of the benzene ring.Biochemistry 3:1160-1164.

334. Sloane, N. H., K. G. Untch, and A. W.Johnson. 1963. Metabolites of p-amino ben-zoic acid. III. A metabolic pathway of PABAresulting in the sequential formation of p-aminobenzyl alcohol and p-hydroxyaniline.Biochim. Biophys. Acta 78:588-593.

335. Smith, L. 1968. The respiratory chain system ofbacteria, p. 55-122. In T. P. Singer (ed.), Bio-logical oxidations. Interscience Publishers,New York.

336. Snow, G. A. 1970. Mycobactins: iron-chelatinggrowth factors from mycobacteria. BacteriolRev. 34:99-125.

337. Snyder, C. D., and H. Rapoport, 1968. The roleof quinone in oxidative phosphorylation. Evi-dence against carbon-oxygen bond cleavage.Biochemistry 7:2318-2326.

338. Sriprakash, K. S. 1970. Studies on the action ofisoniazid and mechanism of isoniazid resist-ance in Mycobacterium tuberculosis H37Rv.Ph.D. thesis. Indian Institute of Science,Bangalore, India.

339. Sriprakash, K. S., and T. Ramakrishnan. 1969.Isoniazid and nicotinamide adenine dinucleo-tide synthesis in Mycobacterium tuberculosis.Indian J. Biochem. 6:49-50.

340. Sriprakash, K. S., and T. Ramakrishnan. 1970.Isoniazid resistant mutants of Mycobac-terium tuberculosis H37Rv: uptake of isoni-azid and the properties of NADase inhibitor.J. Gen. Microbiol. 60:125-132.

341. Stjernholm, R. L., R. E. Noble, and D. Koch-Weser. 1962. Formation of methyl malonylCoA and succinyl CoA by extracts of Myco-bacterium smegmatis. Biochim. Biophys.Acta 64:174-177.

342. Subba Reddy, V. V., H. N. Jayaram, M. Sirsi,and T. Ramakrishnan. 1969. Inhibitory ac-tivity of L-asparaginase from Mycobacteriumtuberculosis on Yoshida ascites sarcoma inrats. Arch. Biochem. Biophys. 132:262-267.

343. Subramanyam, D. 1965. The metabolism ofphosphatides of Mycobacterium 607. IndianJ. Biochem. 2:27-30.

344. Sullivan, P. A. 1968. Crystallization and prop-erties of L-lactate oxidase from Mycobac-terium smegmatis. Biochem. J. 110:363-371.

345. Sundar Raj, C. V., and T. Ramakrishnan. 1970.Transduction in Mycobacterium smegmatis.Nature (London) 228:280-281.

346. Sundar Raj, C. V., and T. Ramakrishnan. 1971.Genetic studies in mycobacteria: isolation ofauxotrophs and mycobacteriophage for My-cobacterium smegmatis and their use intransduction. J. Indian Inst. Sci. 53:126-140.

347. Suryanarayana Murthy, P., E. Bogin, T. Hi-gashi, and A. F. Brodie. 1969. Properties ofthe soluble malate vitamin K and reductaseand associated phosphorylation. J. Biol.Chem. 244:3117-3124.

348. Suryanarayana Murthy, P., and A. F. Brodie.1964. Oxidative phosphorylation in fraction-ated bacterial systems. XV. Reduced nicotin-amide adenine dinucleotide phosphate-linkedphosphorylation. J. Biol. Chem. 239:4292-4297.

349. Suryanarayana Murthy, P., and A. F. Brodie.1966. Phosphorylation coupled to the solublemalate vitamin K reductase of Mycobac-terium phlei. Bacteriol. Proc., p. 70.

350. Suryanarayana Murthy, P. M. Sirsi, and T.Ramakrishnan. 1962. Tricarboxylic acid cycleand related enzymes in cell-free extracts ofMycobacterium tuberculosis H37Rv.Biochem. J. 84:263-269.

351. Suryanarayana Murthy, P., M. Sirsi, and T.Ramakrishnan. 1963. Effects of metabolicinhibitors on the energy supplying reactionsin Mycobacterium tuberculosis. Indian. J.Exp. Biol. 1:16-26.

352. Sutton, W. B. 1963. Isolation of a new electrontransport component with nicotinamide ade-nine dinucleotide phosphate like activity.Biochem. Biophys. Res. Commun. 10:40-44.

353. Sutton, W. B. 1963. Relationship of the hexosemonophosphate shunt to the endogenousmetabolism of cell-free extracts of Mycobac-terium phlei. J. Bacteriol. 85:476-484.

354. Szafranski, P. 1956. Enzymes of the pentosecycle in Mycobacterium tuberculosis H37Rv.Bull. Acad. Polon. Sci. Ser. Sci. Biol. 4:207-210.

355. Szymona, M., and W. Ostrowski. 1964. Inor-ganic polyphosphate glucokinase of Mycobac-terium phlei. Biochim. Biophys. Acta 85:283-295.

VOL. 36, 1972 105

106 RAMAKRISHNAN, MUR

356. Takayama, K., and D. S. Goldman. 1969.Pathway for the synthesis of mannophos-pholipids in Mycobacterium tuberculosis.Biochim. Biophys. Acta 176:196-198.

357. Takayama, K., and D. S. Goldman. 1970. Enzy-matic synthesis of mannosyl-1-phosphoryl-decaprenol by a cell-free system of Mycobac-terium tuberculosis. J. Biol. Chem. 245:6251-6257.

358. Takemori, S., K. Nakazawa, Y. Nakai, K. Su-zuki, and M. Katagiri. 1968. A lactate oxy-genase from Mycobacterium phlei. Improvedpurification and some properties of the en-zyme. J. Biol. Chem. 243:313-319.

359. Tarnok, I., and P. Czanik. 1959. Malachitegreen reducing enzyme in mycobacteria.Nature (London) 183:549-550.

360. Tarshis, M. S. 1963. Further investigation onthe arylsulphatase activity of mycobacteria. I.Group differentiation of strains. Amer. Rev.Resp. Dis. 88:847-851.

361. Tarshis, M. S. 1963. Further investigation onthe arylsulfatase activity of mycobacteria. II.Differentiation of Mycobacterium aviumfrom the unclassified group III, Battey Ba-cilli. Amer. Rev. Resp. Dis. 88:852-853.

362. Tarshis, M. S. 1963. Further investigation onthe aryl sulfatase activity of mycobacteria.III. Differentiation of the rapidly growingMycobacterium balnei, Mycobacterium for-tintum and saprophytic acid-fast bacilli.Amer. Rev. Resp. Dis. 88:854-856.

363. Tasaka, H. 1968. The heat stable acid phospha-tase activity of mycobacteria. Amer. Rev.Resp. Dis. 97:474-476.

364. Tepper, B. S. 1965. Modification of cellularconstituents during growth of Mycobacteriumphlei. Amer. Rev. Resp. Dis. 92:75-82.

365. Tepper, B. S. 1968. Differences in the utilisa-tion of glycerol and glucose by Mycobac-terium phlei. J. Bacteriol. 95:1713-1717.

366. Terai, T., T. Kamahora, and Y. Yamamura.1958. Tellurite reductase from Mycobac-terium avium. J. Bacteriol. 75:535-539.

367. Thacore, H., and H. P. Willett. 1963. Forma-tion of spheroplasts of Mycobacterium tuber-culosis by lysozyme treatment. Proc. Soc.Exp. Biol. Med. 114:43-47.

368. Tirunarayanan, M. O., and Vischer. W. A. 1957.Relationship of isoniazid to the metabolismof mycobacteria. Catalase and peroxidase.Amer. Rev. Tubrc. 75:62-70.

369. Tirunarayanan, M. O., W. A. Vischer, and H.Bruhin. 1960. Some properties of catecholperoxidase of mycobacteria bearing on isoni-azid susceptibility. J. Bacteriol. 80:423-429.

370. Tobari, J. 1964. Requirement of flavin adeninedinucleotide and phospholipid for the activityof malate dehydrogenase from Mycobac-terium avium. Biochem. Biophys. Res.Commun. 15:50-54.

371. Toide, I. 1963. Nicotinamide adenine dinucleo-tide nucleosidase of Mycobacterium butyr-icum. Acta Chem. Scand. 17: suppl. 1, S 161-164.

UT HY, AND GOPINATHAN BACTERIOL. REv.

372. Tokunaga, T., and R. M. Nakamura, 1967.Infection of Mycobacterium tuberculosis withDNA extracted from mycobacteriophage B1.J. Virol. 1:448-449.

373. Tokunaga, T., and R. M. Nakamura. 1968.Infection of competent Mycobacterium smeg-matis with deoxyribonucleic acid extractedfrom bacteriophage B1. J. Virol. 2:110-117.

374. Tokunaga, T., and M. I. Sellers. 1965. Strepto-mycin induction of premature lysis of bacte-riophage-infected mycobacteria. J. Bacteriol.89:537-538.

375. Tokunaga, T., and M. I. Sellers, 1970. Transfec-tion of amino-acid sensitized bacteria, p. 152-165. In S. E. Juhasz and G. Plummer (ed.),Host-virus relationships in Mycobacterium,Nocardia and Actinomyces. Proceedings of asymposium. Charles C Thomas, Publisher,Springfield, Ill.

376. Tomcsanyi, A. 1964. The effect of isoniazid onthe incorporation of amino acids into proteinby a soluble system of mycobacteria. Amer.Rev. Resp. Dis. 92:119-120.

377. Tristanowski, S., and W. Dworak. 1961. Studieson the synthesis of amino acids by mycobac-teria. I. Incorporation of 14C carbon in theform of acetate into amino acids of tuberclebacilli. Arch. Immunol. Ther. Exp. 15:222-227.

378. Trnka, L., and D. W. Smith, 1968. Influence ofcell disruption methods on polyuridylic aciddependent polyphenylalanine synthesis byisolated ribosomes of mycobacteria. Experi-mentia 24:1109-1110.

379. Trnka, L., and D. W. Smith, 1970. Proteosyn-thetic activity of isolated ribosomes of myco-bacteria and its alteration by rifampicin andrelated tuberculostatic drugs. Antibiot.Chemother. 16:369-379.

380. Trnka, L., A. Staflova, J. Nejedly, and B.Volny. 1966. A procedure for disintegration ofmycobacteria in cooled pressure chambers.Amer. Rev. Resp. Dis. 93:291-295.

381. Trnka, L., E. Wiegeshaus, and D. W. Smith,1968. Resolution and identification of ribo-somes from mycobacteria. J. Bacteriol. 95:310-313.

382. Tsukamura, M. 1961. Enzymatic reduction ofpicric acid by mycobacteria. Amer. Rev.Resp. Dis. 84:87-89.

383. Tsukamura, M. 1961. Effect of ultraviolet ra-diation on the permeability and protein syn-thesis of mycobacterium. Jap. J. Tuberc. 9:106-114.

384. Tsukamura, M. 1964. Effect of rifamycin SV onthe incorporation of PO4- 32p;, S04 -315Sand acetate-14C. Jap. J. Tuberc. 12:10-14.

385. Tsukamura, M. 1965. Conversion of salicylateto catechol by Mycobacterium fortitum J.Gen. Microbiol. 41:331-334.

386. Tsukamura, M. 1965. Abnormal protein syn-thesis in mycobacteria occurring in the pres-ence of isonicotinic acid hydrazide. Med. Biol(Tokyo) 63:24-28.

387. Tsukamura, M., and S. Tsukamura. 1964. Ef-


fect of isoniazid on protein synthesis of myco-bacteria. Amer. Rev. Resp. Dis. 89:572-574.

388. Tsumita, T., and E. Chargaff. 1958. Studies onnucleoproteins. VI. The deoxyribonu-cleoprotein and the deoxyribonucleic acid ofbovine tubercle bacilli (BCG). Biochim. Bio-phys. Acta 29:568-578.

389. Urabe, K., H. Saito, H. Tasaka, and A. Matsu-bayashi. 1965. Acid-phosphate activity ofmycobacteria. An aid in the differentiation ofMycobacterium avium from nonphoto-chromogenic acid fast bacilli. Amer. Rev.Resp. Dis. 91:279-283.

390. Virtanen. S. 1960. A study of the nitrate reduc-tion by mycobacteria. Acta Tuberc. Scand.Suppl. 47:1-116.

391. Vitol, M. J., V. N. Shaposhnikov, and Yu P.Shivachkin. 1967. A new path of orotic acidcatabolism in microorganisms (Russian). Dok.Akad. Nauk. SSSR 174:1202-1204.

392. Wakil. 1958. A malonic acid derivative as anintermediate in fatty acid synthesis. J. Amer.Chem. Soc. 80:6465.

393. Wang, L., T. Kusaka, and D. S. Goldman. 1970.Elongation of fatty acids in Mycobacteriumtuberculosis. J. Bacteriol. 101:781-785.

394. Watanabe, T., and A. F. Brodie. 1966. Enzy-matic formation of a phosphorylated deriva-tive of vitamin K. Proc. Nat. Acad. Sci.U.S.A. 56:940-945.

395. Wayne, L. G. 1962. Differentiation of mycobac-teria by their effect on Tween 80. Amer. Rev.Resp. Dis. 86:579-581.

396. Wayne, L. G., and W. M. Gross. 1968. Isolationof deoxyribonucleic acid from mycobacteria.J. Bacteriol. 95:1481-1482.

397. Weber, M. M., T. C. Hollocher, and G. Rosso.1965. The appearance and general propertiesof free radicals in electron transport particlesfrom Mycobacterium phlei. J. Biol. Chem.240:1776-1782.

398. Weber, M. M., and G. Rosso. 1963. Role of anunidentified factor involved in electron trans-port in Mycobacterium phlei. Proc. Nat.Acad. Sci. U.S.A. 50:710-717.

399. Weber, M. M., and M. N. Swartz. 1960. Pyri-dine nucleotide coenzymes of Mycobacteriumphlei. Arch. Biochem. Biophys. 86:233-237.

400. White, A., F. Foster, and L. Lyon. 1962. Altera-tion of colony morphology of mycobacteriaassociated with lysogeny. J. Bacteriol. 84:815-818.

401. White, R. J., G. C. Lancini, and L. G. Silvestri,1971. Mechanism of action of rifampin onMycobacterium smegmatis. J. Bacteriol. 108:737-741.

402. Willett, H. P. 1969. The production of lysinefrom diaminopimelic acid by cell free ex-tracts of Mycobacterium tuberculosis. Amer.Rev. Resp. Dis. 81:653-659.

403. Willett, H. P., and H. Thacore. 1967. Forma-tion of spheroplasts of Mycobacterium tuber-culosis by lysozyme in combination with cer-tain enzymes of rabbit peritoneal monocytes.Can. J. Microbiol. 13:481-488.

404. Winder, F. 1960. Catalase and peroxidase inmycobacteria. Possible relationship to themode of action of isoniazid. Amer. Rev. Resp.Dis. 81:68-78.

405. Winder, F. 1964. The antibacterial action ofstreptomycin, isoniazid and PAS, p. 111-149.In V. C. Barry, (ed.), Chemotherapy of tuber-culosis. Butterworths, London.

406. Winder, F. G., and P. J. Brennan. 1966. Initialsteps in the metabolism of glycerol by Myco-bacterium tuberculosis. J. Bacteriol. 92:1846-1847.

407. Winder, F. G., P. Brennan, and C. Ratledge.1964. Synthesis of fatty acids by extracts ofmycobacteria and the absence of inhibitionby isoniazid. Biochem. J. 93:635-640.

408. Winder, F. G., and P. B. Collins. 1970. Inhibi-tion by isoniazid of synthesis of mycolic acidsin Mycobacterium tuberculosis. J. Gen. Mi-crobiol. 63:41-48.

409. Winder, F. G., and M. P. Coughlan. 1966. Theeffect of iron-deficiency on the deoxyribonu-cleoside-synthesizing activity of cell-free ex-tracts of Mycobacterium smegmatis. Bio-chem. J. 100:39 p.

410. Winder, F. G., and M. P. Coughlan. 1967.Breakdown of DNA stimulated by nucleosidedi- and triphosphate in cell-free extracts ofMycobacterium smegmatis. Biochim. Bio-phys. Acta 134:215-217.

411. Winder, F. G., and M. P. Coughlan. 1969. Anucleoside triphosphate-dependent deoxyri-bonucleic acid-breakdown system in Myco-bacterium smegmatis, and the effect of ironlimitation on the activity of this system.Biochem. J. 111:679-687.

412. Winder, F. G., and M. Lavin. 1969. The reac-tion catalysed by a partially purified nucleo-side triphosphate-dependent deoxyribonu-cleic acid breakdown system from Mycobac-terium smegmatis. Biochem. J. 115:6 p.

413. Winder, F. G., and M. Lavin. 1970. Chainlengths of the oligodeoxyribonucleotidesformed by the nucleoside triphosphate-de-pendent deoxyribonuclease of Mycobacteriumsmegmatis. Biochem. J. 119:25 p.

414. Winder, F. G., and M. S. McNulty. 1970. In-creased DNA polymerase activity accom-panying decreased DNA content in iron-de-ficient Mycobacterium smegmatis. Biochim.Biophys. Acta 209:578-580.

415. Winder, F. G., M. S. McNulty, and L. A. Cal-lender. 1968. Partial purification of DNA po-lymerase from Mycobacterium smegmatis.Biochem. J. llO:lOp.

416. Winder, F. G., and C. O'Hara. 1962. Effects ofiron deficiency and zinc deficiency on thecomposition of Mycobacterium smegmatis.Biochem. J. 82:98-108.

417. Winder, F. G., and C. O'Hara. 1964. Effects ofiron deficiency and of zinc deficiency on theactivities of some enzymes in Mycobacteriumsmegmatis. Biochem. J. 90:122-126.

418. Worcel, A., and D. S. Goldman. 1964. Activa-tion by AMP of the NADH oxidase of Myco-

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RAMAKRISIINAN, MURTHY, AND GOPINATHAN

bacterium tuberculosis. Biochem. Biophys.Res. Commun. 17:559-564.

419. Worcel, A., and D. S. Goldman. 1967. Stereo-specificity of the allosteric NADH dehydro-genase from Mycobacterium tuberculosis.Arch. Biochem. 118:420-423.

420. Worcel, A., D. S. Goldman, and W. W. Cleland.1965. An allosteric reduced nicotinamideadenine dinucleotide oxidase from Mycobac-terium tuberculosis. J. Biol. Chem. 240:3399-3407.

421. Worcel, A., D. S. Goldman, and I. B. Sachs.1968. Properties and fine structure of the ri-bosomes from Mycobacterium tuberculosis.Proc. Nat. Acad. Sci. U.S.A. 61:122-129.

422. Yabu, K. 1967. The uptake of D-glutamic acidby Mycobacterium avium. Biochim. Biophys.Acta 135:181-183.

423. Yabu, K. 1970. Amino acid transport in Myco-bacterium smegmatis. J. Bacteriol. 102:6-13.

424. Yamaki, H. 1965. Inhibition of protein syn-thesis by fusidic acid and helvolinic acids,steroidal antibiotics. J. Antibiot. (Tokyo)Ser. A 18:228-232.

425. Yamane, I. 1957. Growth enhancing factor forhuman tubercle bacilli using minute inocula.J. Bacteriol. 73:334-337.

426. Yamasaki, H., and T. Moriyama. 1970. Inhibi-tory effect of a-ketoglutarate: glyoxylate car-boligase activity on porphyrin synthesis inMycobacterium phlei. Biochem. Biophys.Res. Commun. 38:638-643.

427. Yano, I. and M. Kusunose. 1966. Propionateincorporation into mycocerosic acid byresting cells of Mycobacterium tuberculosisbovis. Biochim. Biophys. Acta 116:593-596.

428. Youatt, J. 1969. A review of the action of isoni-azid. Amer. Rev. Resp. Dis. 99:729-749.

429. Youatt, J., and S. H. Than. 1969. An enzymesystem of Mycobacterium tuberculosis thatreacts specifically with isoniazid. II. Correla-tion of this reaction with the binding andmetabolism of isoniazid. Amer. Rev. Resp.Dis. 100:31-37.

430. Youmans, A. S., J. Millman, and G. P. You-mans. 1956. The oxidation of compounds re-lated to the tricarboxylic acid cycle by wholecells and enzyme preparations of Mycobac-terium tuberculosis var hominis. J. Bacteriol.71:565-570.

431. Youmans, A. S., and G. P. Youmans. 1965.Immunogenic activity of a ribosomal fractionobtained from Mycobacterium tuberculosis.J. Bacteriol. 89:1291-1298.

432. Youmans, A. S., and G. P. Youmans. 1968.Ribonucleic acid, deoxyribonucleic acid, andprotein content of cells of different ages ofMycobacterium tuberculosis and the relationto immunogenicity. J. Bacteriol. 95:272-279.

433. Zatman, L. J., N. 0. Kaplan, S. P. Colowick,and M. M. Ciotti. 1954. Effect of isonicotinicacid hydrazide on diphosphopyridine nucleo-tidases. J. Biol. Chem. 209:453-466.

434. Zeller, E. A., L. S. Van Orden, and W. F. Kirch-heimer. 1954. Enzymology of mycobacteria.VI. Enzymic degradation of guanidine deriva-tives. J. Bacteriol. 67:153-158.

435. Zurack, A. 1969. Vergleichenden Untersu-chungen zum Nikotinsaurestoffwechsel beiMycobacterium bovis Stamm BCG undMycobacterium tuberculosis Zentralbl. Bak-teriol. Parasitenk. Infektionskr. Hyg. Abt. 1Orig. 212:125-136.

436. Zygmunt, W. A. 1963. Antagonism of D-cyclo-serine inhibition of mycobacterial growth byD-alanine. J. Bacteriol. 85:1217-1220.

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