Temperature Sensitive Mutation in the 38 kDaMinor Structural Protein Gene ofPhage MB78 Interferes with Phage Morphogenesis

PINAKI DATTA,1 PRABHAT MALLIK,2 AMAR N. GHOSH2 & MAHARANICHAKRAVORTY1,2,*

1Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi,U.P., India2National Institute of Cholera and Enteric Diseases, P-33, CIT Road, Scheme XM, Beliaghata, Kolkata, India

Received July 12, 2004; Accepted September 5, 2004

Abstract. Temperature sensitive mutation in the gene for the 38 kDa minor structural protein of the phageMB78, a virulent phage of Salmonella enterica serovar typhimurium, interferes with phage development atrestrictive temperature. Electron microscopy of particles produced at non-permissive temperature indicatedthat the particles formed are tailless. Two types of particles are seen: (i) empty capsids, which are not perfecticosahedral (ii) icosahedral particles filled with DNA. The gene for the 38 kDa protein is located in theSalIG fragment of the phage genome. Nucleotide sequence of the SalIG fragment of MB78 as well as itstemperature sensitive mutant has been determined and analysed. Such analysis indicated that in the mutantthe codon GCA has been changed to GTA resulting in substitution of alanine at position 75 of the proteinby valine (A75V). This makes the protein thermolabile. Our results suggest that normal functioning of this38 kDa protein is necessary for attachment of tail fibre to the capsid. Or in other words, this 38 kDaprotein is involved in phage morphogenesis.

Key words: Bacteriophage MB78, defective phage, phage morphogenesis, structural protein, tailless par-ticles, temperature sensitive mutation

Introduction

Bacteriophage MB78 isolated in our laboratory isa long tailed virulent phage of Salmonella entericaserovar typhimurium [1]. It contains a linear,double stranded, circularly permuted and termi-nally redundant DNA, which is 42 kb long. MB78DNA does not have detectable homology witheither phage P22 [2] or 9NA [3] when tested byDNA–DNA filter hybridization method. How-ever, Southern blot hybridization indicated thatMB78 DNA might have homology with a smallpart of P22 genome located in the EcoRIC frag-ment. The DNA replicates as concatamer, which issubsequently converted to full length phage DNA

through headful packaging mechanism [4]. Twostrong promoters of the phage have been studiedin details [5,6]. A number of restriction fragmentsof the phage genome have been cloned in M13mp11 and some of the fragments have been studiedextensively [6–11]. MB78 does not seem to berelated to other phages since the ORFs so faridentified do not show any similarity with reportedphage proteins. This phage may belong to a newfamily. Further, presence of restriction-modifica-tion system in this phage [11] makes the phagemore interesting. Although the locations of severalgenes (ORFs) on the physical map of the genomehave been determined the functions of most of theORFs are yet to be ascertained. Hence, conditionallethal mutants were isolated and attempts weremade to determine their physical locations on the

*Author for all correspondence:

E-mail: c_maharani37@hotmail.com

Virus Genes 30:2, 197–207, 2005� 2005 Springer Science+Business Media, Inc. Manufactured in The Netherlands.

genome and the functions affected due to suchmutation. Here we report that a temperature sen-sitive mutation in the gene for a minor structuralprotein of the phage, located in the SalIG frag-ment of its genome results in the production oftailless particles at non-permissive temperature.Our results suggest that this 38 kDa protein is aminor structural protein of the phage, which isinvolved in phage morphogenesis.

Materials and Methods

Chemicals and Enzymes

All chemicals and enzymes including restrictionendonucleases used were of molecular biologygrade and procured from different companies ofinternational reputation.

Bacteria, Bacteriophages and GeneralManipulations

Escherichia coli strain KK2186 used for transfor-mation was a gift from Prof. P. Berget then at theDepartment of Biochemistry and Molecular Biol-ogy, University of Texas, Houston, USA. The minicell producing strain DS410 of E.coli was providedby Dr. I.B. Holland, University De Paris Sud,Orsay, France. The strain LT2, the restriction+

modification+ strain of S. typhimurium wasobtained from Prof. M.Levine, University ofMichigan, AnnArbor, USA. LB5000, a restriction)

modification + strain of S. typhimurium was kindlyprovided by Dr. K.E. Sanderson, Department ofBiological sciences, University of Calgary, AlbertaCanada. Bacteriophage MB78 was isolated in ourlaboratory [1]. Bacteria were routinely grown in LBbroth [12]. Antibiotics (ampicillin and chloram-phenicol) were added to the media at requiredconcentrations as and when necessary. The tech-niques for preparation of DNA and transformationwere as per standard procedures [13]. Purification ofphage was carried out as described earlier [1].

Mutagenisation of MB78 and Isolation of tsMutants

Mutagenisation was carried out by treatingphage particles with hydroxylamine as per

Silhavy et al. [14] to a survival of 0.1–1.0%. Thesurviving phages were plated on Salmonellastrain LT2. The plates were at first incubated at25�C for a few hours till tiny plaques appeared.Those were then shifted to 40�C. The plaques,which remained tiny even after 12 h or more at40�C, were selected. From these, the phagesforming plaques at 25�C but not at 40�C weretaken as temperature sensitive mutants andpurified.

Expression of Plasmid Coded Genes in Minicells

Experiments were carried out as reported earlier[6]. Minicell producing strain (DS410) of E. coliwas transformed with the desired plasmid.Transformants were grown overnight in 400 mlof Terrific Broth [13] containing relevant antibi-otics. Minicells were purified from the overnightgrown culture as described by Reeve [15] andlabeled in vivo using 35S-methionine. Labeledproteins were analyzed by electrophoresisthrough 12.5% SDS polyacrylamide gel [16]. Thegel was fluorographed and autoradiographed fordetection of labeled proteins. Fluorography ofgel was done using sodium salicylate as scintil-lator as per method of Bonner [17]. Purifiedphage proteins were used as marker. The portionof the gel containing the phage proteins werestained with Coomassie blue while the portioncontaining 35S labelled protein was subjected tofluorography. Later, the autoradiogram wasaligned with the stained gel containing the phageproteins. Alignment was done using positions ofthe slots as marker.

Rate of DNA Synthesis

Cells (2.6 · 108 cells/ml) growing exponentiallyin M9 media [18] were infected with phage at anm.o.i. of 10. At different times followinginfection, cells (0.5 ml) were pulsed for 1 minwith 3H-thymidine (8 nmol containing106 counts/min) at requisite temperature underconstant shaking. The rate of DNA synthesiswas followed by measuring the rate of incorpo-ration of 3H-thymidine into trichloroacetic acidinsoluble fraction as described by Smith andLevine [18].

198 Datta et al.

Protein Synthesis

Pattern of protein synthesis was studied by exposingthe cells growing inM9media to 35S-methionine fora short period (2 min). Incorporation was stoppedby addition of cold methionine. The proteins wereanalysed by SDS-polyacrylamide gel electrophore-sis and autoradiography [16].

Complementation Test

Complementation test was performed in pairwisefashion on plates by spotting �104–105 pfu of eachof the two ts phages to be tested at 40�C ontryptone agar plates seeded with plating bacteria(S. typhimurium). The plates were pre-warmed at40�C for 30 min and incubated at 40�C afterspotting. One set of plates was maintained at 25�Cas control.

Enzyme-Linked Immunosorbent Assay (ELISA)

For ELISA, cells grown overnight (from a singlecolony) in LB media containing 50 lg/ml ofampicillin were harvested. The cells thus collectedwere suspended in PBS (2.7 mM KCl; 4.3 mMNa2HPO4, 7H2O; 1.4 mM KH2PO4) and lysed byfreezing and thawing thrice (flash freezing at)70�C and rapid thawing at 37�C) which wasfollowed by sonication for 1 min. The lysatewas clarified by centrifugation and the supernatantwas used as antigen. The protein content of theantigen was determined as per Bradford [19] andthe samples were stored at )20�C until used.ELISA was performed in 96 well ELISA plate asper Voller et al. [20]. Each well was coated withantigen (cell-free extract of clones in 50 mM Tris–Cl, pH 9.5) by filling the wells with antigen andkeeping for 18 h at 4�C. The wells were washedthoroughly with PBS-T (PBS containing 0.5%Tween 20) after every step. Blocking was carriedout with 5% BSA in PBS-T solution for 2 h at37�C. After washing the plate with PBS-T, MB78antisera (k ¼ 2) was added and incubated for 2 hat 37�C. The excessive antisera were removed bywashing after which horseradish peroxidase(HRP) labeled anti rabbit IgG (1:5000), the sec-ondary antibody, was added in the wells andincubated for 2 h at 37�C. The color was devel-oped with o-phenylenediamine dihydrochloride

(OPD, 1 mg/ml in 0.1 M citrate-phosphate buffer,pH 5.0) and H2O2 (1 ll/ml) in dark. The enzy-matic reaction was stopped by 3 N HCl and theabsorbancy was recorded at 490 nm in an auto-matic ELISA reader (Emax molecular device).

Sequencing of DNA

Sequencing was carried out following Sanger’sdideoxy chain termination method [21] usingdouble stranded templates. When sequencing kitswere used, the protocols recommended by thecompanies were followed.

Electron Microscopy

Samples were negatively stained with uranyl ace-tate. Micrographs were taken in Philips EM 420Telectron microscope with an operating voltage of60 kV.

Results

Isolation of Temperature Sensitive Mutants andDetermination of the Physical Location of theMutated gene on the Phage Genome

Bacteriophage MB78 was mutagenised withhydroxylamine as described under Materials andMethods. Six temperature sensitive mutants werethus isolated. Those were named as MB78ts23,MB78ts30 and so on (vide Table 1). The num-bers denote the isolate numbers. In order toidentify whether the mutations are in the samecistron or different ones complementation testwas conducted as described under Materials andMethods. Out of the six mutants isolated, onlythree, MB78ts23, MB78ts30 and MB78ts42 hadsingle mutation (Table 1). Since the other threemutants did not complement with any of the tsmutants those must be having more than onemutation. The three temperature sensitivemutants having apparently point mutations werefurther tested. These three mutants were able toform plaques at 25�C but not at 40�C. Thereversion frequency is 10)6, which agrees wellwith the reversion rate of point mutation. Todetermine the physical location of this mutationon the phage genome the following strategy was

Temperature sensitive mutant of phage MB78 199

adopted. The mutant phages were plated onLB5000 (r) m+ strain of Salmonella typhimuri-um) transformed with pUC19 carrying differentgenomic fragments of MB78 DNA. A largenumber of genomic fragments cloned in ourlaboratory were tested. Of the three mutantstested only one named as MB78ts23 producedplaques at 40�C (non permissive temperature) onhost carrying SalIG fragment of MB78 genomeconfirming thereby that the mutation inMB78ts23 is located within the SalIG fragment.The recombinant pUC19 having the 2.1 KbSalIG fragment of MB78 DNA will be referredas pSalIG and the gene, which got mutated asg38.

In vivo Expression of Recombinant Plasmid pSalIGin Mini Cells

Since the SalIG fragment of MB78 comple-mented the defective function of MB78ts23 at40�C, it is certain that the SalIG fragment codesfor a protein which when expressed from themutated gene is non-functional at non-permissivetemperature. To ascertain the size of the proteinwhich is thermolabile at 40�C, pSalIG wasexpressed in minicell (as described underMaterials and Methods). The result presented inFig. 1 indicates that the SalIG fragment codesfor a protein which moves as 38 kDa proteinin SDS-PAGE. This protein will be referredas gp38.

Determination of the Function of gp38

Attempt was made to determine the phage func-tions(s) affected in the MB78ts23 by studying dif-ferent parameters such as adsorbtion, DNAreplication, protein synthesis etc.

(a) Adsorbtion to the Host Cell at Non-PermissiveTemperatureTo check whether the mutant phage can adsorb tothe host cell at 40�C or not, LT2 cells were grown

Table 1. Complementation between temperature sensitive mutants of bacteriophage MB78

Strains MB78ts23 MB78ts30 MB78ts42 MB78ts53 MB78ts55 MB78ts60

MB78ts23 ) + + ) ) )MB78ts30 + ) + ) ) )MB78ts42 + + ) ) ) )MB78ts53 ) ) ) ) ) )MB78ts55 - ) ) ) ) )MB78ts60 ) ) ) ) ) )

Fig. 1. In vivo expression of the recombinant plasmid pSalIG in

minicells. Minicells (200 ll) purified from DS410 cells trans-

formed with pSalIG were labeled with 35S-methionine (30 lCi;1000 Ci/mmol) as described by Reeve [15]. Samples were

denatured and analyzed after electrophoresis through 12.5%

SDS-polyacrylamide gel. Samples loaded in each lane have been

indicated at the top of the figure. Positions and molecular

weights (in kDa) of standard proteins have been provided at the

left side of the gel.

200 Datta et al.

at 40�C and then divided into two batches. Thetemperature of one was maintained at 40�C whilethe other was brought down to 26�C. Both thecultures were infected with the mutant phage at anm.o.i. of 10. After allowing the phages to adsorb tothe host by keeping in contact for 5 min, thetemperature of the phage infected culture kept at26�C was raised to 40�C. Simultaneously, thenumber of phages. remaining unadsorbed to thehost was also estimated. Even after 90 min therewas no appreciable difference in turbidity betweenthe two sets of infected cultures suggesting that themutant phage is not defective in adsorbtion atnon-permissive temperature. Otherwise, the cul-ture which was kept at 26�C for 5 min and thentransferred to 40�C would have lysed, which didnot occur. Treatment of the culture with chloro-form did lyse the cells but did not release anyinfective particle ( in both sets of culture). Further,the mutation in MB78ts23 is not in any early geneexpressed within 5 min. Number of phages thatremained unadsorbed at 40�C (which was less than5% of the input phage) also suggest that at non-permissive temperature the phage could absorb tothe host.

(b) SalIG Codes for a Structural ProteinTo ascertain whether gp38, the protein expressedby the SalIG fragment, is a structural proteinor not, Enzyme-linked Immunosorbent Assay(ELISA) was performed according to Voller et al.[20] as elaborated under Materials and Methods.MB78 antisera were use for detecting the antigenspresent in the wells. Characteristic color developedin case of phage MB78 and cell-free extract ofKK2186 transformed with pSalIG but not withcell-free extract of KK2186 transformed withpUC19. The results presented in Table 2 clearlyindicate that the SalIG fragment expressed aprotein, which was recognized as antigen by phageantiserum confirming there by that gp38 is a

structural protein of the phage. Since pUC 19extract did not elicit any reaction it furtherconfirmed that our antisera preparation isfree from any antibody against cellular (host)protein.

The next obvious question was whether gp38 isa major or minor structural protein of MB78. Tohave some idea about the different structuralproteins of the phage, purified phage particles(1012 pfu) were subjected to SDS-PAGE(Fig. 2a). To ascertain whether gp38 is a major orminor phage protein, proteins expressed in mini-cells from pSalIG were labelled with 35S-methio-nine and subjected to electrophoresis throughSDS polyacrylamide gel. Purified phage proteinswere used as marker (Fig. 2b). The portion of thegel containing the structural proteins of the phageand the molecular weight marker was stainedwith Coomassie blue while the 35 S-methioninelabeled protein containing portion (Fig. 2b, lanes3 and 4) was fluorographed and autoradio-graphed. Later, the autoradiogram was alignedwith the stained gel containing the structuralproteins of MB78. Alignment was done usingpositions of the slots as marker for alignment. Inorder to make the 38 kDa minor structural pro-tein distinguishable from the major one, smallamount of phage protein was loaded. This madeother protein bands not so distinctly visible.Results presented in Fig. 2 clearly demonstratethat the movement of the protein coded by pSa-lIG is similar to a minor structural protein of thephage suggesting thereby that gp38 is a minorstructural protein of MB78.

(c) DNA and Protein SynthesisRate of DNA and pattern of protein synthesiswere studied as described under Materials andMethods. DNA and protein synthesis were foundto be normal (data not presented).

Table 2. Quantitative analysis of the ELISA carried out to test antigenicity of the protein expressed by pSalIG against MB78 antisera

Antigen coated Concentration of antigen Absorbance (490 nm)

1. Cell free extract of pUC19 2 lg 0.041 ± 0.002

2. Cell free extract of pSalIG 2 lg 0.525 ± 0.003

3. MB78 phage 2 lg 1.540 ± 0.016

Experiment was repeated six times and data are presented as mean ± SD.

Temperature sensitive mutant of phage MB78 201

Nucleotide Sequence of the SalIG Fragment and ItsAnalysis

To determine the precise location of the gene andthe mutation that made the gene temperaturesensitive, nucleotide sequence of the SalIG frag-ment of phage MB78 as well as the temperaturesensitive mutant MB78ts23 was determined [21].Unidirectional deletion mutants and subcloneswere prepared from the SalIG fragment to facili-tate sequencing. The sequences of consecutivesubclones were aligned to deduce the base sequenceof the fragment. After sequencing the actual lengthof the SalIG fragment was found to be 2058 bp.The data has been deposited in the Gene Bank(Accession No. AYO40866). Nucleotide sequenceof part of the SalIG fragment encoding gp38 hasbeen presented in Fig. 3. From the sequence themolecular weight of the protein coded by SalIG ispredicted to be 36.5 kDa. The ORF, representingthe gene for the above mentioned 38 kDa protein isin the third reading frame and spans from 954 to1955 bp. Comparison of the base sequence of themutant gene with that of the wild type revealed thatin the MB78ts23 the sequence GCA has beenaltered to GTA (the altered codon is marked with

bold letters, the altered base T is written in boldletter and underlined). The resultant amino acidsubstitution is Ala75 fiVal (marked with bold letterin Fig. 3).

Electron Microscopy of the Phage ParticlesProduced at Restrictive Temperature

It was interesting to investigate what type of par-ticles is produced at non-permissive temperature incells infected with the temperature sensitivemutant of the phage. Hence, cells infected atrestrictive temperature with the mutant MB78ts23were treated with chloroform after 2 h and shakenvigorously. The cells lysed but no infective phageparticles were detected in the lysate. The cell lysatewas processed to purify phage particles, if any, bytwo phase system [22], using polyethylene glycoland dextran sulphate and finally through step wisegradient of cesium chloride. The particles sedi-mented at two places. The lighter one was aslightly diffused band while the lower (heavier) onewas sharp and compact. Particles from both thebands were examined under electron microscope.The lighter band contains empty, slightly irregularshaped capsid (Fig. 4a). The relatively heavier

Fig. 2. (a) Protein profile of MB78 particle. Lane 1, Protein bands visualized after SDS-PAGE and Coomassie brilliant blue staining.

Lane 2, Molecular weight marker. (b) Comparison of the mobility of gp38 with proteins present in the phage MB78 particles.

Molecular weight of the protein expressed by the SalIG fragment was compared with that of the Coomassie blue stained proteins of

MB78. Lane 1, Molecular weight marker. Lane 2, Coomassie Blue stained phage proteins. Lane 3, 35S-labeled proteins expressed by

minicells harboring pSalIG. Lane 4, 35S-labeled proteins expressed by minicells harboring pUC19. Very small amount of protein (lysed

phage particles) was loaded in lane 2 so that the minor structural protein of interest marked with an arrow is not masked by the major

structural protein band.

202 Datta et al.

Fig. 3. Nucleotide sequence of part of the 2.1 kb SalI-HindIII fragment covering the ORF and deduced amino acid sequence of the

proteins coded by the fragment: * indicates the termination codon. In the temperature sensitive mutant MB78ts23, the nucleotide ‘C’ in

the codon GCA for the 75th amino acid is changed to ‘T’ (indicated in the figure). The corresponding amino acid change is alanine to

valine (marked in the figure).

Temperature sensitive mutant of phage MB78 203

ones have icosahedral heads filled with DNA(Fig. 4b). However, no tail is attached to any ofthe particles. Normal infective particles are shownin Fig. 4c.

Is gp38 Produced at Non-Permissive Temperature?

Since the mutation is a conditional one (temper-ature sensitive) it was expected that gp38 isproduced at non-permissive temperature but is

non-functional. However, the question remainedhow this protein is involved in attachment of thetail. Does this mutated protein remain associatedwith the capsid or not? In order to get an answer,the defective particles that were examined underelectron microscope were subjected to SDS-PAGE. Normal infective particles were alsotreated similarly (as control). Results presented inFig. 5 clearly indicate that the minor structuralprotein gp38 (marked with arrow) stays associ-

Fig. 3. Continued.

204 Datta et al.

ated with the capsid (vide lane 1, Fig. 5). Or inother words, the mutated gp38 can associate withthe capsid but tail cannot attach to such capsid.

Discussion

The SalIG fragment, a 2.058 kb restriction frag-ment of bacteriophageMB78 DNA, located almostat the middle of the genome expresses a proteinwhich moves as a 38 kDa protein in SDS-PAGE.This fragment can complement a temperaturesensitive mutant (MB78ts23) of the phage. Theopen reading frame for this 38 kDa protein wasidentified. It codes for a protein with 333 aminoacids (Fig. 3), which has been identified as a minorstructural protein of the phage and is required forattachment of the tail to the capsid. The ORFsuggests that the predicted molecular weight of theprotein is 36.5 kDa. The SD sequence that could beidentified is only GAA lying nine nucleotidesupstream of the initiation codon ATG. Such situ-ation has also been reported in case of the Rz geneof k phage [23]. Down stream of the first initiation

Fig. 4. Electron Micrograph of particles produced after

infecting LT2 cells with the temperature sensitive mutant at

restrictive temperature. (a) Particles sedimented at relatively

lower density (lighter particles). Particles are empty, irregular

shaped capsid. (b) Particles sedimented at higher density. Par-

ticles are icosahedral, filled with DNA but tailless. (c) Normal

infective particles. In each case bar marker represents 100 nm.

Fig. 5. Protein profile of defective capsid of phage MB78.

Purified defective capsids and infective particles were subjected

to SDS polyacrylamide gel electophoresis as described under

Materials and Methods. Lane 1, Defective capsids. Lane 2,

Infective complete particles. Lane 3, Molecular weight markers

in kDa. In order to make the 38 kDa protein visible less

amount of sample was loaded. This did not exhibit most of the

small molecular weight proteins. The concerned proteins,

however, are visible.

Temperature sensitive mutant of phage MB78 205

codon there is another ATG codon, which is also inthe same reading frame. Since the putative SDsequence and promoter are present at reasonabledistance upstream of the first ATG codon it is quitelikely that translation starts from the first ATG asshown in Fig. 3. Although a large number of genes(ORFs) of the phage MB78 has been so far iden-tified and cloned, functions of only a few could bedetermined. Computer aided analysis of the basesequence of the ORF for this 38 kDa proteinsuggest homology over a short stretch of bases(50/57) with the sequence of endo-N-acetyl mura-minidase of bacteriophage D. The similarity istowards the C-terminal end of the protein. This isquite interesting as tail-fiber protein of manyphages has endo-N-acetyl muraminidase activity.Although it is tempting to infer that this gene codesfor tail-fiber protein, yet, this does not seem to betrue as some other part of the phage genome has304 nucleotides similar to the sequence of phageP22 tail-fiber gene.

Even this is quite surprising since P22 antiseracannot neutralize phage MB78 [1]. However, itmay throw some light on the evolution of phagetail protein gene. As tail could not be attached tothe capsid, DNA might have leaked out of manyhead particles making those empty which sedi-mented as lighter particles. Nucleotide sequenceanalysis of the mutant gene revealed that in thetemperature sensitive mutant MB78ts23, thesequence GCA has been altered to GTA. This isquite possible as mutation was carried out withhydroxylamine (NH2OH) and hydroxylamineinduces one-way transition mutation from GC toAT. As a result of such mutation valine is incor-porated in place of alanine. A change in 75thamino acid from alanine to valine made the pro-tein temperature sensitive. In case of P22 coatprotein mutant ts7 also such substitution has beenreported; alanine (108) was changed to valine [24].In tumorigenic virus SV40 mutagenization withhydroxylamine produced some mutants of thevirion protein 1 where also alanine (71) waschanged to valine [25]. In most of the cases sub-stitution of alanine by valine makes the proteintemperature sensitive. At restrictive temperaturethe protein is synthesized but is wrongly foldedwhich makes it non-functional. The mutated pro-tein can attach to the capsid but cannot accept thephage tail. It may be concluded that gp38 is a

minor structural protein of MB78 that is requiredfor joining tail to the head and in presence of themutated protein tailless particles are produced

Acknowledgments

The authors express their sincerest thanks to theDepartment of Biotechnology, Government ofIndia, University Grants Commission, IndianNational Science Academy and Indian Council ofMedical Research for financial assistance.

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