Tertiary and quaternary branched polyaminesdistributed in thermophilic Saccharococcus andBacillus

Koei Hamana*, Hiroshi Hamanal, Masaru Niitsu2, Keijiro Samejima2,

Takeshi Sakane3 and Akira Yokota3

College of Medical Care and Technology, Gunma University. Maebashi, Gunma 371,lapai, lFaculty of Engineering, Gunmi University, Kiryu, Gunma 376, Japan, 2Faculty ofPharmaceutical Sciences, Josai University, Sakado, Saitama 350-02, Japan, and3lnstitute for Fermentation, Osaka. Osaka 532. Japan (*Reprint address)

AbstractThe distribution of long linear, tertiary and quaternary branched polyamines in twentythermophilic Gram-positive strains belonging to the genera Saccharococcus, Bacillusor Amphibacillus were analysed by high-performance liquid chromatography and gas

chromatography. The extremely thermophilic S. thermophilus contained a tertiary tetra-amine, Na-aminopropylspermidine, a linear penta-amine, thermopentamine, a tertiarypenta-amine, Na-aminopropylspermine, and a quaternary penta-amine, N*-brc(amino-propyl)spermidine. Na-aminopropylspernins INH2{CH2)3N((CH2)3NH2)(C_H2)1NH(CH2)3NH2]

tirsi iounO in this study, was also detected in the extremely thermophilic R caldolyticus'and'8. caldotenax' and the moderately thermophilic B. stearothermophilus, B. smithiiand 8. thermocatenulatus.

lntroductionThe extremely thermophilic Gram-negative eubacteria belonging to the genera

Thutnuq'Rhodothermus', Thannomirobium, Thnmoleophilum, or Hydrogeno-

bactqubiquitously contained linear penta-amines, linear hexa-amines, teftiary

branched tetra-amines and/or quaternary penta-amines (Hamana et a|.r 1990,

199 1, L992arb).Neither penta-amines, hexa-amines nor branched polyamines

were found in the mderate thermophiles Thrmuq Thnmonem*, Acidothsmus

or mesophiles (HamarLa et al., 1991, 1992b).

These findings indicated that the occurrence of long linear and/or branched

polyamines was closely associated with extreme thermophily. It was suggested

that a chemotanonomic significance existed in the polyamine distribution type

in these thermophiles (Hamana and Matsuzaki r lgg2).In the Gram-positive

Bacillus species tested, the occurrence of a tetra-amine, spermine, was limited

to two moderately thermophilic strains, B. stearothermophilus and

B. acidocaldarius (Hamana et al., 1989). The presence of spermine is

apparently associated with their thermophily. Traditional mesophllic Bacillus

species contained a triamine, spermidine, as the maior polyamine. The

extremely thermophilic B. schlegelii contains linear penta-amines, linear hexa-

amines, a tertiary tetra-amine and a quaternary penta-amine (Hamana et al.,

1992a'). The polyamine distribution pattern is thought to be useful as a

chemotaxonomic marker in a large group of Gram-positive rods, with the

genus Bacillus including phylogenetically heterogeneous organisms.

23 Microbios 75 23-32 1993 Published ano O t993 by The Faculty Press88 Regent Street, Cambridge, Great Britain

Recently, the Gram-positive, extremely thermophilic non-endospore-formingcoccus, Saccharococcus thermopilzs (Nystrand 1984), and the Gram-positivespore-forming rod, Amphibacillus xylans, growing under mesophilic, andanaerobic conditions (Niimura et al.r 1990), were isolated as a new genus.Members including the moderately thermophitic sporulating bacilti, Bacillusthermoglucosidasius (Suzuki et a|.r 1983), B. thqmoleooorans (ZarilLaand Perry,1987), B. smithii (Nakamura et a1.,1988), the slightly thermophilic Bacilluscycloheptamiczs (Deinhard et al.r l987b) and B. tusciae (Bonjour and Aragno,1984), and the thermotolerant acidophile, B. acidotertestris (Deinhard et al.,1987a), were validly established. The extreme thermophile, Bacillus thermo-catenulatu.s (Golovacheva et al., L975) was recently validated by the Inter-national Committee of Systematic Bacteriology (1991). Several other caldo-active Gram-positive rods tentatively assigned as extremely or moderatelythermophilic Bacillus species have been isolated (Heinen, lg7l;Heinen andHeinen, 1972; Heinen et al., 1982; Sharp et a1.,1980; Claus and Berkeley,1986). In order to survey the distribution of unusual long and branchedpolyamines in Gram-positive thermophiles, we analysed polyamines of thesenew members in addition to the re-analysis ofthe three thermophilic Baciltusstrains.

Materials and methodsOrganisms and culture

The bacteria were cultured in a synthetic polyamine-free medium and acomplex medium containing the usual polyamines. S. thermophitus was grownat 70"C in tryptone-glucose-extract broth, pH 7.0 (TGE Broth, Difco Lab.,u.s.A.) supplemented with 0.5vo sucrose (TGE-S medium) (Nystrand 1984),and synthetic 199 medium (polyamine-free) (Flow Lab., Irvine, Scotland)supplemented with 0.5Vo sucrose (199-S medium).

B. thermoglucosidasius was grown in BBl-trypticase soy broth (BectonDickinson and co., u.s.A.) (TSB medium), pH 7.3 and a polyamine freesynthetic medium containing sucrose and glutamate (SG medium) (Suzukiet al., 1983) at 60oC. B. thermoleooorans, a hydrocarbon utilizing bacillus,was cultured in a polyamine-free mineral salt medium supplemented withO.LVo z-heptadecane as subsrrate (MH medium) (zarilla and perry, l98z)and rSB medium at 60oC. Synthetic 199 medium, pH 7.0, was also usedfor-the above three bacilli. The acidophile, B. acidoterrestris was culturedat 50oc in TSB medium at pH 4.3 (TSB-4 medium) and 199 medium atpH 4.3 (199-4 medium) (Deinhard et al., 1987a). B. tusciae was grown in199 medium, pH 5.0, supplemented with 0.2v0 sodium pyruvate (199-5-pmedium) and HCoy'H2 medium, pH 5.0 (for aurotrophic growth) (Bonjourand Aragno, 1984).

24 Microbios K. Hamana ef a/.

Acidophilic B. acidocaldarium and B. cycloheptanl'czs were grown in 199-4medium and TSB medium at pH 4.0 (TSB-4 medium) at 65 or 50oC,respectively. B. smithii, B. stearothtmophilus,'8. thsmodenitifimns'r'Bacilluscaldooelox', B, therrnocatenulatus,'8. flaoothermus','8. caldolyticu.s', and'8. caldotenatc' were cultured at 60, 65, 65, 65, 65, 65, 65, 75 or 75"C,respectively, in 199 medium, p}I7.3, and TSB medium, pH7.3. B. schlegelii

was cultured in polyamine-free ammonium-pyruvate medium, pH 7.0 (APmedium), and HCOJ/H2 medium, pH 7.0 (for autotrophic growth) (Schenkand Aragno, 1979) at 75oC. Facultatively anaerobic alkalophilic Amphibacillusxylans was grown in glucose-yeast extract medium, pH 10 (GY medium),and 199 medium, pH 10 (199-10 medium) at 40oC (Niimura et a1.,1990).For anaerobic cultivation, 0.13 mM titanium (III) citrate was added.

Polyamine analysis

The organisms growing at stationary phase were harvested and homogenizedin equal volumes of cold I M HClOo. The whole HC1O4 extracts wereanalysed by high-performance liquid chromatography (HPLC) on a column(4 mm ID x 50 mm) of cation-exchange resin (Hitachi number 2619F) at70oC. The elution buffers used were 0.045 M sodium citrate-0.061 M citricacid-0.064 M NaCl and 0.20 M sodium citrate-2.O M NaCl. Polyamineseluted on the HPLC were followed by o-phthalaldehyde. The whole extractwas subiected to a Dowex 50 rif column to concentrate the polyamines. Afterwashing with I M HCl, the polyamines were eluted with 6 M HCl and theeluates were evaporated. Gas chromatography (GC) was performed on a

GC-9A gas chromatograph (Shimadzu Co., Ltd) after heptafluorobutyrizationof the samples (Hamana et al., 1992a). The identity of polyamines wasconfirmed by gas chromatography-mass spectrometry (GC-Mass) on a IMS-DEX 300 (JEOL Co. Ltd) (Hamana et al.,l992a). Quaternary branched penta-amines were degraded to tertiary branched tetra-amines during GC analysis(Hamana et al., 1992a). Various linear tetra-amines, penta-amines, hexa-amines, tertiary branched tetra-amines, and quaternary branched penta-amines

were synthesized according to our previous report (Niitsu and Samejima,1e86).

Results

Typical chromatograms of HPLC and GC analysis of Saccharococcus andBacillus strains are shown in Figures I and 2. Cellular concentrations ofpolyamines were calculated by the combination of the HPLC and GC datausing the cultures grown in polyamine-free synthetic media (Table 1).

Saccharococans thennophilus grownin either 199-5 or TEG medium containedputrescine (4), spermidine (34), spermine (343), thermopentamine (3343),Na-aminopropylspermidine 3(314, Na-aminopropylspermine 3(3)43 andNa-Dr'slaminopropyl)spermidine 3(3)Q)a as the major polyamines (Table 1).

25 Polyamines of thermophilic Bacillus

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Homospermidine (44) was the minor polyamine in the thermophile (Figure 2).Since norspermidine (33) and norspermine (333) were not detected, theproduction of a tertiary tetra-amine, Na-aminopropylnorspermidine 3(3)3,cannot be expected. Therefore, the tetra-amine detected in GC was assignedas a product from 3(3N3)4 during GC. The process involved in reaching thisconclusion was reported by Hamana et al. (7992a).

Moderately thermophilic strains such as Bacillus tlwmoglucosidasius, Bacillusthqmoleooorans, B. acidocaldariuE B. cycloheptanicus, B. tusciaer'Bacillusthermodenitrificans'r'8. caldooelox' and'8. flaoothermus'grown in synthetic199 medium and some complex media, contained 34 and 343 as the majorpolyamines. A new tertiary branched penta-aminer 3(3)43, found in S. thermo-philus was also detected in the two extremely thermophilic strains '8. caldo-lyticus' and '8. caldotenax' as a maior polyamine in addition to 34 and 343,This penta-amine was found, as a minor polyamine, in four moderatelythermophilic strains of B. smithii, B. stearothermophilu.r and B. thqmocatenu-latus. This unusual tertiary penta-amine was found when these organisms

--

o10203040 Elution time (min)

Figure 1 HPLC analysis of whole polyamines of (A) Saccharococcus thermophilus grown inTGE-S medium at TOoC; lBl Bacillus tusciae grown in the 199 medium, pH S.O, supplementedwith O.2% sodium pyruvate at 5OoC; lC) Biciltus acidoterrestris grown in 1g9 medium, pH4.0 at 50oc; and (D) 'Bacillus caldolyticus'YT-P grown in ,l99 medium, pH 7.0 at 75oc.Dotted lines indicate the curves at ten times the standard sensitivity. Abbreviations forpolyamines are shown in Table 1.

28 Microbios K. Hamana ef a/.

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were cultivated in either synthetic 199 or complex media. The occurrenceof agmatineinSaccharococcus and these thermophilic Bacillus was confirmedby the alkaline degradation of the peak with I M NaOH at l l0oc for 24hbefore HPLC. Coe:ristence of thermospermine (33a) with 343 nthese thermo-philes was excluded by GC analysis. Neither 33 nor 333 was produced fromdiaminopropane (3) supplemented to the cultures.

Polyamines ofB. schlegelii were re-analysed according to oru previous report(Hamana et al.,L992a).Identification of polyamine peaks on the HPLC and

GC chromatograms was performed as shown in the previous study (Hamana

et al., 1992a). The long or branched polyamines such as 3343, thermohexa-mine (33343) and homothermohexamine (33433), 3(3)4 and 3(3)(3)4 weredetermined (Table l). The polyamine distribution patterns of the faorltativelyH2-oxidizing B. tusciae and B. schlegelii chemolithotrophically grown inHCO;/H2 medium was identical to those of their heterotrophic (chemo-

organotrophic) cultures in the presence of pyruvate (AP medium).

Retention time (min)

Figure 2 GC analysis of the concentrated polyamine fraction from (A) Saccharococcusthermophilus grown in TGE-S medium at 7OoC, and (B) 'Bacillus caldolyticus' YT-P grownin 199 medium, pH 7.0, at 75oC, under GC-column temperature at (a) 120-2gOoC(16oC/min) or (b) 200-28OoC (8oC/min). Abbreviations for polyamines are shown in Table 1.

29 Polyamines of thermophilic Bacillus

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A thermotolerant acidophile, B. acidoterrestris, was grown in either TSBmedium or 199 me&ium and contained only 4 and 34 as the maior polyamine.A mesophlle, Amphibacillus xylans grown in GY medium contained 41 34and 343. The aerobic and anaerobic cultures showed nearly the same

polyamine pattern which was closely related to that of yeast extracts supple-mented to the medium. However, no significant amount of polyamines wasdetected in the organism cultured in the polyamine-free 199-10 medium.These data show the absence of its own polyamines inA. xylans. The poly-amines detected in this organism grown in GY medium may have been derivedfrom the medium.

Discussion

Saccharococcus thetmophilus contained 34 and 343 as the main polyaminecomponents. The tertiary and quaternary branched polyamines produced byaminopropylation into 34 or 343 at the site ofNa must also occur in extremethermophiles. This is the first report on the occurrence of 3p)a3 in nature.In spite of the absence of 33, 333 and 334 which are synthesized by amino-propylation to the aminopropyl moiety of diamines or triamines, theoccurrence of 3343 indicates the presence of aminopropyl-transfer activityto the aminopropyl moiety of 343 in the organism. The guanine-cytosinecontent of the DNA of this thermophilic Gram-positive coccus and its cellwall composition were found to be similar to those of a moderately thermo-philic rod, Bacillus stearothermophilus (Nystrand, 1984).

In Bacillus species, the occurrence of 343 and 34 might be a unique patternof obligate, moderate thermophilic strains including acidophiles, growing atthe optimum growth temperature of 60-65oC. Chemotaxonomic similaritybased on the polyamine pattern between S. thermophilus and thermophilicbacilli is suggested. The occurrence of a tertiary branched penta-aminer3(3)43,is limited in the extreme thermophiles growing at 70-75oC, and some ofthe moderate thermophiles tested. However, the former were rich in penta-amines. The thermophilic actinomycetes growing at 55oC, Thermoactinomyces

sacchari and 7. oulgaris, taxonomically related to moderately thermophilicBacillus species, contained 343 in addition to 4 and 34 which are the majorpolyamines of mesophilic actinomycetes (Hamana and Matsuzaki, 1987).

An extremely thermophilic H2-oxidizing bacillus, B. schlegeftr, produceda pentamine, 3343 and two hexa-amines, 33343 arad 33433. B. schlegelii isalso characterized by the presence of 3Q)a and 3(3)(3)4 formed from 34 and

the absence of 3(3)43 which is produced from 343.The polyamine distributionprofile of the obligately thermophilic, facultatively chemolithoautotrophicb'acterium is unique in the thermophilic bacilli tested.

Acidophilic strains of the thermophilic Bacillu.r genus, namely Bacillusacidocaldarius and B. cycloheptanicus, and the acidophilic, facultatively

30 Microbios K. Hamana er a/.

H2-oxidizing B. tusciae, have the same polyamine pattern as that of neutro-philic, organotrophic strains of moderately thermophilic bacilli. Polyaminetypes were found in the slightly thermophilic B. cychheptanicus and B. tusc'iae,

growing at 50oC, where the optimum growth temperature corresponds tothe typical pattern for the moderate thermophiles growing at 60-65oC. Anacidophilic, slightly-thermophilic, B. acidoterresrrrs, growing at 50oC, was

devoid of 343. The presence of 4 and34 and the absence of 343 are commonto mesophilic bacilli including thermotolerant (facultatively thermophilic)species such as B. coagulaas and B. breois growing at 50oC (Hamana et al.,1e8e).

Recently it has been proposed that the thermoacidophilic B. acidocaldaius,B. acidotenestis and B. cychheptanians should be reclassified in the new genus

Alcyclobacl/zs (tU7isotzkey et al., 1992). However, the polyamine pattern ofB. acidotenesrrrs differs from the other two species, as shown in this study.Amphibacillus xylans is devoid of any polyamines, suggesting that this meso-philic, alkalophilic Gram-positive rod bears no relation in its polyamine dis-tribution profile to mesophilic alkalophilic Bacillus species which ubiquitouslycontain 4 and 34 (Hamalna et al., 1989).

These findings suggest that the occurrence oflinear penta-amines and hexa-

amines or branched polyamines in Gram-positive eubacteria are more closelyassociated in their thermophilic activity rather than in their acidophylic,alkalophilic or chemolithotrophic properties. Distribution profiles ofpolyamines including unusual long and branched types may serve as

phenotypic markers in the classification of Bacillus species and of alliedeubacteria.

AcknowledgementsWe are indebted to the Japan Collection of Microorganisms, Reken, lnstitute ofApplied Microbiology, The University of Tokyo, and the American Type CultureCollection, for supplying bacterial strains.

References

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31 Polyamines of thermophilic Bacillus

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Accepted 18 March 1993

32 Microbios K. Hamana e, a/.