Polyamine Distribution Patterns in Aerobic Gram-Positive Cocci and Some Radio-Resistant Bacteria

J. Gen. App!. Microbiol., 40, 181-195 (1994)

POLYAMINE DISTRIBUTION PATTERNS IN AEROBIC GRAM-POSITIVE COCCI AND SOME RADIO-RESISTANT BACTERIA

KOEI HAMANA*

College of Medical Care and Technology, Gunma University, Maebashi 371, Japan

(Received November 8, 1993; Accepted April 22, 1994)

Cellular polyamines of various aerobic Gram-positive cocci and some radio-resistant eubacteria were analyzed. Staphylococcus and Micrococcus species except for some spermidine-containing or cadaverine-containing strains were lacking in polyamines. Planococcus species ubiquitously contained putrescine, spermidine and agmatine. Marinococcus were divid- ed into two groups, one of which contains no polyamine and another putrescine, spermidine and agmatine. Polyamines were not detected in Pediococcus, Tetragenococcus, Aerococcus, Alloiococcus, Helcococcus and Kineococcus species. Spermidine was found in Stomatococcus mucilaginosus and Salinicoccus hispanicus while no polyamine was detected in Salinicoccus roseus. Sporosarcina halophila and Sporosarcina ureae ubiquitously contained spermidine, however, agmatine was found in the former and spermine in the latter. Polyamine profiles were heterogeneous within aerobic Gram-positive cocci, though each profile was specific for the genera. Different polyamine patterns found in the genera Marinococcus, Salinicoccus and Sporosarcina suggest the heterogeneity of these genera. Radio-resistant Rubrobacter radiotolerans was devoid of polyamines, whereas spermidine was detected in radio-resistant Deinobacter grandis as well as radio-tolerant Deinococcus species.

Polyamine distribution profiles serve as a phenotypic chemotaxonomic marker in various Gram-negative eubacteria belonging to the Proteobacteria (5,16,17,19) and the Flavobacterium-Cytophaga-Flexibacter complex (12,13,16). Distribution patterns of diaminopropane, putrescine, 2-hydroxyputrescine, cadaverine, spermidine, norspermidine and homospermidine in Gram-negative eubacteria are closely associated with their phylogenetic relationships. Information on polyamine profiles

* Address reprint requests to: Dr . Koei Hamana, College of Medical Care and Technology, Gunma University, 3-39-15 Showa-machi, Maebashi 371, Japan.

181 182 HAMANA VOL. 40 in Gram-positive eubacteria are required to evaluate the usefulness of polyamine analysis for the chemotaxonomy of eubacteria. In this study, polyamines were analyzed to determine their distribution patterns in the two major groups of Gram-positive cocci, genus Staphylococcus (22) and genus Micrococcus (23). Sys- tematic polyamine analysis of the other aerobic, Gram-positive cocci belonging to the genera Pediococcus (11), Aerococcus (1,10), Planococcus (21, 24), Marinococcus (20), Stomatococcus (4), and Sporosarcina (6) would be of some help to elucidate chemotaxonomic significance of cellular polyamine composition. Two Gram-positive halophilic cocci belonging to the genus Salinicoccus (29, 30) and a new aerobic, Gram-positive motile coccus, Kineococcus aurantiacus (33) were recently published. Pediococcus halophila was reclassified in a new genus Tetragenococcus (8). Two Aerococcus-like organisms, Alloiococcus otitis and Helco- coccus kunzii, were newly described (2, 7). Polyamines of these new members of microaerobic and facultative-anaerobic Gram-positive cocci were analyzed. A Gram-positive coccal family, Deinococcaceae, contains only a single genus, Deinococcus, which is radiation-resistant. The genus includes D. radiodurans, D. radiophilus, D. proteolyticus, and D. radiopugnans (9,25). Gram-positive Rubro- bacter radiotolerans (27,34) and Gram-negative Deinobacter grandis (26) have been validly published as radio-tolerant bacteria. It would be of great interest to examine polyamine types and cellular polyamine levels of these radio-resistant bacteria because polyamines are considered to stimulate cellular DNA packing to protect it from radiation damage (3).

MATERIALS AND METHODS

Cultivation of organisms. Organisms listed in Tables 1-3 were grown in synthetic 199 medium (Flow Lab., Irvine, Scotland) or in 199 medium supplemented with 8% NaCI (199 NaCI medium) to exclude polyamine uptake from the medium. The synthetic 199 medium prepared for mammalian cell culture contains the usual 21 amino acids, 17 vitamins, 10 components of nucleic acids, sodium acetate, glucose, NaCI, KCI, CaCl2, MgS04, NaH2P04 and Fe(N03)3 but not ornithine, diaminobutyric acid and polyamines. Nutrient broth (NB) (Nissui Pharmaceutical Co., Tokyo, Japan), B medium containing beef extract, peptone, yeast extract and glucose (34), MH medium containing proteose peptone and yeast extract (20), TGY medium containing tryptone, glucose and yeast extract (9), TSB medium (BBL-Trypticase Soy Broth, Becton Dickinson and Co., MD, U.S.A.) and GAM medium (Nissui Pharmaceutical Co.) were used in some cultures. 199SW medium and GAMSW, dissolved in 70% sea water, were used for halophilic strains. Staphylococcus species were cultured at 37°C. Rubrobacter was cultivated at 46 and 30°C. Other organisms were grown at 30°C. Polyamine analysis. Polyamines in the packed wet cells were extracted with 0.5 M cold perchrolic acid (PCA). The PCA extracts and the packed cells were also hydrolyzed with 6 M HCl at 110°C for 24 h. Cellular free polyamines were 1994 Polyamines in Aerobic Gram-positive Cocci 183

Table 1. Cellular concentrations of polyamines in Staphylococcus.

T 1 / 7 1 184 HAMANA VOL. 40

Table 1. (Continued)

pn~~rominn (i,

extracted into cold 0.5 M PCA, whereas polyamines covalently bound to macromolecules in the cells were detected as free polyamines in the hydrolysates of the PCA extracts or the packed cells. Polyamines in the PCA extracts and the acid hydrolysates dissolved in water were analyzed by high performance liquid chro- matography (HPLC) on a column (4 mm ID. X 50 mm) of cation-exchange resin (Hitachi #2619F) at 70°C. Polyamines were eluted by a linear gradient of two buffers, 0.045 M sodium citrate-0.061 M citric acid-0.064 M NaCI and 0.20 M sodium citrate-2.0 M NaCI. Diamines were also analyzed to confirm their identity by a reverse phase HPLC on a column (4.6 mm ID. X 150 mm) of ODS-silica gel (M-15, 5tim, Mitsubishi Kakoki, Tokyo, Japan) at 55°C. A buffer containing 1.75 mM sodium 1-octanesulfonate-3% methanol-10 mM KH2PO4-4 mM NaOH was used for the elution. To assign the agmatine peak, polyamine samples were alkaline- hydrolyzed with 1 M NaOH at 110°C for 24 h before HPLC analysis. Agmatine was decomposed to putrescine by hydrolysis. Polyamines eluted on the HPLC were detected by heating with o-phthalaldehyde reagent. For the assay of amino acid-decarboxylation activity, the cell lysates were added to the reaction mixture containing amino acids and the amount of damines produced during incubation were assayed by HPLC (15).

RESULTS

Although cellular polyamine contents of bacteria were affected somewhat by culture temperature, growth phase and medium composition (16), essentially the same polyamine distribution profile was obtained when the organisms were har- vested at either logarithmically growing phase or stationary phase in the present study (not shown). When the organisms were grown in NB, B, MH, TGY, TSB and GAM media containing polyamines (polyamines of NB is shown in Table 1), uptake of polyamines from the media could not be excluded. The data of the 1994 Polyamines in Aerobic Gram-positive Cocci 185

Table 2. Cellular concentrations of polyamines in Micrococcus.

cultures in the polyamine-free synthetic 199 medium showed the net polyamine compositions synthesized in the cells. Polyamine concentrations in the cells growing at the stationary phase at their optimum growth temperature are shown in Tables 1-3 and profiles of polyamine distribution are summarized in Table 4. Among the cells of 22 strains of Staphylococcus species grown in 199 medium, a significant amount of spermidine was detected only in the cells of S. caseolyticus. A small amount of spermidine was detected in the cells of S. aureus JCM 2151, S haemolyticus JCM 2416, S piscifermentans JCM 6057 and S. warneri JCM 2415 grown in 199 medium. Supplement of 10 mM putrescine into the culture medium 186 HAMANA VOL. 40

Table 3. Cellular concentration of polyamines in Pediococcus, Tetragenococcus, Aerococcus, Alloiococcus, Helcococcus, Planococcus, Marinococcus, Salinicoccus, Sporosarcina, Kineococcus, Stomatococcus, Deinococcus, Deinobacter and Rubrobacter. 1994 Polyamines in Aerobic Gram-positive Cocci 187

Table 3. (Continued) 188 HAMANA VOL. 40

Table 4. Polyamine distribution patterns in Gram-positive cocci.

caused a slight increase of spermidine in the cells of the four strains (Fig. 1 and Table 1). No polyamines were detected in the other 17 strains grown in 199 medium. The polyamine profiles were identical in either the PCA extract or HCl-hydrolysate of the packed cells. Ornithine decarboxylase (ODC) activity to produce putrescine was not detected in the 17 strains by our cell-free assay system. Cadaverine was detected in the PCA extract of Micrococcus aurantiacus (IFO 15364), M, luteus (IFO 12992) and MMroseus (IAM 13593 and IFO 3764) grown in 199 medium, as shown in Figs. 1 and 2 and Table 2. Lysine decarboxylase (LDC) activity to convert lysine to cadaverine was detected in the four strains. Other Micrococcus strains grown in 199 medium were devoid of polyamines in the PCA extracts and acid-hydrolysates. No spermidine was produced in any strains after the supplement of putrescine into the medium (Fig. 1). After the HC1- hydrolysis of the packed cells or PCA extracts, two strains of M. roseus showed three-time higher levels of cadaverine (an example is shown in Fig. 2-A). Pediococcus species, Aerococcus species, Tetragenococcus halophilus, Alloiococ- cus otitis and Helcococcus kunzii, grown in 199 medium, were lacking in polyamines (Table 3). Planococcus citreus, P. kocurii and Marinococcus halophilus contained putrescine, spermidine and agmatine, whereas Marinococcus albus was devoid of 1994 Polyamines in Aerobic Gram-positive Cocci 189

Fig. 1. Ion-exchange HPLC analysis of polyamines in the PCA extract from Staphylococcus aureus JCM 2151 grown in 199 medium (A-a), 199 medium supplemented with 10 mM putrescine (A-b) and nutrient broth (A-c), and Micrococcus roseus IAM 13593 grown in 199 medium (B-a), 199 medium supplemented with 10 mM putrescine (B-b) and nutrient broth (B-c). Abbreviations for polyamines are shown in Table 1. polyamines, in their PCA extracts (Fig. 3 and Table 3). Arginine decarboxylase (ADC) activity assayed by our cell-free system was detected in P. citreus, P. kocurii and M. halophilus. Polyamines were not detected in the PCA extract of Salinicoc- cus roseus and Kineococcus aurantiacus grown in 199 medium. No polyamine was detected in the HCl-hydrolysate of the packed whole cells of M. albus, S. roseus and K. aurantiacus. Putrescine and spermidine were not produced in their respective cultures by supplementation with ornithine and putrescine into the medium, respectively. Salinicoccus hispanicus and Stomatococcus mucilaginosus contained spermidine as the major polyamine. Sporosarcina halophila and Sporosarcina ureae ubiquitously contained spermidine. Agmatine or spermine was the minor polyamine in the two Sporosarcina, respectively (Fig. 4). Four Deinococcus species and Deinobacter grandis contained spermidine as the major polyamine, as shown in Fig. 3 and Table 3. The PCA extracts and HCl-hydrolysates of the packed cells gave nearly the same polyamine profile. Putrescine and spermidine levels of the organisms were increased by supplementation with ornithine into the medium, showing ODC activity. D. erythromyxa contained cadaverine in its PCA extract (Fig. 3). In HCl-hydrolysate of the PCA extract as well as the acid hydrolysate of the packed cells, the cadaverine content expressed per wet cell was five times higher (Fig. 3). LDC activity was found in the cell lysate of D. erythromyxa. Polyamines were not detected in Rubrobacter 190 HAMANA VOL. 40

Fig. 2. Ion-exchange HPLC analysis of polyamines in the PCA extract (A) and its HCl-hydrolysate (A-HC1) of Micrococcus roseus IFO 3764 grown in 199 medium and the PCA extract (B) and its HCl-hydrolysate (B-HC1) of Micrococcus luteus IFO 12992 grown in 199 medium. Abbreviations for polyamines are shown in Table 1.

° C for optimum growth or at 30°C radiotolerans grown in 199 medium either at 46 (Table 3).

DISCUSSION

Absence of appreciable amounts of polyamines was a unique property of the genera Staphylococcus and Micrococcus. However, polyamine analysis does not serve as a chemotaxonomic marker for the genera because many aerobic Gram- positive cocci do not have polyamines. Sporadic occurrence of cadaverine in the genus Micrococcus may be species-specific within the genus. M, roseus contained free cadaverine extractable with 5 % PCA and bound cadaverine to unknown acid-labile macromolecules. Cadaverine in M. aurantiacus and M. luteus was completely extracted with 5 % PCA. Spermidine synthetic activity found in the limited strains of Staphylococcus is possibly significant but insufficient to use for the chemotaxonomy of Staphylococcus species. 1994 Polyamines in Aerobic Gram-positive Cocci 191

Fig. 3. Ion-exchange HPLC analysis of polyamines in the PCA extract from Deinococcus proteolyticus (A), Planococcus kocurii (B) and Deinococcus erythromyxa (C) and HCl-hydrolysate of Deinococcus erythromyxa (C-HC1). Reverse phase HPLC analysis of polyamines in the PCA extract from Planococcus kocurii (B-ODS) and HCl-hydrolysate of Deinococcus erythromyxa (C-HCI-ODS). Abbreviations for polyamines are shown in Table 1.

The phylogeny of Aerococcus and Pediococcus as determined by 16S rRNA sequence analysis showed that P. urinaeequi was closely related to A. viridans, and P. halophilus was reclassified in a new genus Tetragenococcus (1, 8). Two Aerococ- cus-like cocci, Alloiococcus otitis and Helcococcus kunzii (2, 7), as well as Aerococ- cus, Pediococcus and Tetragenococcus, were characterized by the absence of polyamines. These results indicate that polyamine analysis is useless for differentiation of these five genera. The occurrence of putrescine, spermidine and agmatine in the two species of Planococcus and Marinococcus halophilus was quite unique among the Gram- positive cocci tested. The absence of polyamines was observed in another Marino- coccus species, MMalbus, and Salinicoccus roseus. Salinicoccus hispanicus, formerly called Marinococcus hispanicus (30), was also unique in containing spermidine alone. The different polyamine profiles found in the two Marinococcus species, in the two Salinicoccus species and in the two Sporosarcina species, respectively, suggest the heterogeneity of these genera. Since the polyamine pattern would be a 192 HAMANA VOL. 40

Fig. 4. Ion-exchange HPLC analysis of polyamines in the PCA extract from Sporosarcina halophila IAM 13060 grown in 199SW medium (A-a) and GAMSW medium (A-b) and Sporosarcina ureae JCM 2577 grown in 199 medium (B-a) and GAM medium (B-b). Abbreviations for polyamines are shown in Table 1.

chemotaxonomic marker of genus level, analysis of polyamines may be useful for further identification and classification of the Gram-positive cocci belonging to the genera Stomatococcus, Kineococcus, Sporosarcina, Planococcus, Marinococcus and Salinicoccus. As Deinococcus species were separated from the genus Micrococcus (9,25), polyamine profile of authentic Deinococcus species differed from that of Micrococcus species. D. erythromyxa (formerly called "MMerythromyxa"), which has been excluded from the genus Deinococcus and reassessed into genus Micrococcus (9), contained free cadaverine and cadaverine-binding macromolecules. This fact suggests that this organism diverges from typical Deinococcus species and is rather allied to M. roseus. Except for the difference in Gram-staining and morphology, apparent similarities in fatty acid composition, G + C content of DNA, peptidoglycan type and oligonucleotide catalog of 16S rRNA were observed between Deinococcus (Gram-positive coccus) and Deinobacter (Gram-negative rod) (26). By the occurrence of spermidine both in authentic Deinococcus species and Demo- 1994 Polyamines in Aerobic Gram-positive Cocci 193 batter grandis, a close relationship between the two genera was shown. Deinococ- cus-Thermus cluster based on 16S rRNA and 5S rRNA sequence analyses have been proposed (28, 31, 32) . Mesophilic Deinococcus species contained only spermidine, whereas thermophilic Thermus species which grow at 70°C contained tetraamines, pentaamines, hexaamines and branched polyamines in addition to spermidine (18). The occurrence of long and branched polyamines seems to be associated with thermophily and has no connection with the taxonomic relation between genus Deinococcus and genus Thermus. Highly radio-tolerant Rubrobacter radiotolerans was devoid of any polyamines, whereas spermidine occurred in other radio-resistant bacteria, Deinococcus and Deinobacter. Either radio-tolerant Acinetobacter radioresistens or other radio- sensitive Acinetobacter species ubiquitously contained diaminopropane at similar concentrations in the cells (14). These findings indicate that polyamine composition and concentration in the cells are dissociated from the radio-tolerance of these bacteria.

The author is indebted to Institute for Fermentation, Osaka, Institute of Applied Microbiology, The University of Tokyo, Japan Collection of Microorganisms, RIKEN, Department of Microbiology, Gifu University School of Medicine, Culture Collection Center, Tokyo University of Agriculture, American Type Culture Collection, U.S.A. and Dr. S. Satake of College of Medical Care and Technology, Gunma University for supplying bacterial strains. The author is obliged to Dr. S. Matsuzaki, Dokkyo University School of Medicine for valuable suggestions.

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