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Istituto di Chimica Biomolecolare, Comprensorio ex Olivetti, via Campi Flegrei 34, 80078 Pozzuoli, Na, Italy
Correspondence
Assunta Giordano
agiordano{at}icmib.na.cnr.it
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 6AGT is AJ493660.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-D-glucopyranoside (XGlc) (Romano et al., 2003
). In 1985, the isolation of an obligately alkaliphilic, extremely halotolerant Bacillus strain, WN13, was described, later classified by Fritze (1996) as Bacillus haloalkaliphilus. Little effort has been devoted to the study of intracellular ion concentrations in halophilic Gram-positive bacteria. Apparent intracellular cation concentrations as high as those present in the growth medium were reported in Bacillus haloalkaliphilus (Weisser & Trüper, 1985). However, this species is now known to produce ectoine as the main osmotic solute (Oren, 2002). Few Bacillus species that grow in the presence of high NaCl concentrations and are also alkaliphilic have been described (Fritze, 1996; Arahal & Ventosa, 2002). The significance of haloalkaliphiles has prompted the isolation and characterization of such strains from a variety of environments. In this study, a new member of the genus Bacillus is proposed on the basis of polyphasic studies. Strain 6AGT was isolated from a mat of green algae collected from a mineral pool at the Malvizza site (Montecalvo Irpino, Avellino province, Campania region, south Italy). The strain was isolated by the dilution-plating technique from the algal mat (pH 8·0, 18 °C) at the green–red algae boundary in a small pool. Enrichment medium (medium 1) contained the following components (l–1): Na2CO3, 3·0 g; KCl, 2·0 g; MgSO4.7H2O, 1·0 g; NaCl, 160 g, trisodium citrate, 3·0 g; yeast extract, 10·0; MnCl2.4H2O, 0·36 mg; FeSO4, 50 mg. Na2CO3 and NaCl were autoclaved separately. The pH of medium 1 was 9·0. Growth on single carbon sources at 10 g l–1 was tested on liquid medium (medium 2) containing (g l–1) K2HPO4, 7·0; KH2PO4, 2·0; MgSO4.7H2O, 0·1; (NH4)2SO4, 1·0; NaCl, 160; Na2CO3, 3·0; with 500 µl thiamine hydrochloride solution l–1 (100 mg l–1). Solid media were prepared by the addition of 1·8 % agar.
Cell morphology was determined by phase-contrast microscopy (Zeiss). Colony morphology was analysed on solid medium with a Leica M8 stereomicroscope. Tolerance of NaCl and growth at various temperatures and pH was assessed in medium 1. All growth tests were done at the optimal growth temperature for 3 days. Sensitivity of the strain to antibiotics was tested by using medium 1 with agar (1·8 %) and Sensi discs (6 mm; Oxoid); tests were incubated for 48 h. Antibiotic tests were carried out according to Romano et al. (1993). Casein and gelatin hydrolysis, oxidase, tyrosinase, aminopeptidase (Bactident-Merck) and catalase were tested in medium 1. For nitrate reduction, medium 1 plus 0·1 % KNO3 was employed. Hydrolysis of hippurate was assessed in medium 1 plus hippurate (1 %) (Romano et al., 1996). Gram-staining was performed according to Dussault (1955). The KOH test was performed according to Halebian et al. (1981). Hydrolysis of N'-benzoyl-arginine p-nitroaniline (BAPNA) stereoisomers was tested according to Oren & Galinski (1994). The extraction of intracellular solutes, their purification and quantification were done according to Romano et al. (2001).
Cell mass for quinone and lipid analysis was obtained from culture in medium 1 at 37 °C at pH 9·0 at optimal NaCl concentration. Lipid analysis was performed as reported by Romano et al. (2001). Lipid hydrolysis and identification of fatty acid methyl esters were done as reported by Romano et al. (2001). Quinones were analysed by LC/MS on a reverse-phase column, by EI/MS and H1 NMR spectra.
The G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by HPLC. The DNA was isolated as described previously (Romano et al., 2003).
The total 16S rRNA gene sequence was determined by direct sequencing of the PCR product. Genomic DNA extraction, amplification of the 16S rRNA gene and purification of PCR products were carried out as described previously (Romano et al., 2003). Purified PCR products were sequenced by the DSMZ using the ABI PRISM Dye Terminator cycle sequencing ready reaction kit (Applied Biosystems) as directed in the manufacturer's protocol. Sequence reactions were electrophoresed using an Applied Biosystems 373A DNA sequencer.
The multiple sequence alignment program CLUSTAL W (Chenna et al., 2003) was used to align the 16S rRNA gene sequence of our isolate with the sequences of representative organisms belonging to the Bacillus/Staphylococcus group. Sequences of 16S rRNA genes for comparison were obtained from the EMBL database or RDP. Phylogenetic analysis was performed using the PHYLIP package, version 3.5 (Felsenstein, 1993). Distances (distance options according to Kimura's two-parameter model) and clustering with the maximum-parsimony method were determined by using bootstrap values based on 1000 replications.
16S rRNA gene phylogenetic analysis based on the neighbour-joining method (Saitou & Nei, 1987) showed strain 6AGT to be placed in the genus Bacillus (Fig. 1). In particular, the sequence similarity to any other species within the genus Bacillus with validly published names was less than 95·7 %.
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; Nielsen et al., 1995), while our isolate showed less than 96 and 95 % similarity, respectively, to these two latter species. Phylogenetic analysis based on the neighbour-joining and maximum-parsimony methods showed the same position for strain 6AGT and the three undescribed isolates among the genus Bacillus, grouping together and forming a cluster separated from other species (Fig. 1
). The G+C content of the DNA of strain 6AGT was 48·4 mol%.
Strain 6AGT was a Gram-positive non-sporulating coccus measuring 0·8–0·9 µm. It grew as single cells and/or clusters and was non-sporulating, even in the presence of 100 mg MnCl2 ml–1. Colonies on enrichment medium 1 were usually yellow. The main features of the novel isolate are listed in Table 1.
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). Strain 6AGT was able to grow on medium 2 containing glucose. Other carbon sources did not sustain growth. Among the antibiotics tested, strain 6AGT was sensitive to (µg) vancomycin (30), ampicillin (25), novobiocin (30), fusidic acid (10), streptomycin (25), chloramphenicol (10), erythromycin (5 and 30) and bacitracin (10).
The phenotypic features of strain 6AGT and B. agaradhaerens and B. clarkii differ in several aspects (Table 1). Strain 6AGT is a regular non-sporulating coccus, while B. agaradhaerens and B. clarkii are sporulating rods. The most interesting common feature between the above-mentioned Bacillus species and strain 6AGT is their haloalkaliphilicity; in fact, they grew well at high pH values and in the presence of NaCl and they were able to tolerate NaCl concentrations up to 16–25 %. Carbohydrate utilization profiles showed growth on glucose for 6AGT and on several sources for B. agaradhaerens, while no growth was observed in minimal carbohydrate media for B. clarkii (Nielsen et al., 1995).
Strain 6AGT possessed complex lipids based on fatty acids. The total lipid content accounted for 9–11 % of the total dry weight of cells grown under optimal growth conditions in medium 1. Under these conditions, phosphoethanolamine, diphosphatidylglycerol and phosphatidylglycerol were the main lipids (approx. 96 %), identified by 1H and 13C NMR spectra. The chemical shift values of signals present in spectra were similar to those reported by Romano et al. (2001), with the exception of the absence of the signals due to double bonds in the fatty acid chains. The assignments were done by comparison with commercial standards and by distortionless enhanced polarization transfer (DEPT) experiments. Glycolipids were not visualized; this was probably due to their absence or occurrence in very small amounts.
The fatty acid composition determined from cells grown under standard growth conditions was characterized by the abundance of branched saturated acyl chains; in particular, aiC15 : 0 reached 89 % of total fatty acid methyl esters.
LC/MS as well as EI/MS analyses of the quinone content of strain 6AGT showed three major peaks, assigned to MK-7(H4), MK-7(H2) and demethylated MK-7(H2) [DMK-7(H2)] in a relative ratio of 1·5 : 7·0 : 1·5.
Little effort has been devoted to the study of osmoprotectants accumulated by halophilic Gram-positive bacteria; under optimal growth conditions, strain 6AGT accumulated glycine betaine, hydroxyectoine, ectoine and glutamate.
On the basis of these results, we suggest that strain 6AGT represents a novel species, Bacillus saliphilus sp. nov. This affiliation is preliminary, in that the creation of a new genus based on a single strain makes it impossible to assess the limits of variability of the taxon. Finally, it is well known that the genus Bacillus is clearly heterogeneous, and many contributions have been made to the development of Bacillus systematics (Berkeley et al., 2002).
Description of Bacillus saliphilus sp. nov.
Bacillus saliphilus (sal.i.phi'lus. L. n. sal salt; Gr. adj. philos loving; N.L. masc. adj. saliphilus loving salt).
Alkalitolerant and halotolerant. Cells are Gram-positive, regular cocci, 0·8–0·9 µm in diameter, appearing singly and/or in clumps. Colonies are yellow. Mesophilic, exhibiting optimum growth at 37 °C, but is able to grow between 4 and 50 °C and at pH 7·0–10·0 (optimum pH 9·0). Grows at salinity of 1–25 % (w/v), with optimum growth at 16 % (w/v) salts. Able to grow on glucose as a sole carbon source. Positive for oxidase, gelatinase, tyrosine decomposition and nitrate reduction but negative with respect to starch, casein, hippurate and phenylalanine hydrolysis. Catalase reaction is weak. Possesses 
-glucosidase activity. Menaquinones are MK-7(H2) (70 %), MK-7(H4) (15 %) and DMK-7(H2) (15 %) and the predominant polar lipids are phosphatidylglycerol and diphosphatidylglycerol. iC15 : 0, aiC15 : 0 (89 %) and C17 : 0 are the main cellular fatty acids. Accumulates glycine betaine as the major osmoprotectant and hydroxyectoine, ectoine and glutamate as minor components. The G+C content of DNA is 48·4 mol%. Phylogenetically related to three uncharacterized micro-organisms and to B. agaradhaerens (95·4 % similarity) on the basis of 16S rRNA gene sequences. The following antibiotics inhibit growth (µg unless stated): chloramphenicol (10), erythromycin (5, 30), bacitracin (10), vancomycin (30), ampicillin (25), fusidic acid (10), streptomycin (25) and novobiocin (30). Grows in the presence of penicillin G (2, 10 IU), tetracycline (30, 50), kanamycin (30), neomycin (30) and gentamicin (30).
The type strain, strain 6AGT (=DSM 15402T=ATCC BAA-957T), was isolated from an algal mat around a small, bubbling mineral pool at the Malvizza site (Campania, Italy).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Berkeley, R. C. W., Heyndrickx, M., Logan, N. & De Vos, P. (editors) (2002). Applications and Systematics of Bacillus and Relatives. Oxford: Blackwell.
Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. (2003). Multiple sequence alignment with the CLUSTAL series of programs. Nucleic Acids Res 31, 3497–3500.
Duckworth, A. W., Grant, W. D., Jones, B. E. & van Steenbergen, R. (1996). Phylogenetic diversity of soda lake alkaliphiles. FEMS Microbiol Ecol 19, 181–191.
Dussault, H. P. (1955). An improved technique for staining red halophilic bacteria. J Bacteriol 70, 484–485.
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Fritze, D. (1996). Bacillus haloalkaliphilus sp. nov. Int J Syst Bacteriol 46, 98–101.
Halebian, S., Harris, B., Finegold, S. M. & Rolfe, R. D. (1981). Rapid method that aids in distinguishing Gram-positive from Gram-negative anaerobic bacteria. J Clin Microbiol 13, 444–448.
Nielsen, P., Fritze, D. & Priest, F. G. (1995). Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology 141, 1745–1761.
Oren, A. (2002). Halophilic Microorganisms and Their Environments. Boston: Kluwer Academic.
Oren, A. & Galinski, E. A. (1994). Hydrolysis of N'-benzoyl-arginine-p-nitroanilide stereoisomers as a phenotypic test: a study of Gram-positive halotolerant bacteria. Syst Appl Microbiol 17, 7–10.
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Romano, I., Nicolaus, B., Lama, L., Trabasso, D., Caracciolo, G. & Gambacorta, A. (2001). Accumulation of osmoprotectants and lipid pattern modulation in response to growth conditions by Halomonas pantelleriense. Syst Appl Microbiol 24, 342–352.[CrossRef][Medline]
Romano, I., Giordano, A., Lama, L., Nicolaus, B. & Gambacorta, A. (2003). Planococcus rifietensis sp. nov, isolated from algal mat collected from a sulfurous spring in Campania (Italy). Syst Appl Microbiol 26, 357–366.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Weisser, J. & Trüper, H. G. (1985). Osmoregulation in a new haloalkaliphilic Bacillus from the Wadi Natrun (Egypt). Syst Appl Microbiol 6, 7–11.
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