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1 Departamento de Zoologia and Centro de Neurociências de Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal
2 Departamento de Bioquímica and Centro de Neurociências de Biologia Celular, Universidade de Coimbra, 3004-517 Coimbra, Portugal
Correspondence
António Veríssimo
averiss{at}ci.uc.pt
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ABSTRACT |
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type with a cross-linkage containing D-Asp. The major respiratory quinone is menaquinone 7 and the major fatty acids are anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0. The DNA G+C content is 37.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain B22T formed a new branch within the family Bacillaceae. The novel isolate is phylogenetically closely related to members of genera of moderately halophilic bacilli and formed a coherent cluster with species of the genera Salinibacillus, Virgibacillus, Oceanobacillus and Lentibacillus, supported by bootstrap analysis at a confidence level of 71 %. Strain B22T exhibited 16S rRNA gene pairwise sequence similarity values of 94.7–94.3 % with members of the genus Salinibacillus, 95.1–92.8 % with members of the genus Virgibacillus, 94.7–93.2 % with members of the genus Oceanobacillus and 93.1–92.3 % with members of the genus Lentibacillus. On the basis of phylogenetic analysis and physiological and biochemical characteristics, it is proposed that strain B22T represents a novel species in a new genus, Paucisalibacillus globulus gen. nov., sp. nov. Strain B22T (=LMG 23148T=CIP 108857T) is the type strain of Paucisalibacillus globulus.
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), Virgibacillus (Heyndrickx et al., 1998), Oceanobacillus (Lu et al., 2001) and Lentibacillus (Yoon et al., 2002), form a coherent phylogenetic cluster. During an investigation of the presence of Legionella in composts and potting soils used in Portugal, several slightly halophilic Gram-positive bacteria were isolated and further characterized. One of these isolates shared many physiological and biochemical characteristics with species of the genera mentioned above, but had a distinctly lower NaCl requirement for optimal growth and a distinctive peptidoglycan composition. Furthermore, 16S rRNA gene sequence analysis showed that this organism represents a distinct phylogenetic lineage within the family Bacillaceae. In this study, the morphological, physiological, chemotaxonomic and phylogenetic characteristics of strain B22T are described.
Strain B22T was isolated from potting soil on buffered charcoal yeast extract (BCYE) medium (Edelstein, 1981), which is normally used for the isolation and growth of Legionella spp. Cultures were purified by subculturing and preserved at –70 °C in 5 % yeast extract medium with 15 % glycerol. Despite the organism having been isolated on BCYE, the strain was routinely cultured at 37 °C in alkaline buffered medium 2 (ABM2) with 1 % NaCl, adjusted to pH 8.0 (Tiago et al., 2005b). Unless otherwise stated, all morphological examinations and biochemical and tolerance tests were performed on this medium after up to 6 days incubation as described previously (Tiago et al., 2005b). The growth temperature range of the strain was examined in liquid medium in a reciprocal water bath between 20 and 45 °C. The NaCl range for growth of the organism was determined at pH 8.0 and 37 °C. The pH range for growth was determined at 37 °C in the same medium buffered at pH values between pH 6.0 and 9.5 as described previously (Tiago et al., 2005b). Anaerobic growth was assessed at 37 °C in anaerobic chambers with an H2/CO2 atmosphere (bioMérieux). Single carbon source assimilation tests were performed in 20 ml screw-capped tubes as described previously (Tiago et al., 2005a). Acid production from carbohydrates was also determined using API 50CH test strips (Analytab Products; bioMérieux) containing 50 CHB/E medium, as described previously (Tiago et al., 2006). Peptidoglycan analysis was performed according to Schleifer (1985) and Schleifer & Kandler (1972). Respiratory quinone analysis was performed according to Tindall (1989) and the fatty acid profile was determined as described by Tiago et al. (2005b) using the standard MIS library Generation Software (Microbial ID).
The DNA G+C content was determined by HPLC as described by Mesbah et al. (1989). The 16S rRNA gene was sequenced as described by Tiago et al. (2006). Phylogenetic analysis was performed using the ARB software package (Ludwig et al., 2004). Phylogenetic trees were constructed using maximum-likelihood (Felsenstein, 1981) and neighbour-joining (Saitou & Nei, 1987) algorithms. Tree topologies were evaluated by performing bootstrap analysis (Felsenstein, 1985) of a dataset of 1000.
Strain B22T formed small, beige-coloured, spherical colonies. Cells were Gram-positive, motile by two polar flagella and rod-shaped (0.5 µm in width by 3.0–7.0 µm in length), with oval terminal endospores in a non-swollen sporangium. The NaCl concentration for optimum growth was 1.0 %, but growth occurred in the absence of NaCl and in medium containing up to 8.0 % NaCl. Strains belonging to the phylogenetically most related genera had higher NaCl requirements for optimal growth and they could grow at much higher NaCl concentrations. Moreover, only species of the genus Oceanobacillus and strain B22T were able to grow in media without NaCl (Table 1). The novel organism also had a narrower pH range (pH 7.0–9.0) for growth when compared with members of related genera. Strain B22T did not reduce nitrate. Casein, starch, DNA, arbutin, aesculin, hippurate, elastin, gelatin and Tweens 20, 40, 60 and 80 were hydrolysed, but xylan and urea were not. Furthermore, B22T utilized several sugars and proteinaceous substrates.
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; L-lys was the diamino acid at position 3 of the peptidoglycan and the dicarboxylic amino acid present in the cross-linkage was D-Asp. The peptidoglycan composition of the novel strain is unique amongst members of phylogenetically related genera, which are characterized by direct cross-linkage between positions 3 and 4 and by the presence of meso-diaminopimelic acid as diamino acid (Table 1
). Menaquinone-7 (MK-7) was the major respiratory quinone detected. The fatty acid composition of strain B22T was characterized by the predominance of branched fatty acids, namely anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0, which made up 58.9, 18.3 and 15.4 % of the total fatty acids, respectively (Table 2).
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). Furthermore, this tree topology was found using the maximum-likelihood as well as the neighbour-joining algorithms.
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On the basis of these findings, it is proposed that strain B22T represents a novel species in a new genus, Paucisalibacillus globulus gen. nov., sp. nov.
Description of Paucisalibacillus gen. nov.
Paucisalibacillus (Pau'ci.sa.li.ba.cil'lus. L. adj. paucus few, little; L. n. sal, salis salt; L. masc. n. bacillus a small staff, a wand; N.L. masc. n. Paucisalibacillus a rod that needs only small amounts of salt).
Form rod-shaped cells, which stain Gram-positive, are motile by means of two polar flagella at one end and spore-forming. Strictly aerobic, oxidase-negative and catalase-positive. NaCl is not required for growth; small amounts of NaCl improve growth. Peptidoglycan is of the A4 type with L-lys as the diamino acid and D-Asp as the dicarboxylic amino acid present in the cross-linkage. Major respiratory quinone is MK-7. Fatty acids are predominantly saturated and branched. Belongs to the family Bacillaceae. The type species is Paucisalibacillus globulus.
Description of Paucisalibacillus globulus sp. nov.
Paucisalibacillus globulus [glo'bu.lus. L. n. globulus (nominative in apposition) a little ball, a globule, because the bacterium forms colonies that are similar to a little ball, a globule].
Exhibits the following characteristics in addition to those described for the genus. Cells are 0.5 µm in width by 3.0–7.0 µm in length. Oval endospores are formed in a terminal non-swollen sporangium. Heterotrophic. Colonies are small, smooth, spherical and beige-coloured. The optimum temperature for growth is about 37 °C and no growth occurs at 15 or 45 °C; the optimum pH is between 8.0 and 8.5 and no growth occurs at pH 6.0 or 9.5. Grows in 0–8 % NaCl; optimal growth occurs in media containing 1 % (w/v) NaCl. Predominant fatty acids are anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0, which comprise over 90 % of the total fatty acids. Hydrolyses casein, starch, DNA, arbutin, aesculin, hippurate, elastin, gelatin and Tweens 20, 40, 60 and 80. Xylan and urea are not hydrolysed. DNase is detected. Does not reduce nitrate. Assimilates glucose, mannose, galactose, fructose, L-sorbose, D-xylose, sucrose, maltose, lactose, trehalose, L-rhamnose, raffinose, L-fucose, ribitol, xylitol, L-erythritol, mannitol, 2-oxoglutarate, lactate, malate, pyruvate, acetate, L-glutamate, glycine, serine and threonine. Does not utilize arabinose, ribose, melezitose, cellobiose, melibiose, sorbitol, arabitol, myo-inositol, glycerol, succinate, citrate, aspartate, alanine, asparagine, cysteine, phenylalanine, histidine, isoleucine, lysine, methionine, proline, glutamine, arginine, valine or ornithine. Acid is produced from ribose, xylose, glucose, mannose, fructose, mannitol, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, sucrose, starch, glycogen, gentiobiose, turanose, tagatose and 5-ketogluconate, but not from the other substrates in the API 50CH test strips.
The type strain is B22T (=CIP 108857T=LMG 23148T), isolated from potting soil. The DNA G+C content of strain B22T is 37.9 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
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Tiago, I., Mendes, V., Pires, C., Morais, P. V. & Veríssimo, A. (2005a). Phenylobacterium falsum sp. nov., an alphaproteobacterium isolated from a nonsaline alkaline groundwater, and emended description of the genus Phenylobacterium. Syst Appl Microbiol 28, 295–302.[CrossRef][Medline]
Tiago, I., Pires, C., Mendes, V., Morais, P. V., da Costa, M. & Veríssimo, A. (2005b). Microcella putealis gen. nov., sp. nov., a Gram-positive alkaliphilic bacterium isolated from a nonsaline alkaline groundwater. Syst Appl Microbiol 28, 479–487.[CrossRef][Medline]
Tiago, I., Mendes, V., Pires, C., Morais, P. V. & Veríssimo, A. (2006). Chimaereicella alkaliphila gen. nov., sp. nov., a Gram-negative alkaliphilic bacterium isolated from a nonsaline alkaline groundwater. Syst Appl Microbiol 29, 100–108.[CrossRef][Medline]
Tindall, B. J. (1989). Fully saturated menaquinones in the archaebacterium Pyrobaculum islandicum. FEMS Microbiol Lett 60, 251–254.[CrossRef]
Yoon, J. H., Kang, K. H. & Park, Y. H. (2002). Lentibacillus salicampi gen. nov., sp. nov., a moderately halophilic bacterium isolated from a salt field in Korea. Int J Syst Evol Microbiol 52, 2043–2048.[Abstract]
Yoon, J. H., Kang, S. J., Lee, S. Y., Lee, M. H. & Oh, T. K. (2005). Virgibacillus dokdonensis sp. nov., isolated from a Korean island, Dokdo, located at the edge of the East Sea in Korea. Int J Syst Evol Microbiol 55, 1833–1837.
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