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Laboratori de Microbiologia, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
Correspondence
Jesús Guinea
jguinea{at}farmacia.far.ub.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the16S rRNA gene sequence of Paenibacillus antarcticus 20CMT is AJ605292.
A table giving fatty acid composition (Table A) and figures showing electron micrographs and a phylogenetic tree (Figs A and B, respectively) are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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proposed the genus Paenibacillus to accommodate a group of aerobic or facultatively anaerobic rod-shaped, endospore-forming bacteria. Many Bacillus species were transferred to the genus Paenibacillus based on a comparison of their 16S rRNA gene sequences with other members of the Bacillaceae (Ash et al., 1993; Heyndrickx et al., 1995, 1996a, b; Shida et al., 1997a; Pettersson et al., 1999). Traditionally, Gram-positive, rod-shaped, endospore-forming bacteria have been classified in the genus Bacillus (Claus & Berkeley, 1986). However, in recent years, the genus Bacillus has been separated into several distinct genera, such as Alicyclobacillus (Wisotzkey et al., 1992), Aneurinibacillus and Brevibacillus (Shida et al., 1996), Halobacillus (Spring et al., 1996), Paenibacillus (Ash et al., 1993), Amphibacillus (Niimura et al., 1990), Filobacillus (Schlesner et al., 2001), Geobacillus (Nazina et al., 2001), Virgibacillus (Heyndrickx et al., 1998), Gracilibacillus and Salibacillus (Wainø et al., 1999) and Ureibacillus (Fortina et al., 2001). Antarctica is a source of novel Bacillus species (Logan et al., 2000, 2002, 2004a); novel members of the genus Paenibacillus have also been described recently in this environment (Logan et al., 2004b).
According to Ash et al. (1993), members of the genus Paenibacillus produce ellipsoidal endospores in swollen sporangia. The cell wall shows structures typical of Gram-positive bacteria, but usually stains negatively. The DNA G+C contents range from 40 to 54 mol% and anteiso-C15 : 0 is the major cellular fatty acid. Some members of the genus produce antibacterial compounds (Slepecky & Hemphill, 1991) and iturin-like antifungal antibiotics (Chung et al., 2000). One distinctive characteristic of the genus Paenibacillus is the ability to excrete a wide variety of enzymes that degrade natural biopolymers such as alginate, chondroitin, chitin, curdlan, starch (Kanzawa et al., 1995; Nakamura, 1987; Chung et al., 2000; van der Maarel et al., 2000) and other polysaccharides (Priest et al., 1988).
In this study, the taxonomic status of strain 20CMT was investigated using a combination of phenotypic characterization, sequencing of the 16S rRNA gene, DNA base composition, DNA–DNA hybridization and cellular fatty acid composition analysis. Strain 20CMT is proposed as a representative of a novel species, Paenibacillus antarcticus sp. nov.
Strain 20CMT was isolated from sediment collected in Chlorite Lake on the Byers Peninsula of Livingston Island (South Shetland Islands, Antarctica). Sample aliquots were removed with a platinum loop and diluted in a saline solution containing (g l–1, pH 7): NaCl, 0·56; KCl, 0·27; CaCl2, 0·03; and NaHCO3, 0·01. Trypticase soy agar (TSA; ADSA) plates were inoculated with loopfuls of several sample dilutions using the streak-plate method to obtain well-isolated colonies. Plates were incubated for 4 days at 15 °C. Isolates were maintained aerobically on TSA slopes at 4 °C and also at –80 °C on cryo-beads.
Morphology, cell size and shape of spores were determined by scanning (Hitachi model H 2300) and transmission (Hitachi model H 600AB) electron microscope observations of cells grown in trypticase soy broth (TSB; ADSA) at 15 °C. Motility was determined by phase-contrast microscopy (Olympus model CHS). Gram staining was performed according to Hucker & Conn (1923). Two alternative methods, the KOH test and the L-alanine aminopeptidase assay (Manafi & Kneifel, 1990), were also used. Oxidase, catalase and urease activities, methyl red reaction, Voges–Proskauer, nitrate reduction, indole production, citrate utilization, and hydrolyses of casein, lecithin, gelatin, DNA, tyrosine, starch and Tween 80 were determined following Cowan & Steel (1993). Dihydroxyacetone production, phenylalanine deamination and growth in the presence of lysozyme (0·1 and 0·001 %, w/v) were determined as described by Claus & Berkeley (1986). Acid production from carbohydrates and additional tests were determined using the API 50CH and API 20E system (bioMérieux). Tolerance to NaCl was measured on nutrient agar (ADSA) containing 0–10 % (w/v) NaCl. Plates were incubated at 15 °C for 30 days. The temperature range for growth was determined on TSA incubated for 14 days at 4, 10, 15, 20, 25, 30, 31, 32, 33 and 37 °C. Anaerobic growth was determined on TSB plus agar-agar (1·5 %; ADSA) by incubation in an anaerobic chamber at 15 °C for 5 days.
Cells were Gram-variable, rod-shaped (0·7x2·5 µm) and motile by means of peritrichous flagella (see Fig. Aa, b; available as supplementary material in IJSEM Online). Strain 20CMT produced ellipsoidal spores in swollen sporangia in the subterminal or terminal region of the cell (see Fig. Ac, d, e; available as supplementary material in IJSEM Online). Colonies grown on TSA at 15 °C were non-pigmented, circular, slightly convex, bright and cream coloured with a diameter of 1·0–1·5 mm. Cell wall structure was Gram-positive, as demonstrated by electron microscope examinations of ultra-thin sections (see Fig. Af, available as supplementary material in IJSEM Online), although results of the KOH test and the L-alanine aminopeptidase assay indicated a Gram-negative character. The isolate was facultatively anaerobic and grew at 4–31 °C. Growth was optimal at 10–15 °C. It grew in the presence of 4 % (w/v) NaCl and in 0·001 % (w/v) lysozyme, but not in 0·1 % (w/v) lysozyme. The final pH in Voges–Proskauer broth after 7 days incubation at 15 °C was less than pH 6. The isolate was negative for production of acetylmethylcarbinol, but positive for the methyl red reaction. Strain 20CMT did not decompose tyrosine. Phenotypic characteristics of the Antarctic isolate and the closest phylogenetic relatives were compared (Table 1). Of the organisms compared, only strain 20CMT was positive for oxidase production. Phenotypic studies showed that the Antarctic isolate displayed characteristics consistent with those of the genus Paenibacillus (Ash et al., 1993).
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. Fatty acid analysis of 20CMT (Table A, available as supplementary material in IJSEM Online) revealed that anteiso-C15 : 0 (55·32 %), iso-C15 : 0 (15·04 %) and C16 : 1
11c (7·72 %) were predominant. This fatty acid profile was in accordance with that given in the description of the genus Paenibacillus (Ash et al., 1993). anteiso-Branched saturated C15 : 0 is the predominant fatty acid found in all members of the genus Paenibacillus (Shida et al., 1997a).
Genomic DNA was prepared according to the method of Gevers et al. (2001). The G+C content was determined by HPLC as described by Mesbah et al. (1989). DNA–DNA relatedness was measured fluorometrically using the microplate hybridization method described by Ezaki et al. (1989). Determination of the 16S rRNA gene sequence of strain 20CMT and phylogenetic analyses were carried out as described previously by Bozal et al. (2002).
Phylogenetic studies based on 16S rRNA gene sequences confirmed that strain 20CMT is a member of the genus Paenibacillus (Fig. 1; Fig. B, available as supplementary material in IJSEM Online). The 16S rRNA gene sequence of strain 20CMT showed 99·5 % similarity to that of Paenibacillus macquariensis DSM 2T, which is significant enough to suggest possible species relatedness. Stackebrandt & Goebel (1994) suggested that a sequence similarity value greater than 97 % indicated conspecificity of the strains involved. The similarity values shown by 20CMT to other Paenibacillus type strains were under 97 % (Paenibacillus borealis DSM 13188T, 95·5 %; Paenibacillus odorifer LMG 19079T, 94·7 %). To further verify the taxonomic position of isolate 20CMT, DNA–DNA hybridizations were performed with P. macquariensis LMG 6935T (Marshall & Ohye, 1966). The low DNA–DNA reassociation value of 47 % between these two strains and the 16S rRNA gene sequence analysis confirmed the distinct position of strain 20CMT within the genus Paenibacillus. The G+C content of strain 20CMT was 40·7 mol%, which lies within the range observed for members of the genus Paenibacillus (Shida et al., 1997a).
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). Based on polyphasic evidence, it is proposed that strain 20CMT be assigned as the type strain of a novel species in the genus Paenibacillus, Paenibacillus antarcticus sp. nov.
Description of Paenibacillus antarcticus
Paenibacillus antarcticus (ant.arc'ti.cus. L. masc. adj. antarcticus of the Antarctic environment, where the organism was isolated).
Cells are rod-shaped (0·7x2·5 µm) and motile by means of peritrichous flagella. Subterminal or terminal ellipsoidal spores are formed in swollen sporangia. Colonies grown on TSA are non-pigmented, circular, slightly convex, bright and cream coloured. Cells are facultatively anaerobic and stain Gram-variable. Growth is not inhibited by the presence of 4 % NaCl or 0·001 % lysozyme. Growth occurs at 4 and 31 °C, but not at 0 or 32 °C; optimal growth occurs at 10–15 °C. Oxidase, catalase, urease and methyl red reactions are positive. Nitrate reduction, Voges–Proskauer reaction, 
-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilization, dihydroxyacetone production, indole production, H2S production, phenylalanine deamination and tryptophan deaminase are negative. Aesculin, starch and Tween 80 are hydrolysed. Does not hydrolyse casein, lecithin, gelatin, DNA or tyrosine. With API systems, acid is produced from L-arabinose, ribose, D-xylose, methyl -D-xyloside, galactose, D-glucose, D-fructose, D-mannose, methyl 
-D-glucoside, N-acetylglucosamine, amygdalin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, D-raffinose, starch, -gentiobiose and D-turanose. Acid is not produced from glycerol, erythritol, D-arabinose, L-xylose, adonitol, L-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, methyl -D-mannoside, arbutin, inulin, melezitose, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. The predominant fatty acid is anteiso-C15 : 0 (55·32 %).
The type strain is 20CMT (=LMG 22078T=CECT 5836T); its G+C content is 40·7 mol%.
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ACKNOWLEDGEMENTS |
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