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1 Microbial Type Culture Collection and Gene Bank (MTCC), Sector 39A, Chandigarh 160 036, India
2 Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India
Correspondence
T. Chakrabarti
tapan{at}imtech.res.in
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11c (6·16 %), C15 : 0 iso (6·03 %), C17 : 0 anteiso (5·68 %) and C16 : 17c alcohol (5·01 %). The presence of the fatty acid C16 : 17c alcohol distinguishes this strain from other closely related species of the genus Paenibacillus. The strain contains MK-7 as the diagnostic menaquinone. The G+C content of the genomic DNA is 41·2 mol%. Analysis of the 16S rRNA gene sequence (1466 nt) revealed the presence of signature sequences PAEN 515F (5'-GAGTAACTGCTCTCGGAATGACGGTACTTGAGAAGAAAGCCCC-3') and PAEN 862F (5'-TCGATACCCTTGGTGCCGAAGT-3'), which were found in the species of the genus Paenibacillus surveyed by Shida et al. [Shida, O., Takagi, H., Kadowaki, K., Nakamura, L. K. & Komagata, K. (1997)
. Int J Syst Bacteriol 47, 289–298]. The sequence shows closest similarity (95·85 %) to that of Paenibacillus apiarius, followed by Paenibacillus alvei (94·34 %), Paenibacillus cineris (93·87 %), Paenibacillus favisporus (93·80 %), Paenibacillus chibensis (93·47 %) and Paenibacillus azoreducens (93·40 %). Biochemical, physiological, chemotaxonomic and phylogenetic analyses justify placement of the strain in the genus Paenibacillus but not within any existing species. It should, therefore, be considered as representing a novel species, for which the name Paenibacillus assamensis sp. nov. is proposed. The type strain is GPTSA 11T (=MTCC 6934T=JCM 13186T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Paenibacillus assamensis GPTSA 11T is AY884046.
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). On the basis of 16S rRNA-based molecular analysis it became apparent that the genus is very heterogeneous, consisting of different phylogenetic groups (Ash et al., 1991). Later, Ash et al. (1993) moved the members of Bacillus group 3 to the genus Paenibacillus. Thereafter, its description was emended by Shida et al. (1997). At present, more than 60 species (http://www.bacterio.cict.fr/p/paenibacillus.html) are recognized as members of the genus Paenibacillus. Members of this genus produce ellipsoidal endospores in swollen sporangia, possess C15 : 0 anteiso as the major cellular fatty acid and have genomic DNA G+C contents in the range 39–54 mol% (Shida et al., 1997; Montes et al., 2004; Takeda et al., 2005). Species belonging to the genus Paenibacillus have been isolated from various ecological niches such as soil, rhizospheres, water, diseased insect larvae, food (Daane et al., 2002), cow faeces (Velázquez et al., 2004), blood cultures (Roux & Raoult, 2004), Antarctic sediment (Montes et al., 2004) and recently from the phyllosphere of Phoenix dactylifera (Rivas et al., 2005). Examination of such habitats has resulted in the discovery of 23 novel species in the last 3 years (2002–2004).
In this paper, we report the taxonomic characterization of a novel aerobic, mesophilic, Gram-variable, endospore-forming bacterial strain, GPTSA 11T, which produces spreading colonies. Strain GPTSA 11T was isolated from a warm-spring water sample by dilution-plating on tryptic soy broth agar (TSBA; tryptic soy broth, 3 %; agar, 1·5 %). The warm spring is located in a reserve forest inhabited by wild elephants and other animals and is rarely disturbed by human activities. As elephant dung was found in and around the spring, it can be assumed that this is a water hole used by animals living in the forest. The temperature of the spring was 38 °C and the pH was 7·2. The colony morphology, cell morphology, motility and Gram reaction of the strain were determined by using standard methods (Smibert & Krieg, 1994; Murray et al., 1994; Powers, 1995). Physiological characteristics were examined by growing the isolate on basal TSBA medium at different temperatures, pH values and NaCl concentrations. After 36 h growth at 30 °C on TSBA, colonies were round and yellowish white in colour. Within 48 h, the entire plate was occupied by single colonies apparently produced as a result of ‘jumping’ from the primary inoculum. The isolate was able to grow at temperatures between 20 and 37 °C, at pH 6·8–12·0 and was able to tolerate NaCl concentrations of up to 2·5 %. Various biochemical tests were done at 30 °C using standard methods (Claus & Berkeley, 1986; Smibert & Krieg, 1994). Degradation of chitin and xylan was checked on nutrient agar (HiMedia) medium supplemented with 0·4 % (w/v) each compound. Acid production from various carbohydrates were checked on basal medium (beef extract, 0·3 %; tryptose, 1 %; NaCl, 0·5 %; phenol red as acid–base indicator) supplemented with 0·5 % carbohydrate. The biochemical and physiological characteristics of the strain are given in the species description.
For cellular fatty acid analysis, the strain was grown on TSBA medium at 30 °C for 24 h. Extraction and analysis of cellular fatty acids were done using the Sherlock Microbial Identification System (MIDI), as described previously (Pandey et al., 2002). The major fatty acid was found to be C15 : 0 anteiso, followed by C16 : 0 iso, C16 : 111c, C15 : 0 iso, C17 : 0 anteiso and C16 : 17c alcohol (Table 1). The diagnostic cell-wall amino acid was found to be meso-diaminopimelic acid, as determined by TLC (Staneck & Roberts, 1974). Menaquinones were extracted from 200 mg dry cell mass with a 10 % aqueous solution of 0·3 % (w/v) NaCl in methanol and petroleum ether (60–80 °C boiling point) at a ratio of 1 : 1. The upper phase was collected and dried in a Turbo Vap LV evaporator (Zymark). The residue was dissolved in 100 µl acetone. The extract was developed on a TLC plate (20x20 cm Silica gel 60 F254; Merck) using petroleum ether (boiling point 60–80 °C) and diethyl ether (85 : 15, v/v). Purified menaquinones were dissolved in 2-propanol and analysed by reverse-phase TLC according to Collins & Jones (1980). The diagnostic menaquinone of strain GPTSA 11T was found to be MK-7. The genomic DNA of GPTSA 11T was isolated by using Marmur's protocol (Johnson, 1994). The G+C content of the genomic DNA was determined spectrophotometrically (Lambda 35 spectrophotometer; Perkin Elmer), using the thermal denaturation method (Mandel & Marmur, 1968), and was found to be 41·2 mol%. A fragment comprising the full-length 16S rRNA gene from strain GPTSA 11T was amplified by a PCR using primers 8-27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1500r (5'-AGAAAGGAGGTGATCCAGGC-3') (Escherichia coli numbering). The amplification reaction and the purification of amplicons were performed as described previously (Pandey et al., 2002). The purified PCR product was sequenced by using the dideoxy chain terminator method with the Big Dye Terminator kit followed by capillary electrophoresis on an ABI 310 Genetic Analyzer (Applied Biosystems). An almost-complete (1466 nt continuous stretch) 16S rRNA gene sequence of the strain was used as a query to search for homologous sequences in the GenBank database and in the Ribosomal Database Project library (Project II, release 9; http://rdp.cme.msu.edu/index.jsp). Analysis of the sequence indicated that GPTSA 11T contains the consensus signature sequence stretches PAEN 515F (5'-GAGTAACTGCTCTCGGAATGACGGTACTTGAGAAGAAAGCCCC-3') (Shida et al., 1997) and PAEN 862F (5'-TCGATACCCTTGGTGCCGAAGT-3') (Pettersson et al., 1999), which are mostly found among different species within the genus Paenibacillus. Strain GPTSA 11T showed the highest degree of sequence similarity with Paenibacillus apiarius DSM 5581T (95·85 %), followed by Paenibacillus alvei IAM 1258T (94·34 %), Paenibacillus cineris LMG 18439T (93·87 %), Paenibacillus favisporus GMP 01T (93·80 %), Paenibacillus chibensis JCM 11200T (93·47 %) and Paenibacillus azoreducens CM1T (93·40 %). Sequences from type strains of these six and 13 other species of Paenibacillus showing more than 92·50 % similarity were used for phylogenetic analysis. These sequences were aligned with that of strain GPTSA 11T by the CLUSTAL X program (Thompson et al., 1997) and edited manually. Aligned sequences were analysed by the PHYLIP software package, version 3.5c (Felsenstein, 1993). Computation of pairwise evolutionary distances for the aligned sequences was done using the DNADIST program with the Kimura two-parameter model (Kimura, 1980). To obtain the confidence value for the aligned sequence dataset, bootstrap analysis of 100 replications were done using SEQBOOT. Phylogenetic trees showing the relationships between GPTSA 11T and other reference strains were constructed using the neighbour-joining method (Saitou & Nei, 1987) and the unweighted pair group arithmetic averages linkage algorithm. Distance-matrix data obtained from DNADIST were also used to construct a phylogenetic tree by using KITSCH. Consensus trees for each of these methods were generated using CONSENSE from the PHYLIP package. Irrespective of the different tree-generating methods used, strain GPTSA 11T always occurred as a separate clade within the Paenibacillus thiaminolyticus–Paenibacillus popilliae–P. alvei–P. apiarius cluster (Fig. 1). The 16S rRNA gene sequence similarity of these strains with respect to GPTSA 11T lies between 92·98 and 95·85 %. The overall genomic relatedness of GPTSA 11T with respect to these species may not be high because, as Stackebrandt & Goebel (1994) observed, strains with less than 97 % 16S rRNA gene sequence similarity have DNA–DNA relatedness values below 70 %. The latter percentage of genomic relatedness is considered as the ‘gold standard’ for bacterial species definition (Wayne et al., 1987). Thus, in the absence of a close relative with significant 16S rRNA gene sequence similarity, strain GPTSA 11T can be considered as a novel species. The presence of ellipsoidal endospores, C15 : 0 anteiso as a major fatty acid, meso-diaminopimelic acid as a cell-wall amino acid, MK-7 as the diagnostic menaquinone and PAEN 515F and PAEN 862F signature sequences in the 16S rRNA gene suggest that the strain belongs to the genus Paenibacillus. However, it differs from closely related species in terms of several phenotypic characteristics and the DNA G+C content (Table 2). In addition, unlike P. cineris, strain GPTSA 11T cannot produce acid from L-arabinose, myo-inositol, D-melezitose, arbutin, D-fructose, galactose or lactose. Close phylogenetic relatives also differ from GPTSA 11T in terms of cellular fatty acid composition, both qualitatively and quantitatively (Table 1). On the basis of the phenotypic, chemotaxonomic and phylogenetic data, it is evident that GPTSA 11T represents a novel species within the genus Paenibacillus, for which we propose the name Paenibacillus assamensis sp. nov.
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Gram-variable, strictly aerobic, mesophilic, sporulating, motile rods occurring as single cells or in pairs. Cells measure 1·0–2·5 µm in length and 0·5–0·6 µm in width. Colonies are round, convex with undulated margins and light yellowish-white in colour, spreading as single colonies over the entire plate. Endospores are ellipsoidal and subterminal, occurring in bulging sporangia. The strain can grow at temperatures between 20 and 37 °C, at pH values in the range 6·8–12·0 and can tolerate NaCl at concentrations of up to 2·5 %. It is oxidase-, catalase-, gelatinase- and arginine dihydrolase-positive but negative for urease, DNase, phenylalanine deaminase, lysine and ornithine decarboxylase activities. Negative for indole and H2S production, nitrate reduction, in the Voges–Proskauer test and for gas production from glucose but gives a positive result in the methyl red test. Cannot utilize acetate, citrate or propionate. Hydrolyses starch, aesculin and casein but not tyrosine, ONPG or Tweens 20, 40 and 80. Produces acid from D-glucose, glycerol, gentiobiose, glycogen, D-maltose, D-mannose, D-ribose, produces acid weakly from D-amygdalin, cellobiose, sucrose and trehalose, but not from adonitol, L-arabinose, D-arabinose, L-arabitol, arbutin, D-fructose, D-galactose, myo-inositol, inulin, D-lactose, D-mannitol, D-melibiose, D-melezitose, D-raffinose, L-rhamnose, salicin, sorbitol, L-sorbose, xylitol or D-xylose. Can grow in the presence of 0·001 % lysozyme but not 0·01 % lysozyme. Cannot degrade chitin or xylan. The major fatty acids are C15 : 0 anteiso, followed by C16 : 0 iso, C16 : 111c, C15 : 0 iso, C17 : 0 anteiso and C16 : 17c alcohol. The cell-wall amino acid is meso-diaminopimelic acid. The diagnostic menaquinone is MK-7. The DNA G+C content is 41·2 mol%.
The type strain is GPTSA 11T (=MTCC 6934T=JCM 13186T), isolated from a warm-spring water sample from Assam, India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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