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1 Microbiology Laboratory, Department of Biotechnology, North Bengal University, Siliguri-734430, West Bengal, India
2 Department of Microbiology, Bose Institute, CIT-Scheme VII-A, Kolkata-56, India
Correspondence
Ranadhir Chakraborty
rcnbu2003{at}yahoo.com
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ABSTRACT |
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-Proteobacteria and to be related to Serratia marcescens subsp. sakuensis (98·35 %) and S. marcescens subsp. marcescens (98·30 %); however, strain NiVa 51T exhibited only 43·7 % similarity to S. marcescens by DNA–DNA hybridization. The G+C content of the genomic DNA of the isolate was 60 mol%. Both biochemical characteristics and fatty acid analysis data supported the affiliation of strain NiVa 51T to the genus Serratia. Furthermore, strain NiVa 51T was found to utilize urea as nitrogen source. The results of DNA–DNA hybridization as well as physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain NiVa 51T from recognized Serratia species. Strain NiVa 51T therefore represents a novel species, for which the name Serratia ureilytica sp. nov. is proposed, with type strain NiVa 51T (=LMG 22860T=CCUG 50595T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NiVa 51T is AJ854062.
Figures showing the growth curve of strain NiVA 51T and phylogenetic trees constructed by using maximum-parsimony, KITSCH and FITCH algorithms, and tables detailing the cellular fatty acid composition of strain NiVA 51T and related taxa, and similarity coefficients of members of the family Enterobacteriaceae are available as supplementary material in IJSEM Online.
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subclass of the Proteobacteria. Some members of this genus produce pigments identified as prodigiosin (Hearn et al., 1970
; Gerber, 1975). A spore-forming subspecies of Serratia marcescens (S. marcescens subsp. sakuensis) was recently described by Ajithkumar et al. (2003). We have isolated a novel non-pigmented, non-spore-forming strain (isolate NiVa 51T) from water of the River Torsa, West Bengal, India. The Torsa is an international river traversing three countries, China (Tibet), Bhutan and India, before entering Bangladesh. The strain was isolated in February 2001, when the river water had a high free-ammonia nitrogen content (61·2 p.p.m.) owing to sewage contamination (Bhadra et al., 2003). Strain NiVa 51T was able to hydrolyse urea to ammonia and to utilize urea as a sole nitrogen source for its growth.
During characterization of nickel-resistant copiotrophic bacteria isolated from water from the River Torsa, strain NiVa 51T was recovered on nutrient agar (Himedia) containing 3 mM nickel chloride after incubation at 35 °C for 24 h. Subcultivation was done on nutrient agar at 35 °C for 16–18 h. Strain NiVa 51T was able to grow on nutrient agar at 10–42 °C, but not at 4 or 45 °C. Growth was also observed at 28 °C on trypticase soy agar (Difco) after incubation for 16–18 h. Basal medium [(g l–1): MgSO4.7H2O, 0·5; KH2PO4, 0·5; KCl, 0·1; pH 6·5±0·2] containing 50 mM glucose and 0·5 mM ammonium sulfate or urea did not support the growth of NiVa 51T until supplemented with 0·01 % yeast extract. The same basal medium with similar supplementation was used to determine growth curves. In all growth experiments, a 1 ml aliquot of exponentially grown cells in nutrient broth was centrifuged, washed in sterile 0·86 M NaCl, resuspended in 250 µl sterile basal medium and inoculated in 20 ml medium contained in 250 ml Erlenmeyer flasks. Cells inoculated in the same medium, but devoid of urea or ammonium sulfate, served as a negative control. Cultures were grown at 35 °C with shaking. A UV-Vis spectrophotometer (Shimadzu) and a digital pH meter (Systronics) were used to determine OD560 and the pH of the culture medium, respectively, at regular intervals. Generation times, calculated from the growth curves (see Supplementary Fig. S1 in IJSEM Online) obtained from the culture media containing urea and ammonium sulfate as a sole nitrogen source, were 120±2 and 90±0·2 min, respectively. For estimation of free-ammonia ‘nitrogen’, generated in the medium containing urea, a 10 ml aliquot of growing culture (withdrawn at regular intervals) was filtered through a bacterial-filter membrane (diameter 0·45 µm) and then diluted with ammonia-free distilled water. Quantification of free ammonia was done by following standard techniques (APHA, 1985; Manivasakam, 1980). One of the two control sets contained all the chemical ingredients, including urea, without bacterial cells and the other contained heat-killed cells (100 °C for 15 min). In the active culture medium containing 0·5 mM urea as sole nitrogen source and at an initial cell density of 3x108 cells ml–1, the rate of free-ammonia ‘nitrogen’ released into the culture medium in the first 2 h incubation was 0·6 p.p.m. h–1. This reached a maximum rate of 1·4 p.p.m. h–1 after 6–10 h incubation.
Gram staining was performed following the method of Gerhardt et al. (1994). Cell morphology was observed under a phase-contrast microscope (Olympus, model M021) at x1000, with cells grown for 16 h at 35 °C on nutrient agar. The 16S rRNA gene was amplified, cloned and sequenced as described by Carr et al. (2003). The sequence obtained was compared with those in GenBank by using the BLAST N program (Altschul et al., 1997). After multiple alignments of the data using the CLUSTAL X program (Thompson et al., 1997), phylogenetic analysis was performed by using the software package PHYLIP 3.6c (Felsenstein, 2002) according to three different methods [neighbour joining (Saitou & Nei, 1987), maximum parsimony (Felsenstein, 1983) and maximum likelihood (Yang, 1997)]. For the purpose of neighbour-joining, KITSCH and FITCH analyses, distances were calculated by using both two-parameter (Kimura, 1980) and one-parameter (Jukes & Cantor, 1969) models in DNADIST. The 16S rRNA gene sequence of Plesiomonas shigelloides (the most closely related species in the family Enterobacteriaceae; Brenner, 1992) was used to root all trees. To determine the confidence values for individual branches, 100 bootstrap replications were done for each generated tree by using SEQBOOT and CONSENSE from the PHYLIP package. Phylogenetic trees obtained by the maximum-likelihood method showed that strain NiVa 51T branched deeply with the Serratia cluster consisting of S. marcescens, Serratia rubidaea and Serratia odorifera (Fig. 1). Similar patterns of branching were noted in trees derived from maximum-parsimony, KITSCH and FITCH analysis (see Supplementary Fig. S2 in IJSEM Online). Sequence similarity, based on pairwise sequence comparisons, was investigated by using complete 16S rRNA gene sequences. The 16S rRNA gene sequence of strain NiVa 51T showed 96–97 % similarity to many species of the genus Serratia, and 98·30–98·35 % similarity to S. marcescens subsp. sakuensis and S. marcescens subsp. marcescens.
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. The fatty acid profile of strain NiVa 51T consisted of 12 : 0 (1·4 %), 12 : 0 2-OH (0·28 %), an unknown fatty acid with equivalent length of 13·957 (0·97 %), 14 : 0 (6·28 %), an unknown fatty acid with equivalent length of 14·502 (0·85 %), 14 : 0 2-OH (2·21 %), 15 : 0 (0·29 %), 16 : 0 (31·01 %), 17 : 0 cyclo (2·69 %), 17 : 0 (0·35 %), 18 : 1
7c (17·89 %), 18 : 0 (0·46 %), 19 : 0 cyclo 8c (0·25 %), summed feature 2 (comprising 12 : 0 alde and 14 : 0 3-OH/16 : 1 iso I) (8·01 %) and summed feature 3 (comprising 16 : 17c/15 iso 2-OH) (27·07 %). The fatty acid profile of strain NiVa 51T was compared with those of other members of the same Serratia cluster (S. marcescens, S. rubidaea and S. odorifera) (see Supplementary Table S1 in IJSEM Online), and was found to have a maximum similarity index of 0·747 with S. marcescens GC subgroup B.
For determination of the G+C content, DNA was hydrolysed with P1 nuclease, de-phosphorylated with bovine alkaline phosphatase and analysed via HPLC following techniques described by Mesbah et al. (1989). The DNA G+C content of strain NiVa51T was 60 mol%. This value is higher than that reported for S. marcescens subsp. sakuensis (58 mol%; Ajithkumar et al., 2003), but close to that for S. marcescens (57·5–60 %; Grimont & Grimont, 1992).
Results of the physiological characterization are given in the species description below and in Table 1, and were determined with the methods described by Holt et al. (1994) and Ajithkumar et al. (2003). Strain NiVa 51T yielded a maximum similarity coefficient (SSM; 0·87) to S. marcescens (see Supplementary Table S2 in IJSEM Online).
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From the phenotypic, chemotaxonomic and phylogenetic results presented here, we can conclude that strain NiVa 51T forms a separate group within the genus Serratia. The novel species can be differentiated from S. marcescens subsp. marcescens based on phenotypic characteristics, such as pigment production, methyl red test, caseinase, urease, arginine dihydrolase, and growth in the presence of adonitol, xylose, oxalate, DL-threonine and L-tryptophan as sole carbon source. Proportions of the fatty acids 17 : 0 cyclo and 19 : 0 cyclo 8c in NiVa 51T were approximately four to five times lower than in S. marcescens. 16S rRNA gene sequence similarity between S. marcescens subsp. marcescens and strain NiVa 51T was 98·3 % and DNA–DNA relatedness was 43·7 %. The unique urea-utilizing characteristic of strain NiVa 51T therefore supports its classification as a novel species of Serratia, for which the name Serratia ureilytica sp. nov. is proposed.
Description of Serratia ureilytica sp. nov.
Serratia ureilytica [u.re.i.ly'ti.ca. N.L. n. urea -ae urea; N.L. adj. lyticus -a -um (from Gr. adj. lutikos) dissolving; N.L. fem. adj. ureilytica urea-dissolving].
Cells are facultatively aerobic, Gram-negative, motile, non-fluorescent, non-pigmented, straight rods, 0·7–1·0 µm long and 0·8–1·0 µm wide. Grows between 8 and 43 °C over a pH range of 5–11 in nutrient broth media, and can tolerate up to 1·4 M NaCl. Positive for arginine dihydrolase. DNA G+C content is 60 mol% and major cellular fatty acids are 16 : 0 (31·01 %), summed feature 3 (comprising 16 : 17c/15 iso 2-OH) (27·07 %) and 18 : 17c (17·89 %). Can use urea as a sole nitrogen source for growth. Other biochemical characteristics are given in Table 1
. The isolate has a plasmid of 50 kb of unknown function and can grow in nutrient agar media containing 40 µg ampicillin ml–1, 15 µg tetracycline ml–1 and 20 µg chloramphenicol ml–1.
The type strain, NiVa 51T (=LMG 22860T=CCUG 50595T), was isolated from the River Torsa, northern West Bengal, India.
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ACKNOWLEDGEMENTS |
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