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1 Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, Hyderabad 500 085, India
2 Department of Plant Sciences, School of Life Sciences, University of Hyderabad, PO Central University, Hyderabad 500 046, India
3 Leibniz-Institut für Meereswissenschaften IFM-GEOMAR, Marine Mikrobiologie, Düsternbrooker Weg 20, 24105 Kiel, Germany
Correspondence
Ch. Sasikala
r449{at}sify.com
or
sasi449{at}yahoo.ie
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains JA132T and JA130T are AM282559 and AM282561, respectively.
Supplementary figures are available with the online version of this paper.
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). Members of the family Chromatiaceae isolated from such habitats include: Lamprobacter, Halochromatium, Thiohalocapsa, Marichromatium, Rhabdochromatium, Isochromatium, Thiorhodovibrio, Thiococcus, Thiorhodococcus, Thioflavicoccus, Thioalkalicoccus and Thiorhodospira species (Imhoff, 2001). Currently, the genus Thiorhodococcus comprises two species with validly published names Thiorhodococcus minor (Imhoff et al., 1998) (originally described as Thiorhodococcus minus; Guyoneaud et al., 1997) and Thiorhodococcus mannitoliphagus (Rabold et al., 2006) [‘Thiorhodococcus drewsii’ (Zaar et al., 2003) is also a well characterized species but not validated so far] isolated from anoxic sediment of a fishpond (man-made coastal lagoon), a microbial mat of salt marsh and a microbial mat of an estuary, respectively. In this report, we propose two novel species, Thiorhodococcus bheemlicus sp. nov., isolated from anoxic sediment from an aquaculture pond and Thiorhodococcus kakinadensis sp. nov., isolated from marine tidal waters. Based on phenotypic and phylogenetic analyses these bacteria have properties distinct from all other species of Thiorhodococcus.
The sample that yielded strain JA132T was collected on 24 February 2005 from a marine aquaculture pond, Bheemli, Visakhapatnam, India (1 ° 54' N, 8 ° 27' E). The sample that yielded strain JA130T was collected on 25 April 2005 from a beach of Kakinada, near a fishing harbour, Bay of Bengal, eastern coast of India (1 ° 54' N, 8 ° 14' E). Both samples had a pH of 6.8, a salinity of 2–3 % NaCl and a temperature of 30 °C. Both strains were isolated from photolithoautotrophic enrichments of these samples. Purification and polyphasic taxonomic studies were carried out as described previously (Anil Kumar et al., 2007). Cell material for 16S rRNA gene sequencing was taken from 1 to 2 ml of well-grown liquid culture. DNA was extracted and purified by using the Qiagen genomic DNA extraction kit. PCR amplification and 16S rRNA gene sequencing were performed as described previously (Imhoff et al., 1998). Recombinant Taq polymerase was used for PCR with the primers 5'-GTTTGATCCTGGCTCAG-3' and 5'-TACCTTGTTACGACTTCA-3' (positions 11–27 and 1489–1506, respectively, according to the Escherichia coli 16S rRNA numbering of the International Union of Biochemistry). Sequences were obtained by cycle sequencing with the SequiTherm sequencing kit (Biozym) and the chain termination reaction (Sanger et al., 1977) using an automated laser fluorescence sequencer (Pharmacia). Sequences were aligned using the CLUSTAL X program (Thompson et al., 1997) and the alignment was corrected manually. The CLUSTAL X alignment file was used as the input file into the SEQBOOT program in the PHYLIP package (Felsenstein, 1989) and the output file of SEQBOOT was used as the input file for maximum-likelihood analysis with 100 datasets and five times jumbling. One single tree was produced using 100 trees generated during maximum-likelihood analysis using the CONSENSE program. A final dendrogram with evolutionary distances was constructed by taking the alignment.phy file as the infile (input file) and the consensus tree as the intree (input tree) in the maximum-likelihood program within the PHYLIP package (Felsenstein, 1989).
Cells of strains JA132T and JA130T were cocci, with a 3.0–6.0 µm diameter. Cells of strain JA132T are mostly diplococci [Supplementary Fig. S1(a) available in IJSEM Online], while strain JA130T possesses cells that are arranged as chains or as tetrads [Supplementary Fig S1(b)]. Both strains multiply by binary fission and are motile with a single polar flagellum [Supplementary Fig. S2(a) and (b)]. Electron micrograph of ultrathin sections of both bacteria revealed a vesicular type of internal membrane structures [Supplementary Fig. S3(a) and (b)]. The substrates that were utilized by both the strains are shown in Table 1. Ammonium chloride, urea and dinitrogen were utilized as nitrogen sources for growth by strain JA132T, while nitrate, nitrite, glutamate and glutamine did not support growth. Strain JA130T could utilize ammonium chloride and glutamine as nitrogen sources for growth but was unable to utilize nitrate, nitrite, glutamate and dinitrogen. The whole cell absorption spectrum of strain JA132T showed absorption maxima at 371, 460, 491, 530, 590, 803 and 857 nm, whereas strain JA130T had absorption maxima at 374, 390, 460, 494, 527, 590, 803 and 857 nm, confirming the presence of bacteriochlorophyll a, and most probably carotenoids of the spirilloxanthin series [Supplementary Fig. S4(a) and (b)].
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). Apart from 16S rRNA gene sequence dissimilarity, strains JA132T and JA130T also show phenotypic differences to other Thiorhodococcus species (Table 1
) that justify the description of these strains as novel species Thiorhodococcus bheemlicus sp. nov. and Thiorhodococcus kakinadensis sp. nov., respectively.
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Cells are coccoid in shape and mostly diplococci. Cells are 4.0–6.0 µm in diameter, motile with a single flagellum and divide by binary fission. Growth occurs under anaerobic conditions in the light (photolithoautotrophy/photolithoheterotrophy). Internal photosynthetic membranes are of vesicular type. The colour of phototrophic culture is purple–violet. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 371, 460, 491, 530, 590, 803 and 857 nm, confirming the presence of bacteriochlorophyll a and most probably carotenoids of the spirilloxanthin series. The type strain is mesophilic (30 °C), with a pH optimum at 7.0–7.2 and has an obligate requirement of NaCl for growth (1–3 %, w/v). Photolithoheterotrophy with organic compounds is the preferred mode of growth. Good carbon sources for growth are acetate, pyruvate, fumarate, glucose and Casamino acids. Growth on lactate, glycerol, glycolate and valerate is also possible. Chemotrophic (aerobic) growth could not be demonstrated. Fermentative growth is not possible in the presence of pyruvate as a fermentable carbon source. No growth factors are required. The DNA G+C (by HPLC) content is 65.5 mol%. Natural habitats are anoxic sediments of marine aquaculture pond waters.
The type strain, JA132T (=MTCC 8120T=ATCC BAA-1362T=JCM 14149T=DSM 18805T), was isolated from a marine aquaculture pond, Bheemli, Visakhapatnam, India.
Description of Thiorhodococcus kakinadensis sp. nov.
Thiorhodococcus kakinadensis (ka'ki.na.den'sis. N.L. masc. adj. kakinadensis named after Kakinada, the place from which the type strain was isolated).
Cells are coccoid and are arranged as chains or tetrads. Cells are of 3.0–5.0 µm diameter, motile with single flagellum and divide by binary fission. Growth occurs under anaerobic conditions in the light (photolithoautotrophy/photolithoheterotrophy). Internal photosynthetic membranes are of vesicular type. The colour of phototrophic culture is purple–violet. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 374, 390, 460, 494, 527, 590, 803 and 857 nm, confirming the presence of bacteriochlorophyll a and most probably carotenoids of the spirilloxanthin series. The type strain is mesophilic (30 °C), with a pH optimum at 7.2 and requires NaCl for growth (optimum 1–2 %, w/v). Photolithoheterotrophy with organic compounds is the preferred mode of growth. Good carbon sources for growth are pyruvate, propionate, fumarate, succinate and malate. Growth on acetate and lactate also occurs. Chemotrophic (aerobic) growth could not be demonstrated. Fermentative growth is not possible in the presence of pyruvate as a fermentable carbon source. Niacin, biotin and pantothenate are required as growth factors. The DNA G+C (by HPLC) content is 57.5 mol%. Natural habitats are marine tidal waters.
The type strain, JA130T (=ATCC BAA-1353T=DSM 18858T=JCM 14150T), was isolated from a beach of Kakinada, near a fishing harbour, Bay of Bengal, eastern coast of India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Felsenstein, J. (1989). PHYLIP (Phylogenetic inference package) version 3.5.1. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Guyoneaud, R., Matheron, R., Liesack, W., Imhoff, J. F. & Caumette, P. (1997). Thiorhodococcus minus, gen. nov., sp. nov., a new purple sulfur bacterium isolated from coastal lagoon sediments. Arch Microbiol 168, 16–23.[CrossRef][Medline]
Imhoff, J. F. (2001). True marine and halophilic anoxygenic phototrophic bacteria. Arch Microbiol 176, 243–254.[CrossRef][Medline]
Imhoff, J. F., Süling, J. & Petri, R. (1998). Phylogenetic relationships among the Chromatiaceae, their taxonomic reclassification and description of the new genera Allochromatium, Halochromatium, Isochromatium, Marichromatium, Thiococcus, Thiohalocapsa and Thermochromatium. Int J Syst Bacteriol 48, 1129–1143.
Rabold, S., Gorlenko, V. M. & Imhoff, J. F. (2006). Thiorhodococcus mannitoliphagus sp. nov., a purple sulfur bacterium from the White Sea. Int J Syst Evol Microbiol 56, 1945–1951.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Zaar, A., Fuchs, G., Golecki, J. R. & Overmann, J. (2003). A new purple sulfur bacterium isolated from a littoral microbial mat, Thiorhodococcus drewsii sp. nov. Arch Microbiol 179, 174–183.[Medline]
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