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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. V. Ramana
r449{at}sify.com
or
sasi449{at}yahoo.ie
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ABSTRACT |
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A phase-contrast micrograph, an electron micrograph and whole-cell and pigment absorption spectra of strain JA123T are available as supplementary material with the online version of this paper.
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). At present, the genus Rhodobacter comprises five species [Rhodobacter massiliensis (Greub & Raoult, 2003) has been reclassified recently as Haematobacter massiliensis (Helsel et al., 2007)]; the currently recognized species are Rhodobacter capsulatus (Imhoff et al., 1984), Rba. sphaeroides (Imhoff et al., 1984), Rba. blasticus (Kawasaki et al., 1993), Rba. veldkampii (Hansen & Imhoff, 1985) and Rba. azotoformans (Hiraishi et al., 1996). The marine representatives of the genus, Rhodobacter adriaticus (Imhoff et al., 1984), Rba. euryhalinus (Kompantseva, 1985) and Rba. sulfidophilus (Imhoff et al., 1984), have been transferred to the genus Rhodovulum based on their habitat, salt requirement and separate clustering from their freshwater counterparts and formation of a distinct clade based on 16S rRNA gene analysis (Hiraishi & Ueda, 1994). Six more species in the genus Rhodovulum have also been proposed: Rhodovulum iodosum (Straub et al., 1999), Rdv. imhoffii (Srinivas et al., 2007a), Rdv. marinum (Srinivas et al., 2006), Rdv. robiginosum (Straub et al., 1999), Rdv. strictum (Hiraishi & Ueda, 1995) and Rdv. visakhapatnamense (Srinivas et al., 2007b). Other marine phototrophic members of the Alphaproteobacteria (growing with 1–12 % NaCl) belong to the genera Rhodospira, Roseospirillum, Roseospira, Rhodobium, Rhodobaca, Rhodothalassium and Rhodovibrio (Imhoff, 2001). In this study, a strain from tidal seawater collected from the seashore of Visakhapatnam, India, was isolated. This strain, JA123T, required NaCl for growth and tolerated up to 10 % NaCl and clustered in the genus Rhodobacter based on 16S rRNA gene sequence analysis. Based on phenotypic and phylogenetic analysis, it is proposed that strain JA123T represents a novel species.
Strain JA123T was isolated from enrichments of tidal seawater collected from Ramakrishna beach, Bay of Bengal, Visakhapatnam, India, on 30 March 2004 (GPS position of the site 1 ° 42' N 8 ° 18' E). The sample yielding strain JA123T had a pH of 6.8 and a temperature of 30 °C. Purification and polyphasic taxonomic analyses were carried out as described previously (Srinivas et al., 2007a).
Individual cells of strain JA123T were rod-shaped, 0.8–1.2 µm wide and 1.5–3.0 µm long, non-motile and multiplied by binary fission (see Supplementary Fig. S1 available in IJSEM Online). Electron microphotographs of ultrathin sections of the cells revealed vesicular internal membrane structures (Supplementary Fig. S2). Strain JA123T was able to grow photo-organoheterotrophically [anaerobic conditions, in the light (2400 lx)] and chemo-organoheterotrophically [aerobic conditions, in the dark and in the presence of pyruvate (0.3 %, w/v)]. Photolithoautotrophic growth [anaerobic, light (2400 lx), Na2S (0.5 mM), Na2S2O3 (0.5 mM) and NaHCO3 (0.1 %, w/v)], chemolithoautotrophic growth [aerobic, dark, Na2S2O3 (0.5 mM) and NaHCO3 (0.1 %, w/v)] and fermentative growth [anaerobic, dark, pyruvate (0.3 %, w/v)] could not be demonstrated. Substrates that were utilized as carbon sources and electron donors under photo-organoheterotrophic conditions included acetate, butyrate, lactate, pyruvate, fumarate, oxoglutarate, succinate, malate, glucose, glycerol, sorbitol, cysteine, peptone and Casamino acids (Table 1). Those that could not be utilized included formate, propionate, valerate, crotonate, caproate, caprylate, glycollate, benzoate, tartrate, citrate, fructose, sucrose, mannitol, gluconate, methanol, ethanol, propanol, methionine, aspartate, glutamate, ascorbate and thioglycollate. Ammonium chloride, glutamate, glutamine and molecular nitrogen were utilized as nitrogen sources, whereas urea, nitrate and nitrite did not support growth. Salt (0.5–10 % w/v NaCl) was required for growth of strain JA123T; optimum growth occurred in 1–4 % (w/v) NaCl. Strain JA123T grew at pH 6.0–8.0 and 20–30 °C; optimum growth was observed at pH 6.0–7.5 and 30±2 °C. It was able to grow photo-organoheterotrophically under fluorescent light (optimum light intensity 
2400 lx; range 1000–4000 lx). Furthermore, the strain required biotin as a growth factor. The colour of photosynthetically grown cell suspension was yellowish brown. The whole-cell absorption spectrum of strain JA123T gave absorption maxima at 377, 452, 479, 512, 590, 803 and 851 nm, thus confirming the presence of bacteriochlorophyll a and probably carotenoids of the spheroidene series (Supplementary Fig. S3).
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) and the alignment was corrected manually. The CLUSTAL X alignment file was used as the input file to the program SEQBOOT 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 program CONSENSE. A final dendrogram with evolutionary distances was constructed by taking the alignment .phy file as the infile and the consensus tree as the intree in the maximum-likelihood program of the PHYLIP package. Data showed that the novel isolate branched separately, but clustered with type strains of species of the genus Rhodobacter and was distinct from other genera of purple non-sulfur bacteria. The highest sequence similarities of strain JA123T were found with the type strains of Haematobacter massiliensis (95.7 %), Rba. veldkampii (95.2 %), Rba. sphaeroides (95 %), Rba. capsulatus (94.7 %), Rba. azotoformans (94.6 %) and Rba. blasticus (94.2 %) (Fig. 1). Apart from 16S rRNA gene sequence dissimilarity, strain JA123T clearly differed phenotypically from other Rhodobacter species (Table 1
), which justifies the description of a novel species to accommodate this strain; the name Rhodobacter vinaykumarii sp. nov. is proposed. An additional strain, JA249, which also represents Rhodobacter vinaykumarii, was isolated from marine water in Kiel, Germany (5 ° 21' N 1 ° 08' E).
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Cells are rod-shaped, 0.8–1.2x1.5–3.0 µm, non-motile and divide by binary fission. Growth occurs under anaerobic conditions in the light (photo-organoheterotrophy) or under aerobic conditions in the dark (chemo-organoheterotrophy). Internal photosynthetic membranes are of the vesicular type. The colour of photosynthetically grown cultures is yellowish brown. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 377, 452, 479, 512, 590, 803 and 851 nm, thus confirming the presence of bacteriochlorophyll a and probably carotenoids of the spheroidene series. Mesophilic (30 °C), with optimum growth at pH 6.0–7.5. Requires NaCl for growth (optimum 1–4 %, w/v). Photoheterotrophy with organic compounds is the preferred mode of growth. Good growth is obtained on pyruvate, fumarate, oxoglutarate and malate. Growth on acetate, succinate, lactate, glucose, glycerol, sorbitol, cysteine, peptone and Casamino acids also occurs. Photolithoautotrophic and chemolithoautotrophic growth is not possible in the presence of thiosulfate/hydrogen as electron donor and NaHCO3 as carbon source. Fermentative growth is not possible in the presence of pyruvate as the fermentable carbon source. Biotin is required for growth.
The type strain is JA123T (=DSM 18714T =JCM 14544T =CCUG 54311T), isolated from marine tidal waters on the east coast of India. The DNA G+C content of the type strain is 68.8 mol% (by HPLC). An additional strain of this species, JA249, has been isolated from marine water in Germany.
Emended description of the genus Rhodobacter Imhoff et al. 1984
The description is as given previously (Imhoff et al., 1984; Imhoff, 2005) with the modification that most species of the genus Rhodobacter are freshwater bacteria and do not require salt, but some species may be adapted to the marine environment and require salt.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Greub, G. & Raoult, D. (2003). Rhodobacter massiliensis sp. nov., a new amoebae-resistant species isolated from the nose of a patient. Res Microbiol 154, 631–635.[Medline]
Hansen, T. A. & Imhoff, J. F. (1985). Rhodobacter veldkampii, a new species of phototrophic purple nonsulfur bacteria. Int J Syst Bacteriol 35, 115–116.
Helsel, L. O., Hollis, D., Steigerwalt, A. G., Morey, R. E., Jordan, J., Aye, T., Radosevic, J., Jannat-Khah, D., Thiry, D. & other authors (2007). Identification of "Haematobacter", a new genus of aerobic Gram-negative rods isolated from clinical specimens, and reclassification of Rhodobacter massiliensis as "Haematobacter massiliensis" comb. nov. J Clin Microbiol 45, 1238–1243.
Hiraishi, A. & Ueda, Y. (1994). Intrageneric structure of the genus Rhodobacter: transfer of Rhodobacter sulfidophilus and related marine species to the genus Rhodovulum gen. nov. Int J Syst Bacteriol 44, 15–23.
Hiraishi, A. & Ueda, Y. (1995). Isolation and characterization of Rhodovulum strictum sp. nov. and some other purple nonsulfur bacteria from colored blooms in tidal and seawater pools. Int J Syst Bacteriol 45, 319–326.
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Imhoff, J. F. (2005). Genus Rhodobacter. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part C, pp. 161–167. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
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Kawasaki, H., Hoshino, Y., Hirata, A. & Yamasato, K. (1993). Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria. Arch Microbiol 160, 358–362.[Medline]
Kompantseva, E. I. (1985). Rhodobacter euryhalinus sp. nov., a new halophilic purple bacterial species. Mikrobiologiia 54, 974–982.
Srinivas, T. N. R., Anil Kumar, P., Sasikala, Ch., Ramana, Ch. V., Süling, J. & Imhoff, J. F. (2006). Rhodovulum marinum sp. nov., a novel phototrophic purple non-sulfur alphaproteobacterium from marine tides of Visakhapatnam, India. Int J Syst Evol Microbiol 56, 1651–1656.
Srinivas, T. N. R., Anil Kumar, P., Sasikala, Ch. & Ramana, Ch. V. (2007a). Rhodovulum imhoffii sp. nov. Int J Syst Evol Microbiol 57, 228–232.
Srinivas, T. N. R., Anil Kumar, P., Sasikala, Ch., Ramana, Ch. V. & Imhoff, J. F. (2007b). Rhodovulum visakhapatnamense sp. nov. Int J Syst Evol Microbiol 57, 1762–1764.
Straub, K. L., Rainey, F. A. & Widdel, F. (1999). Rhodovulum iodosum sp. nov. and Rhodovulum robiginosum sp. nov., two new marine phototrophic ferrous-iron-oxidizing purple bacteria. Int J Syst Bacteriol 49, 729–735.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
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