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1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad, PO Central University, Hyderabad 500 046, India
2 Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, Hyderabad 500 085, India
Correspondence
Ch. V. Ramana
r449{at}sify.com
or
sasi449{at}yahoo.ie
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ABSTRACT |
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Phase contrast images and transmission electron micrographs of cells of strains JA131T and JA135T and plots of the absorption spectra of extracted pigments from the two strains are available with the online version of this paper.
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MAIN TEXT |
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) (originally described as ‘Rhodospirillum mediosalinum’; Kompantseva & Gorlenko, 1984), Roseospira marina and Roseospira navarrensis. Another species of the genus Roseospira, ‘Roseospira thiosulfatophila’, has been proposed (Guyoneaud et al., 2002) but, to date, the name has not been validly published. In this communication, we propose two novel species of the genus Roseospira.
Strain JA131T was isolated from a water sample [pH 
6.8, 30 °C, 2–3 % (w/v) salinity] which was collected on 25 March 2004 from the fishing harbour at Visakhapatnam, India (1 ° 41' N 8 ° 18' E). Strain JA135T was isolated from a sediment sample [pH 6.8; 30 °C, 6–7 % (w/v) salinity] collected on 12 February 2005 from Kurka saltern, Goa, India (1 ° 29' N 7 ° 49' E). Original enrichments of both strains were from photolithoheterotrophic media [anaerobic, 1 mM Na2S . 9H2O+0.3 % (w/v) pyruvate/malate]. Strain JA131T was isolated from an enrichment culture containing 2 % NaCl and strain JA135T was isolated from an enrichment containing 8 % NaCl. Subsequent culturing, purification and characterization were performed in Biebl & Pfennig (1981) medium with the following modifications (l–1); 1 g MgSO4 . 7H2O, 0.15 g CaCl2 . 2H2O and 20 g NaCl supplemented with Na2S2O3 . 5H2O (2 mM). Strain purification and a taxonomic study based on a polyphasic approach were carried out as described earlier (Srinivas et al., 2007).
Cell material for 16S rRNA gene sequencing was taken from 1–2 ml 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., 1998b). Recombinant Taq polymerase was used for PCR. The PCR was started 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 system 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 to the SEQBOOT program in the PHYLIP package (Felsenstein, 1989) and the output file from 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 and the consensus tree as the intree in the maximum-likelihood program of the PHYLIP package
While cells of strain JA131T are vibrioid, 0.5–0.9 µm wide and 2–6 µm long (see Supplementary Fig. S1a in IJSEM Online), cells of strain JA135T are vibrioid to crescent-shaped (short to very long spirals of 12–30 µm occur rarely), 0.8–1.0 µm wide and 3–8 µm long (see Supplementary Fig S1b in IJSEM Online). Both strains multiply by binary fission. Strain JA131T is non-motile. Strain JA135T is motile by means of a pair of monopolar flagella (see Supplementary Fig. S2 available with the online version of this paper). Transmission electron micrographs of ultrathin sections of both novel strains revealed a vesicular type of internal membrane structure (see Supplementary Fig. S3a, b in IJSEM Online). Both strains were able to grow photoorganoheterotrophically [anaerobic, light (2400 lx), with pyruvic acid (0.3 % w/v)]. Chemoorganoheterotrophic [aerobic, dark and pyruvate (0.3 % w/v)] growth was observed in both strains, however, the biomass yields were poor. Photolithoautotrophy [anaerobic, light (2400 lx), Na2S . 9H2O, Na2S2O3 . 5H2O (0.5 mM) and NaHCO3 (0.1 % w/v)], chemolithoautotrophy [aerobic, dark, Na2S2O3 . 5H2O (0.5 mM) and NaHCO3 (0.1 % w/v)] and fermentative growth (anaerobic, dark, with pyruvate [0.3 % w/v]) could not be demonstrated. Organic substrates utilized/not utilized by the novel strains are given in Table 1. Both novel strains could utilize ammonium chloride, glutamate and glutamine as nitrogen sources, while urea, nitrate and nitrite did not support growth. Diazotrophic growth and acetylene reduction could not be demonstrated. Niacin, thiamine and p-aminobenzoic acid are required as growth factors. Sulfate assimilation is absent and sulfide, thiosulphate and thioglycolate are used as sulfur sources by both novel strains; in addition, strain JA135T could utilize elemental sulfur. Salt (NaCl) is essential for the growth of both novel strains; the salinity range was from 1–5 % (w/v) and the optimum level was 2 % (w/v) for strain JA131T and 1–3 % (w/v) for strain JA135T. The pH range for growth of strain JA131T is 6.5–8.0, with an optimum at pH 7.0. For strain JA135T, the pH range for growth is 7.0–8.0, with an optimum at pH 7.5. The temperature range for growth for both strains is 25–35 °C and the optimum is 30 °C.
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909 nm was observed with strain JA131T as previously observed with ‘Roseospira thiosulfatophila’ (Guyoneaud et al., 2002).
The DNA G+C contents of strains JA131T and JA135T were 67 and 71 mol% (by HPLC), respectively. The phylogenetic relationships of strains JA131T and JA135T to other purple nonsulfur bacteria were examined using 16S rRNA gene sequences. The data obtained revealed that the novel isolates clustered with the type strains of species of the genus Roseospira but were distinct from other genera of purple nonsulfur bacteria. Strains JA131T and JA135T showed the highest gene sequence similarities to the type strains of Roseospira navarrensis (95.9 %), Roseospira marina (95.5 %), Roseospira mediosalina (94.2 %) and ‘Roseospira thiosulfatophila’ (96.1 %). The sequence similarity between strains JA131T and JA135T is 96.6 % (Fig. 1). Apart from differences in 16S rRNA gene sequences, strains JA131T and JA135T also showed clear phenotypic differences to other species of the genus Roseospira (Table 1
) that justify their description as representatives of two separate novel species, Roseospira visakhapatnamensis sp. nov. and Roseospira goensis sp. nov., respectively.
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Cells are vibrioid, 0.5–0.9 µm wide and 2–6 µm long. Cells are non-motile and divide by binary fission. Growth occurs under anaerobic conditions in the light (photoorganoheterotrophy) or under aerobic conditions in the dark (chemoorganoheterotrophy). Internal photosynthetic membranes are of the vesicular type. Phototrophic cultures are red–brown. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 371, 473, 503, 540, 590, 803, 847 and 900 nm, confirming the presence of bacteriochlorophyll a, with an unusual absorption shoulder at 900 nm. The type strain is mesophilic (30 °C), with a pH optimum of 7.0 and requires 2 % NaCl (w/v) for optimal growth. The preferred mode of growth is photoorganoheterotrophy with a few organic compounds. Pyruvate, aspartate and peptone are good carbon sources. Growth also occurs on mannitol and cysteine. Photoautotrophic and chemoautotrophic growth are not possible in the presence of sulfide/thiosulfate/hydrogen as electron donors and NaHCO3 as the carbon source. Fermentative growth is not possible in the presence of pyruvate as a fermentable carbon source. Sulphate assimilation is absent. Sulfide, thiosulfate and thioglycolate are used as sulfur sources. Niacin, p-aminobenzoic acid and thiamine are required as growth factors. The DNA G+C base composition is 67 mol% (by HPLC). Natural habitat is marine waters.
The type strain, strain JA131T (=ATCC BAA-1365T=JCM 14190T), was isolated from water samples from a fishing harbour at Visakhapatnam, a city which faces into the Bay of Bengal and is on the East coast of India.
Description of Roseospira goensis sp. nov.
Roseospira goensis (go'en.sis. N.L. fem. adj. goensis pertaining to Goa, a state in India known for its beautiful beaches, from where the type strain was isolated).
Cells are vibrioid to crescent-shaped, 0.8–1.0 µm wide and 3.0–8.0 µm long. Cells are motile by means of a pair of monopolar flagella and divide by binary fission. Growth occurs under anaerobic conditions in the light (photoorganoheterotrophy) or under aerobic conditions in the dark (chemoorganoheterotrophy). Internal photosynthetic membranes are of the vesicular type. Phototrophic cultures are red–brown. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 377, 473, 503, 540, 590, 803 and 857 nm confirming the presence of bacteriochlorophyll a and most probably rhodovibrine. The type strain is mesophilic (30 °C), with a pH optimum at 7.5 and requires 1–3 % NaCl (w/v) for optimal growth. The preferred mode of growth is photoorganoheterotrophy with a few organic compounds. Butyrate, valerate, lactate, pyruvate, fumarate, glutarate, glycerol, 2-oxoglutarate and peptone are good carbon sources. Growth also occurs on formate, acetate, malate and glucose. Photoautotrophic and chemoautotrophic growth does not take place in the presence of sulfide/thiosulfate/hydrogen as the electron donor and NaHCO3 as the carbon source. Fermentative growth does not take place in the presence of pyruvate as the fermentable carbon source. Sulphate assimilation is absent. Sulfide, thiosulphate, thioglycolate and elemental sulfur are used as sulfur sources. Niacin, thiamine and p-aminobenzoic acid are required as growth factors. The DNA G+C content is 71 mol% (by HPLC). Natural habitats are marine salterns.
The type strain, JA135T (=ATCC BAA-1364T=JCM 14191T) was isolated from sediment samples from a marine saltern, Goa, facing the Arabian sea on the West coast of India.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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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., Sophie, M., Eatock, C., Bothorel, V., Hirschler-Réa, A., Willison, J., Robert, D., Liesack, W., Herbert, R. & other authors (2002). Characterization of three spiral-shaped purple non-sulfur bacteria isolated from coastal lagoon sediments, saline sulfur springs, and microbial mats: emended description of the genus Roseospira and description of Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov. Arch Microbiol 178, 315–324.[CrossRef][Medline]
Imhoff, J. F., Süling, J. & Petri, R. (1998a). Reclassification of species of the spiral-shaped phototrophic purple non-sulfur bacteria of the 
-Proteobacteria: description of the new genera Phaeospirillum gen. nov., Rhodovibrio gen. nov., Rhodothalassium gen. nov. and Roseospira gen. nov. as well as transfer of Rhodospirillum fulvum to Phaeospirillum fulvum comb. nov., of Rhodospirillum molischianum to Phaeospirillum molischianum comb. nov., of Rhodospirillum salinarum to Rhodovibrio salinarum comb. nov., of Rhodospirillum sodomense to Rhodovibrio sodomensis comb. nov., of Rhodospirillum salexigens to Rhodothalassium salexigens comb. nov. and of Rhodospirillum mediosalinum to Roseospira mediosalina comb. nov. Int J Syst Bacteriol 48, 793–798.
Imhoff, J. F., Süling, J. & Petri, R. (1998b). 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.
Kompantseva, E. I. & Gorlenko, V. M. (1984). A new species of moderately halophilic purple bacterium Rhodospirillum mediosalinum sp. nov. Mikrobiologiia 53, 954–961 (in Russian).
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci U S A 74, 5463–5467.
Srinivas, T. N. R., Anil Kumar, P., Sasikala, Ch. & Ramana, Ch. V. (2007). Rhodovulum imhoffii sp. nov. Int J Syst Evol Microbiol 57, 228–232.
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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