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Int J Syst Evol Microbiol 57 (2007), 932-935; DOI  10.1099/ijs.0.64843-0
© 2007 International Union of Microbiological Societies

Rhodobium gokarnense sp. nov., a novel phototrophic alphaproteobacterium from a saltern

T. N. R. Srinivas1, P. Anil Kumar1, Ch. Sasikala1, Ch. V. Ramana2 and J. F. Imhoff3

1 Environmental Microbial Biotechnology Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, Hyderabad 500 072, 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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A pink-pigmented, phototrophic, purple nonsulfur bacterium, strain JA173T, was isolated in pure culture from a saltern in Gokarna, India, in a medium containing 2 % (w/v) NaCl. Strain JA173T was a non-motile Gram-negative rod that multiplied by budding. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JA173T clusters with the class Alphaproteobacteria; highest sequence similarity (98 %) was to the type strain of Rhodobium orientis and 94 % similarity was observed to the 16S rRNA gene sequence of the type strain of Rhodobium marinum. However, DNA–DNA hybridization with R. orientis DSM 11290T revealed a relatedness value of only 35.1 % with strain JA173T. Strain JA173T contained lamellar internal membranes, bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Strain JA173T had an obligate requirement for NaCl (optimum growth at 2–6 %, w/v) and grew photoheterotrophically with a number of organic compounds as carbon source or electron donor. Photoautotrophic, chemoautotrophic and fermentative growth could not be demonstrated. Yeast extract was required for growth. Based on 16S rRNA gene sequence analysis, DNA–DNA hybridization data and morphological and physiological characteristics, strain JA173T is sufficiently different from other species of the genus Rhodobium to be recognized as a representative of a novel species, Rhodobium gokarnense sp. nov. The type strain is JA173T (=ATCC BAA-1215T=DSM 17935T=JCM 13532T).

Abbreviations: FTIR, Fourier-transform infrared

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JA173T is AM180706.

A phase contrast micrograph, an electron micrograph, absorption spectra and FTIR spectra of strain JA173T are available as supplementary material in IJSEM Online.


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The genus Rhodobium is composed of marine species that are capable of photosynthesis, multiply by budding and possess lamellar internal membrane structures (Hiraishi et al., 1995Go). At present, the genus Rhodobium comprises two species, both of which were isolated from marine sediments: Rhodobium marinum (originally described as Rhodopseudomonas marina, Imhoff, 1983Go); and Rhodobium orientis (Hiraishi et al., 1995Go). Strain JA173T, which was isolated from a saltern, is described in this report and, based on its phylogenetic and phenotypic properties, which are distinct from those of the other Rhodobium species, it is proposed that this strain represents a novel species in this genus.

Soil and water, including salt crystals, were collected on 27 December 2003 at around midday from a saltern located in Gokarna, India. GPS positioning of the sample collection site was 14° 32' N 74° 19' E. The sample yielding strain JA173T had a pH of 6.8 and a temperature of 30 °C. Strain JA173T was isolated from photoheterotrophic enrichments of this soil sample. Purification and polyphasic taxonomic studies were carried out as described previously (Srinivas et al., 2006Go). Fourier-transform infrared (FTIR) spectroscopic analysis data (Ramana et al., 2006Go) of strain JA173T were compared with those of cells of Rhodobium orientis DSM 11290T. DNA–DNA hybridization was carried out at the DSMZ (Braunschweig, Germany) using a spectrophotometric method (De Ley et al., 1970Go; Huß et al., 1983Go) after chromatographic (hydroxyapatite) purification of DNA (Cashion et al., 1977Go).

Individual cells of strain JA173T were rod-shaped, 0.5–0.6 µm wide and 1.0–2.0 µm long, non-motile and multiplied by budding (see Supplementary Fig. S1 in IJSEM Online). An electron micrograph of ultrathin sections of the cells revealed lamellar internal membrane structures (see Supplementary Fig. S2 in IJSEM Online). Strain JA173T was able to grow photoorganoheterotrophically in the presence of incandescent light (optimum light intensity, 1000–4000 lux) (anaerobic; light, 2400 lux) and chemoorganoheterotrophically and aerobically in the dark with pyruvate (0.3 %, w/v). Photolithoautotrophic growth (anaerobic; light, 2400 lux; 20 % H2, v/v; 0.5 mM Na2S; 0.5 mM Na2S2O3; and 0.1 % NaHCO3, w/v), chemolithoautotrophy (aerobic; dark; 0.5 mM thiosulfate; and 0.1 % NaHCO3, w/v) and fermentative growth (anaerobic; dark; 0.3 % glucose, w/v; 0.3 % fructose, w/v) could not be demonstrated. Substrates that were utilized as carbon sources/electron donors under photoorganoheterotrophic conditions included acetate, butyrate, pyruvate, citrate, succinate, fumarate, malate, glucose, mannitol, sorbitol and Casamino acids (Table 1Go). Formate, propionate, valerate, caproate, caprylate, lactate, tartrate, benzoate, fructose, glycerol, methanol, ethanol, glutamate, peptone and yeast extract could not be utilized. Thiosulfate, sodium sulfide and H2 (with 0.1 % NaHCO3) were not utilized as electron donors under photolithoautotrophic conditions. Ammonium chloride, molecular nitrogen and glutamine were utilized as nitrogen sources, whereas urea, glutamate, nitrate and nitrite did not support growth. Strain JA173T required yeast extract as a growth factor. Salt (NaCl) was obligatory for the growth of strain JA173T; this strain grew in 0.5–10.0 % (w/v) NaCl, with optimum growth at 2.0–6.0 % (w/v) NaCl. Strain JA173T grew at pH 5.0–9.0, with optimum growth at pH 6.5–8.0. The temperature optimum for growth was 30±2 °C.


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  		Table 1. Characteristics that differentiate species of the genus Rhodobium

Data for Rhodobium orientis are from Hiraishi et al. (1995)Go. Data for Rhodobium marinum are from Imhoff (1983)Go and Imhoff & Hiraishi (2005)Go; organic substrate utilization for this species was tested during photoheterotrophic growth. Acetate, butyrate, fumarate, D-glucose, malate, pyruvate, sorbitol and succinate are utilized by all taxa, whereas benzoate, methanol and tartrate are not utilized. All taxa exhibited sessile budding and are pink–red in culture. +, Substrate utilized or present; –, substrate not utilized or absent; +/–, variable reaction in different strains; (+), weak growth or microaerobic growth only; [+], weak growth; ND, not determined; NI, no information.

 
The colour of the phototrophically grown cell suspension was pink to pink–red. The whole cell absorption spectrum of strain JA173T showed absorption maxima at 370, 402, 488, 530, 590, 803 and 872 nm, thus confirming the presence of bacteriochlorophyll a and most probably carotenoids of the spirilloxanthin series (see Supplementary Fig. S3a, b in IJSEM Online). The cellular components (metabolomes) of strain JA173T were compared with those of Rhodobium orientis DSM 11290T under identical growth conditions using whole cell FTIR spectra (see Supplementary Fig. S4 in IJSEM Online), which clearly indicated differences between the two strains. Variations were recorded in the proteins (1650–1580 cm–1), carbohydrates (1200–900 cm–1) and aromatic compounds (870–675 cm–1). No major differences were observed in lipids and fatty acids (3100–2800 cm–1). An ester peak (1728 cm–1) was observed in strain JA173T alone, which is similar to that observed in Rubrivivax gelatinosus DSM 17011T (Ramana et al., 2006Go). The DNA G+C content of strain JA173T was 72.4 mol% (determined by HPLC). The phylogenetic relationship of strain JA173T to other purple nonsulfur bacteria was examined by 16S rRNA gene sequencing (Fig. 1Go). The data obtained revealed that the novel isolate branched separately, but was affiliated with the type strains of Rhodobium species. The highest sequence similarities to strain JA173T were found with the type strains of Rhodobium orientis (98 %) and Rhodobium marinum (94 %). However, DNA–DNA hybridization with Rhodobium orientis DSM 11290T revealed a relatedness value of only 35.1 % with strain JA173T. Apart from 16S rRNA gene sequence dissimilarity and DNA–DNA hybridization studies, strain JA173T clearly differed from other Rhodobium species in its phenotypic properties (Table 1Go); these results justify the description of this strain as a representative of a novel species, Rhodobium gokarnense sp. nov.


Figure 1
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  		Fig. 1. Dendrogram depicting the phylogenetic relationships of strain JA173T within the family Rhodobiaceae determined using 16S rRNA gene sequence analysis. Bar, 2 substitutions per 100 nucleotides.

 
Description of Rhodobium gokarnense sp. nov.
Rhodobium gokarnense (go'kar.nense. L. neut. adj. gokarnense pertaining to Gokarna, the place from which the type strain was isolated).

Cells are rod-shaped, 0.5–0.6x1.0–2.0 µm, non-motile and divide by budding. Growth occurs under anaerobic conditions in the light (photoorganoheterotrophy) or under aerobic conditions in the dark (chemoorganoheterotrophy). Internal photosynthetic membranes have a lamellar structure. Phototrophic cultures are pink to pinkish-red. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 370, 402, 488, 530, 590, 803 and 872 nm, confirming the presence of bacteriochlorophyll a and probably the spirilloxanthin series. Mesophilic (30 °C), with optimum growth at pH 6.5–8.0. Requires 2.0–6.0 % (w/v) NaCl for optimal growth. Photoorganoheterotrophy with various organic compounds is the preferred mode of growth. Good carbon sources are pyruvate and fumarate. Growth also occurs on acetate, butyrate, citrate, succinate, malate, glucose, mannitol, sorbitol and Casamino acids. Photoautotrophic and chemoautotrophic growth is not possible in the presence of sulfide/thiosulfate/hydrogen as electron donor and NaHCO3 as carbon source. Yeast extract is required as a growth factor. The DNA G+C content of the type strain is 72.4 mol% (by HPLC).

The type strain, JA173T (=ATCC BAA-1215T=DSM 17935T=JCM 13532T), was isolated from a saltern in Gokarna, India.


   ACKNOWLEDGEMENTS
 
Financial assistance received from Department of Biotechnology and Department of Ocean Development, Government of India is acknowledged. P. A. K. and T. N. R. S. acknowledge the CSIR, Government of India, for the award of JR & SR fellowships. The skilful assistance of F. Lappe (IFM-GEOMAR, Kiel) in molecular analysis is kindly acknowledged. Financial assistance received under DST-DAAD exchange program (grant 422-PPP-34105) is acknowledged.


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Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Hiraishi, A., Urata, K. & Satoh, T. (1995). A new genus of marine budding phototrophic bacteria, Rhodobium gen. nov., which includes Rhodobium orientis sp. nov. and Rhodobium marinum comb. nov. Int J Syst Bacteriol 45, 226–234.[Abstract/Free Full Text]

Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184–192.

Imhoff, J. F. (1983). Rhodopseudomonas marina sp. nov., a new marine phototrophic purple bacterium. Syst Appl Microbiol 4, 512–521.

Imhoff, J. F. & Hiraishi, A. (2005). The phototrophic bacteria. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part C, pp. 571–574. Edited by D. J. Brenner, N. R. Krieg & J. T. Staley. New York: Springer.

Ramana, Ch. V., Sasikala, Ch., Arunasri, K., Anil Kumar, P., Srinivas, T. N. R., Shivaji, S., Gupta, P., Süling, J. & Imhoff, J. F. (2006). Rubrivivax benzoatilyticus sp. nov., an aromatic hydrocarbon-degrading purple betaproteobacterium. Int J Syst Evol Microbiol 56, 2157–2164.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]




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