| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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, Marine Mikrobiologie, Düsternbrooker Weg 20, 24105 Kiel, Germany
Correspondence
Ch. V. Ramana
r449{at}sify.com
sasi449{at}yahoo.ie
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
, b; Imhoff & Pfennig, 2001), coastal lagoons with elevated salt concentrations (Imhoff et al., 1998a, b), tidal waters, brackish waters (Pfennig & Trüper, 1989; Imhoff, 1988) and marine coastal sediments (Imhoff, 1983). They have even been found in the extreme marine habitats of Antarctica (Madigan et al., 2000; Karr et al., 2003).
Previously, the taxonomy of photosynthetic bacteria was based exclusively on phenotypical characteristics (Pfennig & Trüper, 1974, 1989). A 16S rRNA gene sequence comparison of these bacteria revealed phylogenetic differences among freshwater, true marine and halophilic bacteria (Imhoff et al., 1998a). On the basis of these results, a number of taxa have been reclassified. The marine representatives of the genus Chromatium were transferred to the genera Allochromatium, Marichromatium, Isochromatium and Halochromatium (Imhoff et al., 1998a), while the marine Rhodobacter species were reclassified as species of the genus Rhodovulum (Hiraishi & Ueda, 1994) and those of Rhodospirillum to Rhodothalassium and Roseospira (Imhoff et al., 1998b).
The marine anoxygenic phototrophic Gammaproteobacteria are distributed among the families Chromatiaceae and Ectothiorhodospiriaceae. In the Chromatiaceae, the genera Halochromatium, Thiohalocapsa, Thiococcus, Rhabdochromatium, Isochromatium, Thiorhodococcus, Marichromatium and Thiorhodovibrio (Imhoff et al., 1998a) grow in NaCl concentrations from 1 to 11 %. The Ectothiorhodospiraceae genera include Halorhodospira, Thiorhodospira and Ectothiorhodospira, growing in NaCl concentrations from 1 to 32 % (Imhoff, 2001). Marine representatives of phototrophic Alphaproteobacteria that can grow in 1–12 % NaCl are found in the genera Rhodospira, Roseospirillum, Roseospira, Rhodobium, Rhodovulum, Rhodobaca, Rhodothalassium and Rhodovibrio (Imhoff, 2001). No marine representatives of anoxygenic phototrophic Betaproteobacteria have been reported so far. Apart from phylogenetic differences based on 16S rRNA gene sequences of marine and freshwater species of anoxygenic phototrophic bacteria, good correlations were also observed in the major quinone and fatty acid compositions, which were in accordance with the requirement of NaCl or sea salt for growth of some of these bacteria (Imhoff et al., 1998b).
We have isolated several strains of marine purple bacteria from coastal areas of Andhra Pradesh, India. Strain JA128T, isolated from photoheterotrophic enrichments of seawater from a tidal beach, had 95 % 16S rRNA gene sequence homology with the type strains of Rhodovulum iodosum and Rhodovulum sulfidophilum, and 93 % with Rhodovulum robiginosum. This isolate, based on phenotypic characteristics and significant differences in the 16S rRNA gene sequence, is described here as a novel species, Rhodovulum marinum sp. nov.
Tidal water samples from a beach at Visakhapatnam on the east coast of India (Bay of Bengal) were collected in polypropylene bottles during March 2004. The medium of Pfennig & Trüper (1992) supplemented with NaCl (2 % w/v), pyruvate (0.3 % w/v) as carbon source and ammonium chloride (0.12 %) as nitrogen source was used for photoheterotrophic growth under light (2400 lux) at 30±2 °C. Purification was done by using the repeated agar shake dilution method (Pfennig & Trüper, 1992; Imhoff, 1988). Purified cultures were grown in completely filled screw cap test tubes (10x100 mm) for photoheterotrophic growth.
Morphological properties (cell shape, cell division, cell size, flagella) were observed by phase-contrast light microscopy (Olympus BH-2), ultrastructure of flagella was studied after staining with 1 % phosphotungstic acid and ultrathin sections for intracytoplasmic structures, such as the internal membrane system, were viewed through a transmission electron microscope (H-7500; Hitachi). In vivo absorption spectra were measured with a Spectronic Genesys 2 spectrophotometer in sucrose solution (Trüper & Pfennig, 1981). Absorption spectra were also recorded from pigments extracted with acetone after eluting the cell suspension with acetone through a 10x200 mm column packed with aluminium oxide. Utilization of other inorganic and organic compounds as electron donors for phototropic growth was tested without any additional electron donor in the presence of yeast extract (0.03 %, w/v). Formic acid, propionate, butyrate, caproate, valerate, lactate, glycerol, methanol and ethanol were used at a concentration of 0.1 % (v/v); other compounds tested were used at 0.3 % (w/v), with benzoate (1 mM) and NaHCO3 (0.1 %). For testing sulfur sources, MgSO4.7H2O was replaced by MgCl2.5H2O (0.01 %); sulfur sources (Na2S.9H2O, Na2S2O3, sodium thioglycolate, cysteine, MgSO4.7H2O, all at 0.5 mM, and FeSO4, 10 mM) were added to the medium in addition to NaHCO3 (0.1 %). Nitrogen source utilization was tested by replacing ammonium chloride with different nitrogen sources at 0.12 % (w/v). Vitamin requirements were tested by replacing yeast extract with single and also combinations of vitamins as growth factors. Chemotrophy was determined by growing the cultures in Erlenmeyer flasks placed in an orbital shaker in the dark at 30 °C. Diazotrophy of the cultures was determined by growth under an N2 atmosphere and was confirmed by repeated subculturing (four times). Growth was measured turbidometrically at 660 nm.
Genomic DNA was extracted and purified according to the method of Marmur (1961) and the G+C content of the DNA was determined by thermal denaturation (Marmur & Doty, 1962). Cell material for 16S rRNA gene sequencing was taken from 1–2 ml of well grown liquid cultures. DNA was extracted and purified by using the Qiagen genomic DNA buffer set. PCR amplification and 16S rRNA gene sequencing was done as described previously (Imhoff et al., 1998a; Imhoff & Pfennig, 2001). Sequences were aligned using the CLUSTAL W program (Thompson et al., 1994) and the alignment was corrected manually. The distance matrix was calculated on the basis of the algorithm according to Jukes & Cantor (1969) with the DNADIST program within the PHYLIP package (Felsenstein, 1989). The FITCH program in the PHYLIP package was used to fit a tree to the evolutionary distances.
Samples were collected during March 2004 from Ramakrishna beach, Visakhapatnam, Bay of Bengal, on the east coast of India (17.7° longitude, 83.32° latitude). The sample yielding strain JA128T had a pH of 6.8 and a salinity of 2–3 % NaCl, and the temperature was 30 °C. The sample was inoculated into sterile screw cap tubes filled with medium for photoheterotrophic growth and incubated in the light for 1 week with intermittent shaking. Yellowish-brown enrichments were obtained and subcultured into the same medium. This culture was used for subsequent agar shake dilution series for purification. After 4 days incubation, small, convex, yellowish-brown colonies were observed. The colony and cell morphology were the same in all dilution series. The culture was maintained by repeated subculturing in the same medium and also in stabs. The stabs were preserved in a refrigerator at 4 °C. Individual cells of strain JA128T were oval to rod-shaped (Fig. 1a), 0.6–0.8 µm wide and 1–2 µm long. The cells were non-motile and multiplied by binary fission (Fig. 1b). Electron microphotographs of ultrathin sections of the cells revealed vesicular internal membranes (Fig. 1c).
|
) as carbon/electron donor under photo-organoheterotrophic conditions include acetate, pyruvate, lactate, succinate, fumarate, oxaloacetate, tartrate, glucose, fructose, mannitol, sorbitol, glycerol, ethanol, yeast extract and Casamino acids. Those which could not be utilized include formate, propionate, butyrate, valerate, caproate, oleic acid, citrate, 
-ketoglutarate, malate, sucrose, lactose, maltose, methanol, glutamate and benzoate. Thiosulfate, sodium sulfide and H2 (with 0.1 % NaHCO3) were not utilized as electron donors under photolithoautotrophic conditions. Ammonium chloride, molecular nitrogen, nitrate, glutamate and glutamine were utilized as nitrogen sources, while urea and nitrite did not support growth. Salt (NaCl) was obligatory for growth, and growth occurred from 0.05 to 8 % (w/v), the optimum NaCl concentration being 1–3 % (w/v). This is similar to the observed growth of another marine isolate from India, Marichromatium indicum JA100T (Arunasri et al., 2005), which also grows in a range of 0.05–8 % NaCl, reflecting adaptation to a natural habitat in which large variations in the salt concentration during various seasons occurs. The pH range for growth of strain JA128T was 5.5–7.5 with the optimum at 6.0–6.8. The temperature range was from 25 to 35 °C and the optimum was at 30 °C. Thiamine was required as growth factor. The colour of photosynthetically grown cell suspensions was yellowish-brown to beige. The cell absorption spectrum (Fig. 2a) of strain JA128T gave maxima at 378, 402, 488, 520, 590, 802, 884 nm, confirming the presence of bacteriochlorophyll a and most probably the carotenoids spheroidene and spheroidenone (Fig. 2b).
|
|
), but was distinct from other genera of purple non-sulfur bacteria. The highest sequence similarities to strain JA128T were found with the type strains of Rhodovulum iodosum, Rhodovulum sulfidophilum (95 %) and Rhodovulum robiginosum (93 %). Apart from 16S rRNA gene sequence dissimilarity, strain JA128T showed phenotypic differences to other Rhodovulum species (Table 1
) that justify the description of this strain as a novel species.
|
Cells are ovoid to rod-shaped, 0.6–0.8 µm wide, 1.0–2.0 µm long and form chains. Non-motile and division by binary fission. Gram-negative. Growth occurs under anaerobic conditions in the light (photo-organoheterotrophy) or under aerobic conditions in the dark (chemo-organoheterotrophy). Internal photosynthetic membranes are vesicular. The colour of phototrophic cultures is yellow–green to brown, while aerobic cultures are pink. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 375, 479, 590, 804 and 857 nm. Photosynthetic pigments are bacteriochlorophyll a and most probably carotenoids of the spheroidene series. The type strain is mesophilic (range 25–35 °C, optimum 30 °C), slightly acidophilic (pH range 5.5–7.5, optimum 6.0–6.8) and slightly halophilic with NaCl concentrations of 0.5 to 3.0 % required for optimal growth. Photo-organotrophy with various organic compounds is the preferred mode of growth. Good carbon sources are sorbitol, mannitol, pyruvate, lactate, intermediates of the citric acid cycle and some sugars. Growth on acetate, glycerol and ethanol also occurs. Photoautotrophic and chemoautotrophic growth is not possible in the presence of sulfide, thiosulfate or hydrogen as electron donor and NaHCO3 as carbon source. Thiamine is required as growth factor. DNA G+C base composition: 62 mol% (Tm). Natural habitats are marine surface and tidal waters exposed to light. The EMBL accession number for the 16S rRNA gene sequence of strain JA128T is AJ891122. The type strain, JA128T, has been deposited as ATCC BAA 1215T=JCM 13300T=CCUG 52183T.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Felsenstein, J. (1989). PHYLIP – Phylogenetic Inference Package, version 3.5.1. Distributed by the author. University of Washington, Seattle, USA.
Hansen, T. A. & Veldkamp, H. (1973). Rhodopseudomonas sulfidophila, nov. spec., a new species of the purple nonsulfur bacteria. Arch Microbiol 92, 45–58.
Heising, S., Dilling, W., Schnell, S. & Schink, B. (1996). Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus). Arch Microbiol 165, 397–401.[CrossRef][Medline]
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.
Imhoff, J. F. (1983). Rhodopseudomonas marina sp. nov., a new marine phototrophic purple bacterium. Syst Appl Microbiol 4, 512–521.
Imhoff, J. F. (1988). Anoxygenic phototrophic bacteria. In Methods in Aquatic Bacteriology, pp. 207–240. Edited by B. Austin. Chichester, New York: Wiley.
Imhoff, J. F. (2001). Transfer of Rhodopseudomonas acidophila to the new genus Rhodoblastus as Rhodoblastus acidophilus gen. nov., comb. nov. Int J Syst Bacteriol Microbiol 51, 1863–1866.
Imhoff, J. F. & Pfennig, N. (2001). Thioflavicoccus mobilis gen. nov., sp. nov., a novel purple sulfur bacterium with bacteriochlorophyll b. Int J Syst Bacteriol Microbiol 51, 105–110.
Imhoff, J. F. & Trüper, H. G. (1992). The genus Rhodospirillum and related genera. In the Prokaryotes, 2nd edn, pp. 2141–2155. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. Berlin, Heidelberg, New York: Springer.
Imhoff, J. F., Süling, J. & Petri, R. (1998a). 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.
Imhoff, J. F., Süling, J. & Petri, R. (1998b). 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.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Karr, E. A., Sattley, W. M., Jung, D. O., Madigan, M. T. & Achenbach, L. A. (2003). Remarkable diversity of phototrophic purple bacteria in a permanently frozen Antarctic lake. Appl Environ Microbiol 69, 4910–4914.
Kompantseva, E. I. (1985). New halophilic purple bacteria Rhodobacter euryhalinus sp. nov. Mikrobiologiia 54, 974–982.
Madigan, M. T., Jung, D. O., Woese, C. R. & Achenbach, L. A. (2000). Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple non-sulfur bacterium isolated from an Antarctic microbial mat. Arch Microbiol 173, 269–277.[CrossRef][Medline]
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Neutzling, O., Imhoff, J. F. & Trüper, H. G. (1984). Rhodopseudomonas adriatica sp. nov., a new species of the Rhodospirillaceae, dependent on reduced sulfur compounds. Arch Microbiol 137, 256–261.[CrossRef]
Pfennig, N. & Trüper, H. G. (1974). The phototrophic bacteria. In Bergey's Manual of Systematic Bacteriology, 8th edn, pp. 24–75. Edited by R. E. Buchanan & N. E. Gibbons. Baltimore: Williams & Wilkins.
Pfennig, N. & Trüper, H. G. (1989). Family Chromatiaceae. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 1637–1653. Edited by J. T. Staley, M. P. Bryant. N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
Pfennig, N. & Trüper, H. G. (1992). The family Chromatiaceae. In the Prokaryotes. A Handbook on the Biology of Bacteria. Ecophysiology, Isolation, Identification, Applications, 2nd edn, pp. 3200–3221. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. Berlin, Heidelberg & New York: Springer.
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., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Trüper, H. G. & Pfennig, N. (1981). Isolation of members of the families Chromatiaceae and Chlorobiaceae. In the Prokaryotes: a Handbook on Habitats, Isolation, and Identification of Bacteria, pp. 279–289. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin: Springer.
This article has been cited by other articles:
|
|
 |
|
  S. K. Chakravarthy, K. Sucharitha, Ch. Sasikala, and Ch. V. Ramana Rhodovulum lacipunicei sp. nov., an obligate sulfide-demanding phototrophic alphaproteobacterium isolated from a purple pond in India Int J Syst Evol Microbiol, July 1, 2009; 59(7): 1615 - 1619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Kumar, P. Aparna, T. N. R. Srinivas, Ch. Sasikala, and Ch. V. Ramana Rhodovulum kholense sp. nov. Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1723 - 1726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Anil Kumar, T. N. R. Srinivas, Ch. Sasikala, and Ch. V. Ramana Rhodobacter changlensis sp. nov., a psychrotolerant, phototrophic alphaproteobacterium from the Himalayas of India Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2568 - 2571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. R. Srinivas, P. Anil Kumar, Ch. Sasikala, Ch. V. Ramana, and J. F. Imhoff Rhodobacter vinaykumarii sp. nov., a marine phototrophic alphaproteobacterium from tidal waters, and emended description of the genus Rhodobacter Int J Syst Evol Microbiol, September 1, 2007; 57(9): 1984 - 1987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. R. Srinivas, P. Anil Kumar, Ch. Sasikala, Ch. V. Ramana, and J. F. Imhoff Rhodovulum visakhapatnamense sp. nov. Int J Syst Evol Microbiol, August 1, 2007; 57(8): 1762 - 1764. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Kumar, T. S. S. Jyothsna, T. N. R. Srinivas, Ch. Sasikala, Ch. V. Ramana, and J. F. Imhoff Marichromatium bheemlicum sp. nov., a non-diazotrophic, photosynthetic gammaproteobacterium from a marine aquaculture pond Int J Syst Evol Microbiol, June 1, 2007; 57(6): 1261 - 1265. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. R. Srinivas, P. A. Kumar, Ch. Sasikala, Ch. V. Ramana, and J. F. Imhoff Rhodobium gokarnense sp. nov., a novel phototrophic alphaproteobacterium from a saltern Int J Syst Evol Microbiol, May 1, 2007; 57(5): 932 - 935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. R. Srinivas, P. A. Kumar, Ch. Sasikala, and Ch. V. Ramana Rhodovulum imhoffii sp. nov. Int J Syst Evol Microbiol, February 1, 2007; 57(2): 228 - 232. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
