Description of Ectothiorhodospira Salini Sp. Nov
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J. Gen. Appl. Microbiol., 56, 313‒319 (2010) Full Paper Description of Ectothiorhodospira salini sp. nov. Vemuluri Venkata Ramana,1 Chintalapati Sasikala,1,* Eedara Veera Venkata Ramaprasad,2 and Chintalapati Venkata Ramana2 1 Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J.N.T. University Hyderabad, Kukakatpally, Hyderabad 500 085, India 2 Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O. Central University, Hyderabad 500 046, India (Received October 26, 2009; Accepted April 28, 2010) Strain JA430T is a Gram-negative, vibrioid to spiral shaped phototrophic purple sulfur bacterium isolated from anoxic sediment of a saltern at Kanyakumari in a mineral salts medium that con- tained 2% NaCl (w/v). Strain JA430T grows optimally at 5‒6% NaCl and tolerates up to 12% NaCl. Intracellular photosynthetic membranes were of the lamellar type. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series are present as photosynthetic pigments. Major cellular T fatty acids are C18:1ω7c, C16:0, C19:0cycloω8c and C16:1ω7c/C16:1ω6c. Strain JA430 exhibits pho- toorganoheterotrophy and chemoorganoheterotrophy and requires para-aminobenzoic acid, pantothenate and pyridoxal phosphate for growth. Phylogenetic analysis on the basis of 16S rRNA gene sequence analysis showed that strain JA430T forms monophyletic group in the genus Ectothiorhodospira. The highest sequence similarity for strain JA430T was found with the type strains of Ectothiorhodospira variabilis DSM 21381T (96.1%) and Ectothiorhodospira haloalka- liphila ATCC 51935T (96.2%). Morphological and physiological characteristics discriminate strain JA430T from other species of the genus Ectothiorhodospira, for which we describe this as a novel species, Ectothiorhodospira salini sp. nov. ( = NBRC 105915T = KCTC 5805T). Key Words—Ectothiorhodospira; Gammaproteobacterium; purple sulfur bacterium; 16S rRNA gene based phylogeny Introduction dosinus, Ectothiorhodospira, Halorhodospira and Thiorhodospira) are represented by members of Members of the family Ectothiorhodospiraceae form true phototrophic species (http://www.bacterio.cict.fr/ a distinct lineage from Chromatiaceae on the basis of classifgenerafamilies.html#Ectothiorhodospiraceae). 16S rRNA gene sequence analysis (Imhoff and Süling, All the phototrophic members of the family Ectothi- 1996). Among the 13 genera recognized in the family orhodospiraceae accumulate sulfur granules outside Ectothiorhodospiraceae, only 4 genera (Ectothiorho- the cell and thus are phenotypically distinct from the members of the Chromatiaceae (Imhoff, 2005). Mem- bers of the genus Halorhodospira (Halorhodospira * Address reprint requests to: Dr. Chintalapati Sasikala, Bac- halophila, Halorhodospira halochloris and Halorho- terial Discovery Laboratory, Centre for Environment, Institute of dospira abdelmalekii) are represented by most halo- Science and Technology, J.N.T. University, Kukatpally, Hydera- bad 500 085, India. philic phototrophic species, while the genera Ectothi- E-mail: [email protected]; [email protected] orhodosinus (Ectothiorhodosinus mongolicus; Gorlenko The GenBank/EMBL/DDBJ accession number for the 16S et al., 2004) and Thiorhodospira (Thiorhodospira sibir- rRNA gene sequence of strain JA430T is FM244738. ica; Bryantseva et al., 1999) are represented by single 314 RAMANA et al. Vol. 56 species. nal or external structures) was observed by phase Revision of species delineation in the genus Ectothi- contrast microscopy (Olympus BH-2) and transmis- orhodospira left only 4 species (Ectothiorhodospira sion electron microscopy (Hitachi H-7500 Model). For mobilis, Ectothiorhodospira shaposhnikovii, Ectothio- TEM examination, cells were subjected to negative rhodospira marina and Ectothiorhodospira haloalka- staining with phosphotungstic acid and grids were ex- liphila [while Ectothiorhodospira vacuolata and Ecto- amined under an H-7500 Hitachi model electron mi- thiorhodospira marismortui are heterotypic synonyms]; croscope. Gram staining was done using a 24-h old Ventura et al., 2000), and a newly described Ectothio- culture grown in nutrient broth. Motility was examined rhodospira variabilis (Gorlenko et al., 2009) brings the by the hanging drop method. validly published names to fi ve. Members of the genus Physiological and biochemical characteristics. Ectothiorhodospira are widely distributed in habitats Growth at various concentrations of NaCl (0‒20%, w/v, like brackish, marine, saline, hypersaline and soda at intervals of 0.5%) different pH (pH 5.0‒10.0, at inter- lakes. In this communication, we propose strain vals of 0.5 pH units) and temperature range (5‒45°C, JA430T, a novel species of this genus that was isolated at intervals of 5°C) was investigated in the medium de- from a saltern. scribed above. Catalase activity of the strains was de- termined as previously described (Cowan and Steel, Materials and Methods 1965). Hydrolysis of gelatin, starch, urea and nitrate reduction was studied as previously described (Lanyi, Isolation of strain JA430T. Strain JA430T was iso- 1987). For indole/phenol production from L-tryptophan/ lated from a phototrophic enrichment of a sediment L-phenylalanine, the organisms were grown in Biebl sample [pH ～7.0, 30°C, 5.0% (w/v) salinity] collected and Pfennig’s mineral medium (Biebl and Pfennig, from a saltern at Kanyakumari, Tamil Nadu, India (GPS 1981), supplemented with malate (22 mM) and L- positioning of the sample collection site is, 8° 05′ N, tryptophan/L-phenylalanine (3 mM) as carbon and ni- 77° 31′ E). Pure culture was obtained by repeated trogen sources, respectively and indole was tested streaking on agar slants (25×150 mm test tubes after 24 h of growth using para-dimethylaminobenzal- sealed with butyl rubber stoppers and the gas phase dehyde (PDAB) (Kupfer and Atkinson, 1964), or phe- was replaced with argon). Purifi ed cultures were grown nol reagent (Swain and Hills, 1959), respectively. Utili- in Biebl and Pfennig’s medium (Biebl and Pfennig, zation of organic compounds as carbon source/ 1981) with 5.0% (w/v) NaCl in completely fi lled screw electron donors under photolithoheterotrophic growth cap test tubes (10×100 mm) for phototrophic growth. was tested in Pfennig’s medium (Biebl and Pfennig, Culture purity was examined microscopically and by 1981) containing the specifi c organic compound streaking the culture on nutrient agar plates incubated (0.35%, w/v) in the presence of yeast extract (0.01%, aerobically in the dark at 30°C for 3 days. w/v). Na2S･9H2O (0.1, 0.5, 1 mM) and 1 mM each of 0 - Media and growth conditions. For routine culturing MgSO4, Na2S2O3, S , SO3 , cysteine, thioglycolate and for physiological tests, strains were grown pho- were tested for the sulfur source. Nitrogen source utili- totrophically (anaerobic, light [2,400 lux]) in a mineral zation was tested by replacing ammonium chloride -1 salts medium containing [g･L ; pH 7.0]: KH2PO4 with urea, nitrate, nitrite, glutamate and glutamine at (0.5), MgSO4･7H2O (2.0), NaCl (20.0), NH4Cl (0.6), 0.06% (w/v). Vitamin requirement was tested by re- CaCl2･2H2O (0.15) with yeast extract (0.3), ferric cit- placing yeast extract with different vitamins (Vitamin -1 rate [5 ml･L from 0.1% (w/v) stock], and trace ele- B12, biotin, niacin, PABA, pantothenate, pyridoxal ment solution SL7 (1 ml･L-1; Biebl and Pfennig, 1981). phosphate, ribofl avin, thiamine and a mixture of all the Pyruvate (25 mM), NaHCO3 (12 mM) and Na2S･9H2O above vitamins [0.02%, w/v]) as growth factors. The (2 mM) were used as carbon source/electron donor. optical density of the culture was measured after incu- Pure cultures were maintained by repeated sub-cultur- bation for 2, 4 and 6 days. ing into fresh medium or as stab cultures and as lyo- Cellular fatty acid composition. Cellular fatty acid philized vials. Strain JA430T was deposited at NBRC methyl esters were analyzed from photoheterotrophi- and KCTC. cally growing cells on agar slants according to the in- Morphological characteristics. Cell morphology (cell structions for the Microbial Identifi cation System (Mi- shape, cell division, cell size, fl agella and visible inter- crobial ID; MIDI) (Sasser, 1990) and the analysis was 2010 Ectothiorhodospira salini sp. nov. 315 outsourced. Results and Discussion Pigment analysis. In-vivo absorption spectra were measured with a Spectronic Genesys 2 spectropho- Morphological and biochemical characteristics tometer in sucrose solution (Trüper and Pfennig, 1981). Cells of strain JA430T were Gram-negative, 1.0‒1.5 μm For pigment analysis, 1 L of the 48 h grown culture wide and 2.0‒3.5 μm long and vibrioid to spiral in was harvested by centrifugation (16,000 rpm × 10 min). shape. Cell suspension was reddish brown when The supernatant was discarded and the pellet was grown photosynthetically. Cells of strain JA430T were washed twice with 0.8% NaCl solution and lyophilized. motile by means of polar fl agella (Fig. 1) and multi- Pigments were extracted with methanol:acetone (7:2) plied by binary fi ssion. A transmission electron micro- from the lyophilized cells. Carotenoid composition was photograph of an ultrathin section of strain JA430T re- determined by C18-HPLC analysis, (eluted with aceto- vealed lamellar type internal membrane structures nitrile:methanol:ethylacetate, 5:4:1; fl ow rate 1 ml･min-1; arranged parallel to the cytoplasmic membrane (Fig. 2). absorption at 450 nm) with a PDA detector. Strain JA430T was able to grow photoorganoheterotro- Quinones. Quinones were analyzed by HPLC us-