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- phically [anaerobic, light (30 μE･m-2･s-1), with pyru- ing the protocol of Hiraishi and Hoshino (1984) and vate (0.3%, w/v)] and chemoorganoheterotrophically Hiraishi et al. (1984). [aerobic, dark and pyruvate (0.3%, w/v)]. Photolithoau- -2 -1 Phylogeny. Cell material for DNA isolation was tak- totrophy [anaerobic, light (30 μE･m ･s ), Na2S･9H2O en from 1‒2 ml of well grown liquid culture. DNA was (1 mM), Na2S2O3･5H2O (2 mM), and NaHCO3 (0.1%, extracted and purifi ed by using the QIAGEN genomic DNA buffer set. Recombinant Taq polymerase was used for PCR, which was started with the primers 5′-GTTTGATCCTGGCTCAG-3′ and 5′-TACCTTGTTAC GACTTCA-3′ (Escherichia coli positions 11‒27 and 1489‒1506, respectively). Sequences were obtained by cycle sequencing with the SequiTherm sequencing kit (Biozym) and the chain termination reaction (Sanger et al., 1997) using an automated laser fl uorescence sequencer (Pharmacia). The identifi cation of phyloge- netic neighbors and calculation of pairwise 16S rRNA gene sequence similarity was achieved using the NC- BI-BLAST search (Altschul et al., 1990) and EzTaxon server (Chun et al., 2007). The CLUSTAL_W algorithm Fig. 1. Electron micrograph of negatively stained cell of of MEGA 4 was used for sequence alignments and strain JA430T showing polar tuft of fl agella. MEGA 4 (Tamura et al., 2007) software was used for Bar, 667 nm. phylogenetic analyses of both individual and concat- enated sequences. Distances were calculated by using the Jukes and Cantor correction in a pairwise deletion procedure. Unweighted pair group with math- ematical average (UPGMA), neighbor-joining (NJ), minimum evolution (ME) and maximum parsimony (MP) methods in the MEGA4 software were used to construct phylogenetic trees. Percentage support val- ues were obtained using a bootstrap procedure. Determination of G+C content of DNA. For DNA G+C, chromosomal DNA was isolated and purifi ed (Marmur, 1961). DNA was hydrolyzed and resultant nucleotides were analyzed by reverse phase HPLC us- Fig. 2. TEM showing lamellar ICM structures in strain ing the methods of Mesbah et al. (1989). JA430T. Bar, 250 nm. 316 RAMANA et al. Vol. 56 d ‒ 5 ‒ 3 ± ± ± ± ± + + red none 25 ‒ 30 7.6 ‒ 8.0 67.3 (Bd) MK-7; Q-8 MK-7; E. mobilis , thermal denatur- m T d ) m + + + + nd red 0 ‒ 71 none 30 ‒ 35 8.0 ‒ 8.5 62.0 ( T MK-7; Q-7 MK-7; shaposhnikovii (Cherni et al., 1969). Ecto- E. shaposhnikovii d ‒ 632 - + nd red none 30 ‒ 40 0.5 ‒ 10 7.5 ‒ 8.5 62.8 (Bd) MK-7; Q-8 MK-7; E. marina Ectothiorhodospira Ectothiorhodospira . d ) m ( T d++ ++++ nd nd nd red none Ectothiorhodospira marismortui (Oren et al., 1989). 26 ‒ 40 2.5 ‒ 15 8.5 ‒ 10.0 MK-7; Q-8 MK-7; E. haloalkaliphila Organic substrate utilization was tested during photoheterotrophic growth and substrates b , variable; (+), weak growth; nd = not determined; Bd, buoyant density; c ± ) 63.5 m +n +++++ +++++ +nd++ +++++ +++++ nd nd nd 5 ‒ 852 2 ‒ 20 none 30 ‒ 35 9.0 ‒ 9.5 variabilis 62.7 ( T E. b T, 5 ------25 ‒ 35 0.5 ‒ 12 7.0 ‒ 10.0 MK-7; Q-7 MK-7; 63 (HPLC) Table 1. Differentiating characteristics of species the genus Table - , substrate not utilized or absent; PABA, PA, PY PA, PABA, reddish brown purple red/deep red Strain JA430 1.0 ‒ 1.5 × 2.0 3.5 0.8 ‒ 1.2 × 1.5‒ 1.2 × 2.0 3.0 0.7 0.8 ‒ 1.2 × 1.5 4.0‒ 0.9 × 1.5 2.5 0.8 0.7 ‒ 1.0 × 2.0 2.6 (Imhoff et al., 1981) is a later heterotypic synonym (Ventura et al., 2000) of vacuolata (Imhoff et al., 1981) is a later heterotypic synonym (Ventura Imhoff (2005). d a Characteristic Ectothiorhodospira Values for type strains. Substrates in parentheses are utilized by some Values a Propionate Sulfate assimilation Photolithoautotrophy Quinones (Mol%) G+C of DNA Sulfur te Sulfi Vitamin requirement Substrates used as carbon/e-donor Hydrogen pH range NaCl optimum (%, w/v) Salinity range (%, w/v) Temperature optimum ( ° C) Temperature Cell size ( μ m) Color of cell suspensions Butyrate Lactate Fumarate Succinate Fructose Glucose Gorlenko et al. (2009); -aminobenzaldehyde; PA = pantothenate; PY = pyridoxal phosphate. = pantothenate; PY = para -aminobenzaldehyde; PA ation; PABA Symbols: +, substrate utilized or present; c Note: used as electron donors was tested in the presence of bicarbonate (0.1%, w/v). Data taken from: de and thiosulfate as an electron donor. All strains are motile and utilize acetate, malate pyruvate as organic carbon sources. sulfi (Pelsh, 1936) is an earlier heterotypic synonym (Ventura et al., 2000) of 1936) is an earlier heterotypic synonym (Ventura thiorhodospira mobilis (Pelsh, 2010 Ectothiorhodospira salini sp. nov. 317 w/v)], chemolithoautotrophy [aerobic, dark, Na2S2O3･ Photosynthetic pigments and quinones T 5H2O (2 mM) and NaHCO3 (0.1%, w/v)] could not be The whole cell absorption spectra of strain JA430 demonstrated. Strain JA430T could utilize only acetate, exhibited absorption maxima at 311, 377, 512‒518, malate and pyruvate as carbon sources under photo- 590‒593, 797 and 860 nm and the absorption spec- lithoheterotrophic or photoorganoheterotrophic growth trum for pigments extracted with acetone gave absorp- conditions, while the vast majority of organic sub- tion maxima at 493‒496 nm, confi rming the presence strates could not support growth (Table 1). Fermenta- of bacteriochlorophyll a and carotenoids of the spiril- tive growth [anaerobic, dark, with glucose/glutamate/ loxanthin series. HPLC analysis indicated the pres- pyruvate (0.3%, w/v)] is absent. ence of spirilloxanthin (53 mol%), rhodopin (22 mol%), Ammonium chloride and urea were used as nitro- anhydrorhodovibrin (13 mol%), tetrahydrolycopene gen sources, while nitrate, nitrite, glutamate and glu- (7 mol%) and rhodovibrin (5 mol%) as major carote- tamine were not utilized by strain JA430T. Diazotrophic noids. MK-7 and Q-7 are the major quinones of strain growth could not be demonstrated in strain JA430T. JA430T. Gelatin, starch and urea were not hydrolyzed by strain JA430T. Catalase reaction was positive. Production of Cellular fatty acid composition indole from L-tryptophan and phenol from L-phenylala- Whole-cell fatty acid analysis of strain JA430T re- nine was absent. Salt (NaCl) was essential for the vealed the predominance of C18:1ω7c (56%) with suffi - T growth of strain JA430 , ranging from 0.5‒12% (w/v) cient amounts of C16:0 (18%), C19:0cycloω8c (12%) and with an optimum of 5% (w/v). The pH range for growth C16:1ω7c/C16:1ω6c (8%). of strain JA430T was 7.0‒10.0, with an optimum at 7.5. PABA, pantothenate and pyridoxal phosphate were re- 16s rRNA gene sequence comparison and DNA G+C quired for the growth of strain JA430T, while all other content species of the genus Ectothiorhodospira have no vita- The neighbor joining (NJ) phylogenetic tree con- min requirement (Table 1). structed by using an almost complete (1,348 nt) 16S rRNA gene sequence of strain JA430T demonstrated that it belongs to the genus Ectothiorhodospira and

Fig. 3. Phylogenetic tree based on 16S rRNA gene sequences showing the position of strain JA430T within the family Ectothiorhodospiraceae. Tree was constructed using the neighbor joining (NJ) method [other tree topologies, unweight- ed pair group with mathematical average (UPGMA), minimum evolution (ME) and maximum par- simony (MP) are similar to NJ] within the MEGA4 software and rooted by using Allochromatium vinosum as the out-group. Numbers at nodes represent bootstrap values (based on 100 resam- plings). The GenBank accession numbers for 16S rRNA gene sequences are shown in parenthe- ses. Bar, one nucleotide substitution per 100 nucleotides. Bootstrap values below 50 have been removed from the dendrogram. 318 RAMANA et al. Vol. 56 showed closest similarity with the type strain E. variabilis ral habitats are solar salterns. The type strain, JA430T WN22T (96.1% sequence similarity; Fig. 3). The phylo- (=NBRC 105915T = KCTC 5805T), was isolated from a genetic distances from other type strains of the genus sediment sample from a solar saltern at Kanyakumari, Ectothiorhodospira are; E. haloalkaliphila BN 9903T Tamil Nadu, India. (96.2%), E. marina DSM 241T (95.6%), E. shaposhnik- ovii DSM 239T (95.1%) and E. mobilis DSM 237T (95%). Acknowledgments The DNA G+C content of the strain JA430T is 63 mol%. The 16S rRNA gene sequence similarity (96.1%) of We thank Prof. J. Euzéby for his expert suggestion for correct strain JA430T with its nearest neighbor E. variabilis species epithet and Latin etymology. Financial support received from Department of Biotechnology, Govt. of India, is acknowl- WN22T is within the recommended standards to delin- edged. V. Venkata Ramana. acknowledges the CSIR, Govern- eate a bacterial species (Stackebrandt and Ebers, ment of India, for the award of a SR fellowship. Facilities used T 2006; Stackebrandt and Goebel, 1994). Strain JA430 under the DST, FIST-II at University of Hyderabad are acknowl- also differs phenotypically (Table 1) from other mem- edged. bers of the genus Ectothiorhodospira in 1. Color of cell suspension, 2. Utilizing limited organic substrates for References growth, 3. Lack of sulfate assimilation, 4. Lack of pho- tolithoautotrophic growth and 5. vitamin requirements. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. These clear differences of strain JA430T from other J. (1990) Basic local alignment search tool. Mol. Biol., 215, 403‒410. validated Ectothiorhodospira spp. necessitated the de- Biebl, H. and Pfennig, N. (1981) Isolation of members of the scription of the novel strain as a new species for which family Rhodospirillaceae. In The Prokaryotes: A Handbook the name Ectothiorhodospira salini sp. nov. is pro- on Habitats, Isolation, and Identifi cation of Bacteria, Vol. 1, posed. ed. by Starr, M. P., Stolp, H., Trüper, H. G., Balows, A., and Schlegel, H. G., Springer, New York, pp. 267‒273. Description of Ectothiorhodospira salini sp. nov. Bryantseva, I., Gorlenko, V. M., Kompantseva, E. I., Imhoff, J. F., Ectothiorhodospira salini (sa.li’ni. L. gen. n. salini, of Suling, J., and Mityushina, L. (1999) Thiorhodospira sibirica a salt-cellar). Cells are vibrioid to spiral shaped, gen. nov., sp. nov., a new alkaliphilic purple sulfur bacteri- um from a Siberian soda lake. Int. J. Syst. Bacteriol., 49, 1.0‒1.5 μm wide and 2.0‒3.5 μm long. Cells are motile 697‒703. by means of polar fl agella and divide by binary fi ssion. Cherni, N. E., Soloveva, J. V., Fedorova, V. D., and Kondrateva, Growth occurs under anaerobic conditions in the light. E. N. (1969) The ultrastructure of cells of the species of Internal photosynthetic membranes are of the lamellar purple sulfur bacteria. Mikrobiologiya, 38, 479‒484. type arranged parallel to the cytoplasmic membrane. Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K., and Phototrophic cultures are reddish brown. The in vivo Lim, Y. W. (2007) EzTaxon: A web-based tool for the identi- absorption spectrum of intact cells exhibits maxima at fi cation of prokaryotes based on 16S ribosomal RNA gene 311, 377, 512‒518, 590‒593, 797 and 860 nm. Bacte- sequences. Int. J. Syst. Evol. Microbiol., 57, 2259‒2261. riochlorophyll a and carotenoids; spirilloxanthin, rho- Cowan, S. T. and Steel, K. J. (1965) Manual for the Identifi cation of Medical Bacteria, Cambridge University Press, London. dopin, anhydrorhodovibrin, tetrahydrolycopene and Gorlenko, V. M., Bryantseva, I. A., Panteleeva, E. E., Tourova, T. rhodovibrin are present. The type strain is mesophilic P., Kolganova, T. V., Makhneva, Z. K., and Moskalenko, A. (30°C), growing at a pH optimum of 7.5 (range, A. (2004) Ectothiorhodosinus mongolicum gen. nov., sp. pH 7.0‒10.0) and requiring 5% NaCl for optimal growth nov., a new purple bacterium from a soda lake in Mongolia. (NaCl range, 0.5‒12.0%). 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