Thiorhodococcus Modestalkaliphilus Sp. Nov. a Phototrophic Gammaproteobacterium from Chilika Salt Water Lagoon, India

J. Gen. Appl. Microbiol., 56, 93‒99 (2010) Full Paper

Thiorhodococcus modestalkaliphilus sp. nov. a phototrophic gammaproteobacterium from Chilika salt water lagoon, India

Kodali Sucharita,1 Chintalapati Sasikala,1,* and Chintalapati Venkata Ramana2

1 Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, 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 August 21, 2009; Accepted October 5, 2009)

A phototrophic gammaproteobacterium designated strain JA395T was isolated from a sediment sample collected from the coast of Birds’ Island in the southern sector of Chilika Lagoon, India. The bacterium is a Gram-negative, motile coccus with a single polar ﬂ agellum. Bacteriochloro- phyll a, and lycopene as major carotenoid. C16:0 , C16:1ω7c/C16:1ω6c and C18:1ω7c are the major cellular fatty acids of strain JA395T. The 16S rRNA gene sequence of strain JA395T clusters with those of species of the genus Thiorhodococcus belonging to the class Gammaproteobacteria. The highest sequence similarities of strain JA395T were found with the type strains of Thiorho- dococcus minor (96.8%), Thiorhodococcus mannitoliphagus (96.3%), Thiorhodococcus bheemlicus (95.8%), “Thiorhodococcus drewsii” (95.4%), and Thiorhodococcus kakinadensis (95.0%). The genomic DNA base composition of strain JA395T (=KCTC 5710T=NBRC 104958T) was 57.8 mol% G + C (by HPLC). Based on the 16S rRNA gene sequence analysis, morphological and physiological characteristics, strain JA395T is sufﬁ ciently different from other Thiorhodo- coccus species and we describe this as a new species, Thiorhodococcus modestalkaliphilus sp. nov.

Key Words—anoxygenic phototrophic bacteria; bacteriochlorophyll a; brackish water lagoon; 16S rRNA gene based phylogeny; Thiorhodococcus

Introduction iphagus (Rabold et al., 2006), Thiorhodococcus minor (Guyoneaud et al., 1997)) to four, with the description of With the recent two additions (Thiorhodococcus “Thiorhodococcus drewsii” (Zaar et al., 2003) still not bheemlicus and Thiorhodococcus kakinadensis; Anil validated. During the study of diversity of anoxygenic Kumar et al., 2007) the number of validly published phototrophic bacteria from Chilika Lagoon, India, we species names of the genus Thiorhodococcus has have isolated a number of purple sulfur and purple increased from two (Thiorhodococcus mannitol- nonsulfur bacteria. In this report, we describe a novel purple sulfur bacterium, strain JA395T isolated from a sediment sample of a coastal brackish water lagoon, * Address reprint requests to: Dr. Chintalapati Sasikala, Bac- Chilika. terial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, J. N. T. University, Kukatpally, Hydera- Materials and Methods bad 500 085, India. E-mail: [email protected]; [email protected] The EMBL accession number for the 16S rRNA gene se- Media and pure cultures. The medium of Pfennig quences of strain JA395T is AM 993156. (Pfennig and Trüper, 1992) supplemented with sodium 94 SUCHARITA, SASIKALA, and RAMANA Vol. 56 chloride (1%, w/v), pyruvate (0.3%, w/v) as carbon ent vitamins (Vitamin B12, biotin, niacin, PABA, pan- source/electron donor and ammonium chloride (0.12%) tothenate, pyridoxal phosphate, ribofl avin, thiamine as nitrogen source was used for photoheterotrophic individually and a cocktail of all the above vitamins growth under fl uorescent light (2,400 lux) at 30 ± 2°C. [0.02%, w/v]) as growth factors. Growth was measured Purifi cation from the enrichment was achieved by re- turbidometrically at 660 nm. Fatty acid methyl esters peated streaking on agar slants (25 mm × 150 mm were prepared, separated and identifi ed according to test tubes sealed with butyl rubber stoppers and the the instructions for the Microbial Identifi cation System gas phase was replaced with argon). Purifi ed cultures (Microbial ID; MIDI; Agilent: 6850) (Sasser, 1990) which were grown in completely fi lled screw cap test tubes was outsourced to Royal Research Labs, Secundera- (10×100 mm) for photoheterotrophic growth. Pure bad, India. cultures of the strain were maintained under refrigera- Genetic properties. Genomic DNA was obtained tion at 4°C and preserved by lyophilization in vials us- from 1‒2 ml well-grown liquid culture using the Ge- ing skim milk powder (20%, w/v) as a cryoprotectant. nomic DNA Extraction Kit (Qiagen). Recombinant Taq Microscopy and pigment analysis. Morphological polymerase was used for PCR. The PCR amplifi cation properties (cell shape, cell division, cell size, fl agella) of 16S rRNA gene and puf gene (pufL, M) was done were observed by phasecontrast light microscopy separately. The primers used for 16S rRNA gene am- (with an Olympus BH-2 microscope). To study the ultra plifi cation started with the sequence 5′-GTTTGATCC structure of the fl agella, cells were stained with 1% TGGCTCAG-3′ (F-27) and 5′-TACCTTGTTACGACTTC phosphotungstic acid; ultra thin sections were viewed A-3′(R-1489) (Positions 11‒27 and 1489‒1506 respec- through a transmission electron microscope (H-7500; tively, according to the Escherichia coli 16S rRNA Hitachi) to examine intracytoplasmic structures such numbering system of the International Union of Bio- as the internal membrane system. chemistry). The specifi c primers pufL (5′-CTKTTCGAC In vivo absorption spectra were measured with a TTCTGGGTSGG-3′) and pufM (5′-CCAKSGTCCAGC Spectronic Genesys 2 spectrophotometer using su- GCCAGAANA-3′) were used for puf gene (pufL, M) crose solution for cell suspension (Pfennig and Trüper, amplifi cation (Nagashima et al., 1997). PCR amplifi ca- 1992). Absorption spectra were also recorded of pig- tion was done as described previously (Imhoff et al., ments extracted with acetone from the cell suspen- 1998). Sequencing of the 16S rRNA gene and puf sion. gene (pufL and M) was outsourced to MWG, Biotech Physiological and biochemical characterization. Pvt. Ltd., Bangalore, India. The resultant 16S rRNA Utilization of organic compounds as carbon source/ gene sequence (1,432 nt) of strain JA395T was aligned electron donors was tested in Pfennig’s media (Pfen- against sequences obtained from the GenBank data- nig and Trüper, 1992) containing the specifi c organic base using the CLUSTAL_X program (Thompson et compound (0.35%, w/v), 1 mM Na2S･9H2O and in the al., 1997) and the alignment was corrected manually. presence of yeast extract (0.01%, w/v). Nitrogen source Phylogenetic trees were constructed by the neighbor utilization was tested by replacing ammonium chloride joining and maximum parsimony methods using with different nitrogen sources (NaNO3, NaNO2, MEGA 4.0 software (Tamura et al., 2007). T CO(NH2)2 [Urea], NH4Cl [at 0.1%, w/v] and N2 gas The puf gene (pufL, M) sequence (1,508 nt) of JA395 [gas phase was replaced with nitrogen in the tube was aligned against puf gene sequences obtained sealed with butyl rubber stoppers]). Exponential cul- from the GenBank database using CLUSTAL_X pro- tures of strain JA395T grown photoheterotrophically gram (Thompson et al., 1997) and the alignment was (pyruvate, sulfi de and dinitrogen as carbon, electron corrected manually. The dendrogram was constructed and nitrogen sources, respectively) were used for using the PhyML (Guindon and Gascuel, 2003) pro- acetylene reduction activity as described earlier gram similarly as it was constructed with 16S rRNA (Sasikala et al., 1990). Sulfur source utilization was sequences. Genomic DNA was extracted and purifi ed tested by replacing MgSO4 with different sulfur sourc- according to the method of Marmur (1961) and the es (sodium sulfi te [0.2%, w/v], elemental sulfur [0.05%, mol% G+C of the DNA was determined by HPLC w/v], sodium sulfi de and sodium thiosulfate [5 mM], (Mesbah et al., 1989). thioglycolate and cysteine [1 mM]). Vitamin requirement was tested by replacing yeast extract with differ- 2010 Description of Thiorhodococcus modestalkaliphilus sp. nov. 95

Results and Discussion

Habitat, enrichment and isolation Strain JA395T was isolated from the enrichment cultures of a sediment sample. Sediment samples were collected on 14th November 2007 from Birds’ Island coast in the southern sector of the Chilika Lagoon, in Orissa, India. GPS positioning of the sample collection site is 19°31.081′N 085°08.182′E. The sample yielding strain JA395T had a pH of 7.0, a salinity of 0.8% NaCl and a temperature of 25°C. Brown color enrichment was observed after 6 days of incubation in Pfennig’s medium (Pfennig and Trüper, 1992), which resulted in small convex, brown colored isolated colonies on the agar slants after 1 week of incubation. A single isolated (a) colony is used as pure culture and designated as JA395T.

Morphology and pigments Individual cells of strain JA395T were coccoid in shape (Fig 1a), 1.5‒2.5 μm in diameter and multiplied by binary fi ssion. The cells were highly motile with a single polar fl agellum (Fig. 1b). Electron microphoto- graphs of ultra thin sections of the cells revealed a ve- (b) sicular type of internal membranes (Fig. 1c). The color of photosynthetically grown cultures of strain JA395T is brown. The whole cell absorption spectrum (Fig. 2) of strain JA395T showed absorption maxima at 378, 460, 490, 527, 594, 803 and 858 nm, confi rming the presence of bacteriochlorophyll a and the absorption spectrum of pigments extracted with acetone gave absorption maxima at 360, 474, 504 and 579 nm, indicating the presence of lycopene as major carotenoid.

General physiology Strain JA395T grows well photoorganoheterotrophi- cally (optimum light intensity is 2,400 lux). Strain (c) JA395T was able to grow photolithoautotrophically Fig. 1. (a) Phase contrast micrograph of strain JA395T. Bar (anaerobically in the light [2,400 lux] with Na2S･9H2O = 10 μm. (b) Negatively stained transmission electron micro- [1.0 mM] or Na2S2O3 [5.0 mM] as electron donors with photograph of strain JA395T. (c) Transmission electron micro- NaHCO [0.1%, w/v] as carbon source) and photo- 3 photograph of ultra thin section of strain JA395T showing ve- lithoheterotrophically (anaerobically in the light sicular intracellular membrane structures. [2,400 lux] with Na2S･9H2O [1.0 mM] or Na2S2O3 [5.0 mM] as electron donors and organic substrates [0.3%, w/v] as carbon source). Chemolithoautotrophy mode of growth (anaerobically in the dark, pyruvate as (aerobically in the dark with thiosulfate [5.0 mM] as fermentable substrate [0.3%, w/v]) could not be dem- electron donor and NaHCO3 [0.1%, w/v] as carbon onstrated. The substrates, which were utilized as car- source), chemoorganoheterotrophy and fermentative bon/electron donor under photolithoheterotrophic 96 SUCHARITA, SASIKALA, and RAMANA Vol. 56

gene sequence similarity (96.8%) of strain JA395T with its nearest neighbor, Thiorhodococcus minor, is with in the recommended standards to delineate a bacterial species (Stackebrandt and Ebers, 2006; Stackebrandt and Goebel, 1994). Phylogenetic affi liation of strain JA395T to other members of the genus Thiorhodococ- cus was also examined by pufL, M gene sequencing (1,508 nt). The data obtained revealed that the sequence of the new isolate was included in the cluster Fig. 2. Whole cell absorption spectrum. of the genus Thiorhodococcus (Fig. 4) but was distinct from other species of the genus. The phenotypic char- conditions, include lactate, succinate, fructose, malate, acters (Table 1), particularly the pH optima (pH 8.5) for glucose, ethanol, propanol, glycolate and casamino- growth and organic substrate utilization, together with acids (Table 1). Those which could not be utilized, in- the genomic difference (about 9% G+C mol%) with T. clude glycerol, butyrate, valerate, propionate, croto- minor, justify the description of this strain as a novel nate, acetate, formate and caprylate. Thiosulfate, species as Thiorhodococcus modestalkaliphilus sp. elemental sulfur and sodium sulfi te were utilized as nov. sulfur sources under phototrophic conditions. Ammo- nium chloride, glutamate, glutamine, and nitrate were Description of Thiorhodococcus modestalkaliphilus utilized as nitrogen sources, while molecular nitrogen, sp. nov. (mo.dest.al.ka.li.phi.lus. L. masc. adj. modes- urea and nitrite did not support growth. Acetylene re- tus, moderate, N.L. n. alkali, alcali; Gr. adj. philos, lov- duction activity for the enzyme nitrogenase was also ing; N.L. masc. adj. modestalkaliphilus, moderately not observed in strain JA395T. Salt is obligatory for the alkaliphilic) isolated from a sediment sample collected growth of strain JA395T and growth occurs from from Birds’ Island coast in the southern sector of Chi- 0.5‒4.0% NaCl (w/v) with an optimum at 1.5% (w/v). lika Lagoon, Orissa, India. Cells are coccoid shaped, The pH range of strain JA395T is 6.5‒9.5 with an opti- 1.5‒2.5 μm in diameter, motile and multiply by binary mum at 8.5. The temperature range is from 25‒30°C fi ssion. Gram-negative. Growth occurs under anaero- and the optimum is at 30°C. Vitamins are not obligate- bic conditions in the light (photolithoautotrophy, pho- ly required for the growth of strain JA395T, while addi- tolithoheterotrophy and photoorganoheterotrophy). tion of yeast extract (0.02%) enhances growth. The Internal photosynthetic membranes are of the vesicu- predominant components of cellular fatty acids are lar type. The color of phototrophic cultures is brown.

C16:0, C16:1ω7c/C16:1ω6c and C18:1ω7c. The in vivo absorption spectrum of intact cells in su- crose exhibits maxima at 378, 460, 490, 527, 594, 803, Phylogeny and 858 nm. Photosynthetic pigments are bacteri- The DNA base composition of strain JA395T was ochlorophyll a and most probably lycopene as the ma- 57.8 mol% G + C (by HPLC). The phylogenetic rela- jor carotenoid. The type strain is mesophilic (tempera- tionship of strain JA395T to other purple sulfur bacteria ture range 25‒30°C), with a pH-optimum at 8.5 (range was examined by 16S rRNA gene sequencing 6.5‒9.5) and require NaCl (range 0.5 to 4.0%) for (1,432 nt). The data obtained revealed that the se- growth. Photolithoheterotrophy with various organic quence of the new isolate was included in the cluster compounds is the preferred mode of growth. Good of the genus Thiorhodococcus (Fig. 3) but was distinct carbon sources are malate, fructose and succinate. from other species of the genus. The highest sequence Thiosulfate, elemental sulfur and sodium sulﬁ te were similarities [Neighbor joining tree (NJ), Fig. 3 and Max- utilized as sulfur sources under phototrophic condi- imum parsimony (MP; Data not shown)] of strain tions. Diazotrophic growth and acetylene reduction JA395T were found with the type strains of Thiorhodo- activity are absent. Whole cell fatty acid analysis re- coccus minor (96.8%), Thiorhodococcus mannitoli- vealed that C16:0, C16:1ω7c/C16:1ω6c and C18:1ω7c are phagus (96.3%), Thiorhodococcus bheemlicus predominant in strain JA395T. The DNA base compo- (95.8%), “Thiorhodococcus drewsii” (95.4%), and Thi- sition is 57.8 mol% G + C (by HPLC). The type strain orhodococcus kakinadensis (95.0%). The 16S rRNA JA395T has been deposited with KCTC (=5710T) and 2010 Description of Thiorhodococcus modestalkaliphilus sp. nov. 97

Table 1. Differentiating characteristics of species of the genus Thiorhodococcus.

Characteristic 1 2 3 4 5 6

Cell size (μm) 1.5‒2.5 1.0‒2.0 2.0‒3.5 1.5‒2.0 3.0‒5.0 4.0‒6.0 Motility + + + + + + Color of cell suspensions B Bo Br Pv Pv Pv Major carotenoid Ly Rp Rp Ra Ra Ra Mol% G+C of DNA 57.8 (HPLC) 66.9 (Tm) 64.5 (Tm) 61.8 (Tm) 57.5 (HPLC) 65.5 (HPLC) Vitamin requirement ---B12 n, b, p - Sulfate assimilation --+ --- Chemolithotrophic growth - +nd--- pH optimum 8.5 7.0‒7.2 6.5‒6.7 7.0‒7.5 7.2 7.0‒7.5 (range) (6.5‒9.5) (6.0‒8.0) (5.2‒8.5) (7.0‒8.5) (6.5‒7.2) (6.5‒8.0) Temperature optimum (°C) 25‒30 30‒35 30‒35 25‒30 25‒30 25‒30 Salinity range (%) 0.5‒40.5‒90‒80.1‒3.0 0.5‒5 0.5‒6 NaCl optimum (%) 1.5 2 2.4‒2.6 0.5‒2.0 1‒21‒3 Carbon/ e- donor Hydrogen - + + nd nd nd Thiosulfate + + + + - + Sulfur + + + + -- Sulﬁ te + - ++-- Formate --+ --- Acetate + + + + (+) + Propionate - ++++- Butyrate --+ --- Lactate + + + + (+) + Succinate + + + + + - Malate + (+) + + + - Fructose + + + (+) -- Glucose + - ++- + Ethanol + + + i -- Propanol + + + (i) -- Glycerol --+ --+ Glycolate + + + (i) - + Crotonate --+ --- Valerate --+ (i) - (+) Casamino acids + - ++- +

Source: Anil Kumar et al. (2007) and this study (data in rows 1, 5 and 6). Symbols: B, brown; Bo, brown orange; Br, brown red; Pv, purple violet; Rp, rhodopin; Ra, rhodopinal; Ly, lycopene; +, substrate utilized or present; -, substrate not utilized or absent; (+), weak growth; i, decrease in growth of ＞50% compared with control. (Data taken from Rabold et al., 2006); (i), decrease of growth up to more than 50% of control; n = niacin, b = biotin, p = pantothenate; nd, not determined. All cells are coccoid in shape; Na2S is utilized by all the strains. *Organic substrate utilization was tested during photoheterotrophic growth; pyruvate, fumarate were utilized by all the strains. Caproate, caprylate, tartrate, methionine were not utilized by any of the strains. 1, Strain JA395T; 2, Thiorhodococcus minor; 3, “Thiorhodococcus drewsii”; 4, Thiorhodococcus mannitoliphagus; 5, Thiorhodococcus kakinadensis; 6, Thiorhodococcus bheemlicus. Data for Thiorhodococcus drewsii (Zaar et al., 2003) Thiorhodococcus minor (Guyoneaud et al., 1997) and Thiorhodococcus mannitoliphagus (Guyoneaud et al., 1997) was taken from the literature.

NBRC (=104958T), and was isolated from a sediment Acknowledgments sample on the coast of Birds’ Island in the southern sector of the salt water Chilika Lagoon, in Orissa, In- Financial assistance received from Ministry of Earth Scienc- dia. es, Government of India is acknowledged. Infrastructural facili- 98 SUCHARITA, SASIKALA, and RAMANA Vol. 56

Fig. 3. Neighbor-joining tree based on near complete 16S rRNA gene sequences showing relationships between strain JA395T and members of the genus Thiorhodococcus. The tree was computed with MEGA ver 4.0 (Tamura et al., 2007). Numbers at nodes are bootstrap values. Bar, 2 nucleotide substitution per 100 nucleotides.

Fig. 4. Phylogenetic tree based on pufL, M gene sequences showing the relationship of strain JA395T within the members of the genus Thiorhodococcus. The tree was constructed using the PhyML program. Numbers at nodes are bootstrap values. Bar, 5 nucleotide substitutions per 100 nucleotides. 2010 Description of Thiorhodococcus modestalkaliphilus sp. nov. 99 ties used under the DST, FIST Level-II support to HCU is grate- In The Prokaryotes. A Handbook on the Biology of Bacte- fully acknowledged. ria. Ecophysiology, Isolation, Identifi cation, Applications, 2nd edition, ed. by Balows, A., Trüper, H. G., Dworkin, M., References Harder, W., and Schleifer, K.-H., Springer, Berlin, Heidel- berg and New York, pp. 3200‒3221, chapter 170. Anil Kumar, P., Sasi Jyothsna, T. S., Srinivas, T. N. R., Sasikala, Rabold, S., Gorlenko, V. M., and Imhoff, J. F. (2006) Thiorhodo- Ch., Ramana, Ch. V., and Imhoff, J. F. (2007) Two novel coccus mannitoliphagus sp. nov., a purple sulfur bacterium species of marine phototrophic Gammaproteobacteria: from the White sea. Int. J. Syst. Evol. Microbiol., 56, 1947‒ Thiorhodococcus bheemlicus sp. nov. and Thiorhodococcus 1951. kakinadensis sp. nov. Int. J. Syst. Evol. Microbiol., 57, Sasikala, K., Ramana, Ch. V., Raghuveer Rao, P., and Subrah- 2458‒2461. manyam, M. (1990) Photoproduction of hydrogen, nitrogen- Guindon, S. and Gascuel, O. (2003) A simple, fast, and accu- ase and hydrogenase activities of free and immobilized rate algorithm to estimate large phylogenies by maximum whole cells of Rhodobacter sphaeroides OU 001. FEMS Mi- likelihood. Syst. Biol., 52, 696‒704. crobiol. Lett., 72, 23‒28. Guyoneaud, R., Matheron, R., Liesack, W., Imhoff, J. F., and Sasser, M. (1990) Identifi cation of Bacteria by Gas Chromatog- Caumette, P. (1997) Thiorhodococcus minus, gen. nov., sp. raphy of Cellular Fatty Acids, MIDI Inc., Newark, DE. nov., a new purple sulfur bacterium isolated from coastal Stackebrandt, E. and Ebers, J. (2006) Taxonomic parameters lagoon sediments. Arch. Microbiol., 168, 16‒23. revisited: Tarnished gold standards. Microbiol. Today, 33, Imhoff, J. F., Süling, J., and Petri, R. (1998) Phylogenetic rela- 152‒155. tionships among the Chromatiaceae, their taxonomic re- Stackebrandt, E. and Goebel, B. M. (1994) A place for DNA- classifi cation and description of the new genera Allochro- DNA reassociation and 16S rRNA sequence analysis in the matium, Halochromatium, Isochromatium, Marichromatium, present species defi nition in bacteriology. Int. J. Syst. Bac- Thiococcus, Thiohalocapsa and Thermochromatium. Int. J. teriol., 44, 846‒849. Syst. Bacteriol., 48, 1129‒1143. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Marmur, J. (1961) A procedure for the isolation of deoxyribo- Molecular evolutionary genetics analysis (MEGA) software nucleic acid from microorganisms. J. Mol. Biol., 3, 208‒ version 4.0. Mol. Biol. Evol., 24, 1596‒1599. 218. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Mesbah, M., Premachandran, U., and Whitman, W. B. (1989) and Higgins, D. G. (1997) The CLUSTAL_X windows inter- Precise measurement of the G+C content of deoxyribo- face: Flexible strategies for multiple sequence alignment nucleic acid by high-performance liquid chromatography. aided by quality analysis tools. Nucleic Acids Res., 25, Int. J. Syst. Bacteriol., 39, 159‒167. 4876‒4882. Nagashima, K. V. P., Hiraishi, A., Shimada, K., and Matsuura, K. Zaar, A., Fuchs, G., Golecki, J. R., and Overmann, J. (2003) A (1997) Horizontal transfer of genes coding for the photo- new purple sulfur bacterium isolated from a littoral micro- synthetic reaction centers of purple bacteria. J. Mol. Evol., bial mat, Thiorhodococcus drewsii sp. nov. Arch. Microbi- 45, 131‒136. ol., 179, 174‒183. Pfennig, N. and Trüper, H. G. (1992) The family Chromatiaceae.