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Marichromatium bheemlicum sp. nov., a non-diazotrophic, photosynthetic gammaproteobacterium from a marine aquaculture pond

¹ Bacterial Discovery Laboratory, Centre for Environment, Institute of Science and Technology, JNT University, Kukatpally, Hyderabad 500 085, 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. Sasikala
r449{at}sify.com or
sasi449{at}yahoo.ie

	ABSTRACT

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A rod-shaped, phototrophic, purple sulfur bacterium, strain JA124^T, was isolated in pure culture from a marine aquaculture pond, located near Bhimunipatnam, in a medium that contained 3 % NaCl (w/v). Strain JA124^T is a Gram-negative, motile rod with a single polar flagellum. Strain JA124^T has a requirement for NaCl, with optimum growth at 1.5–8.5 %, and tolerates up to 11 % NaCl. Intracellular photosynthetic membranes are of the vesicular type. Bacteriochlorophyll a and probably carotenoids of the spirilloxanthin series are present as photosynthetic pigments. Strain JA124^T was able to utilize sulfide, sulfate, thiosulfate, sulfite, thioglycollate and cysteine as sulfur sources. Strain JA124^T was able to grow photolithoautotrophically, photolithoheterotrophically and photo-organoheterotrophically. Chemotrophic and fermentative growth could not be demonstrated. Strain JA124^T lacks diazotrophic growth and acetylene reduction activity. Pyridoxal phosphate is required for growth. During growth on reduced sulfur sources as electron donors, sulfur is deposited intermediately as a number of small granules within the cell. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA124^T clusters with species of the genus Marichromatium belonging to the class Gammaproteobacteria. The highest sequence similarities of strain JA124^T were found with the type strains of Marichromatium indicum (98 %), Marichromatium purpuratum (95 %) and Marichromatium gracile (93 %). However, DNA–DNA hybridization with Marichromatium indicum DSM 15907^T revealed relatedness of only 65 % with strain JA124^T. The DNA base composition of strain JA124^T was 67 mol% G+C (by HPLC). Based on 16S rRNA gene sequence analysis, morphological and physiological characteristics and DNA–DNA hybridization studies, strain JA124^T (=ATCC BAA-1316^T=JCM 13911^T) is sufficiently different from other Marichromatium species to merit its description as the type strain of a novel species, Marichromatium bheemlicum sp. nov.

Phase-contrast and electron micrograph images of cells of strain JA124^T and absorption spectra of a whole-cell extract and acetone-extracted pigments are available as supplementary material with the online version of this paper.

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The genus Marichromatium was established to separate the true marine species of the genus Chromatium from their freshwater counterparts (Imhoff et al., 1998). At present, the genus Marichromatium comprises three described species, Marichromatium gracile (Imhoff et al., 1998) (originally described as Chromatium gracile; Strzeszewski, 1913), Marichromatium purpuratum (Imhoff et al., 1998) (originally described as Chromatium purpuratum; Imhoff & Trüper, 1980) and Marichromatium indicum (Arunasri et al., 2005). In this communication, we propose a novel species of this genus which has properties distinct from all other Marichromatium species based on phenotypic and phylogenetic analysis.

Strain JA124^T was isolated from enrichments of a sediment and water sample from an aquaculture pond near Bheemli, Visakhapatnam (India). The medium of Pfennig (Pfennig & Trüper, 1992) supplemented with NaCl (3 %, w/v), pyruvate (0.3 %, w/v) as a carbon source/electron donor and ammonium chloride (0.12 %) as a nitrogen source was used for photoheterotrophic growth under fluorescent light (2400 lx) at 30±2 °C. Purification was achieved by repeated agar-shake dilution series (Pfennig & Trüper, 1992; Imhoff, 1988; Trüper, 1970). Purified cultures were grown in completely filled screw-capped test tubes (10x100 mm) for photoheterotrophic growth. Morphological properties (cell shape, cell division, cell size, flagella) were observed by light microscopy (with an Olympus BH-2 microscope). To study the ultrastructure of the flagella, cells were stained with 1 % phosphotungstic acid; ultrathin sections were viewed through a transmission electron microscope (H-7500; Hitachi) to examine intracytoplasmic structures such as the internal membrane system. In vivo absorption spectra were measured with a Spectronic Genesys 2 spectrophotometer using sucrose solution for cell suspension (Pfennig & Trüper, 1992). Absorption spectra were also recorded of pigments extracted with acetone from the cell suspension. Cellular components (fatty acids, intracellular and membrane proteins, polysaccharides, photosynthetic pigments and nucleic acids) of strain JA124^T were compared with cells from Mch. indicum DSM 15907^T based on information obtained from Fourier-transform infrared (FTIR) spectroscopy as described previously (Ramana et al., 2006).

The total cellular protein profile of strain JA124^T was compared with that of Mch. indicum DSM 15907^T as follows. The strains were grown under similar conditions, cells were lysed and protein profiles were obtained by SDS-PAGE (Sorokin et al., 2004). After electrophoresis, the gels were silver stained to visualize the protein bands. The utilization of different carbon substrates and electron donors (0.3 %, w/v or v/v, unless otherwise mentioned) was tested in the medium of Pfennig (Pfennig & Trüper, 1992) containing 5 mM Na₂S.9H₂O. Ammonium chloride was replaced with different nitrogen sources in order to test for utilization of nitrogen sources (0.12 %, w/v). Diazotrophy of the culture was determined by growing strain JA124^T under a N₂ atmosphere and was confirmed by repeated subculturing (four times). Cultures of strain JA124^T in the exponential phase grown photoheterotrophically (pyruvate, sulfide and ammonium chloride as carbon, electron and nitrogen sources, respectively) were used to assay for acetylene reduction activity as described earlier (Sasikala et al., 1990). 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 HPLC (Mesbah et al., 1989). 16S rRNA gene sequence analysis was carried out as described by Srinivas et al. (2006). Briefly, sequences were aligned using the CLUSTAL W program (Thompson et al., 1994) and the alignment was corrected manually. The CLUSTAL W alignment file was used as input file to the SEQBOOT program in the PHYLIP package (Felsenstein, 1989) and the output file of SEQBOOT was used as the input file for maximum-likelihood analysis with 100 datasets and five times jumbling. One single tree was generated using 100 trees generated during maximum-likelihood analysis using the CONSENSE program. The distance matrix was calculated on the basis of the algorithm of Jukes & Cantor (1969) with the DNADIST program within the PHYLIP package. The FITCH program in the PHYLIP package was employed to fit a tree to the evolutionary distances using the DIST file as infile and the CONSENSE file as intree. DNA–DNA hybridization was conducted by the Identification Service of the DSMZ (Braunschweig, Germany), using a spectrophotometric method (De Ley et al., 1970; Huß et al., 1983) after chromatographic (hydroxyapatite) purification of DNA (Cashion et al., 1977).

Sediment and water samples were collected on 24 February 2005 at around midday from an aquaculture pond near the sea (Bay of Bengal) at Bheemli, India. GPS positioning of the sample collection site is 17° 51' 41.03'' N 83° 25' 16.81'' E. The sample yielding strain JA124^T had a pH of 6.8, a salinity of 2–3 % NaCl and a temperature of 30 °C. Individual cells of strain JA124^T were rod-shaped (Supplementary Fig. S1, available in IJSEM Online), 0.8–1.0 µm wide and 2–4 µm long. The cells were highly motile with single polar flagella and multiplied by binary fission. Electron photomicrographs of ultrathin sections of the cells revealed a vesicular type of internal membranes (Supplementary Fig. S2).

Strain JA124^T grows well photo-organoheterotrophically (optimum light intensity is 2400 lx). Strain JA124^T was able to grow photolithoautotrophically [anaerobically in the light (2400 lx) with Na₂S.9H₂O or Na₂S₂O₃ (1.0 mM) as electron donors with NaHCO₃ (0.1 %, w/v) as carbon source] and photolithoheterotrophically [anaerobically in the light (2400 lx) with Na₂S.9H₂O or Na₂S₂O₃ (1.0 mM) as electron donors and organic substrates (0.3 %, w/v) as carbon source]. Chemolithoautotrophy [aerobically in the dark with thiosulfate (1.0 mM) as electron donor and NaHCO₃ (0.1 %, w/v) as carbon source], chemo-organoheterotrophy and a fermentative mode of growth [anaerobically in the dark, pyruvate as fermentable substrate (0.3 %, w/v)] could not be demonstrated. Substrates that were utilized as carbon sources/electron donors under photolithoheterotrophic conditions included acetate, pyruvate, succinate, fumarate, glycerol, ethanol, methanol, malate, valerate and caproate (Table 1). Those that could not be utilized included citrate, glucose, mannitol, lactate, tartrate, benzoate and glutamate. Thiosulfate, sulfate, sulfite, thioglycollate, cysteine and sodium sulfide were utilized as sulfur sources under phototrophic conditions. Ammonium chloride, glutamate and glutamine were utilized as nitrogen sources, while nitrate, nitrite, urea and molecular nitrogen did not support growth. Acetylene reduction activity for the enzyme nitrogenase was also not observed in strain JA124^T. Salt is obligatory for growth of strain JA124^T, and growth occurs in 1.5–11 % NaCl (w/v), with optimum growth at 1.5–8.5 % (w/v). The pH range for growth of strain JA124^T is pH 6.0–8.5, with optimum growth at pH 6.5–8.5. The temperature range for growth is 20–35 °C, and the optimum is at 30–35 °C. Yeast extract is required as a growth factor (0.03 %, w/v). In the absence of yeast extract, pyridoxal phosphate (100 µg l^–1) is required for growth of strain JA124^T.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Protein banding patterns of cell lysates, visualized with silver staining after SDS-PAGE, of Mch. indicum DSM 15907T (lane 1) and strain JA124T (lane 2). Lane M, molecular mass standards (kDa). Arrows indicate differences.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Dendrogram depicting the phylogenetic relationships of strain JA124T within the family Chromatiaceae determined using 16S rRNA gene sequence analysis. See text for details. Bar, 2 nucleotide substitutions per 100 nucleotides.

Cells are rod-shaped, 0.8–1.0 µm wide and 2–4 µm long, motile and multiply by binary fission. Gram-negative. Growth occurs under anaerobic conditions in the light (photolithoautotrophy, photolithoheterotrophy and photo-organoheterotrophy). Internal photosynthetic membranes are of the vesicular type. The colour of phototrophic cultures is reddish brown. The in vivo absorption spectrum of intact cells in sucrose exhibits maxima at 374, 488, 590, 797 and 851 nm. Photosynthetic pigments are bacteriochlorophyll a and probably carotenoids of the spirilloxanthin series. The type strain is mesophilic (temperature range 20–35 °C), with a pH optimum at 6.5–8.5, and requires NaCl (range 1.5–11 %) for growth. Photolithoheterotrophy with various organic compounds is the preferred mode of growth. Good carbon sources are acetate, pyruvate and intermediates of the citric acid cycle except citrate. Growth also occurs on glycerol and caproate. Thiosulfate, sulfate, sulfite, thioglycollate, cysteine and sodium sulfide are utilized as sulfur sources under phototrophic conditions. Photolithoautotrophic growth occurs. Diazotrophic growth and acetylene reduction activity are absent. Pyridoxal phosphate is required for growth of the type strain. The DNA base composition is 67 mol% G+C (by HPLC).

The type strain, JA124^T (=ATCC BAA-1316^T=JCM 13911^T), was isolated from a marine aquaculture pond exposed to light at Bheemli, on the Bay of Bengal in India.

	ACKNOWLEDGEMENTS

 
Financial assistance received from Department of Biotechnology, 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 and Mercy Rose Stella (JNTU) for SDS-PAGE analysis is gratefully acknowledged. Financial assistance received under the DST-DAAD exchange program (grant 422-PPP-34105) is acknowledged.

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