Abstract
Abbreviations: BChl a, bacteriochlorophyll a; FT-IR, fourier-transform infrared
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JA2T is AJ888903.
Micrographs of cells of strain JA2T and figures showing whole-cell absorption and FT-IR spectra and a graph showing growth of and utilization of benzoate by strain JA2T are available as supplementary material in IJSEM Online.
A small number of low molecular mass aromatic hydrocarbons support the growth of certain purple non-sulfur bacteria (Sasikala & Ramana, 1998) as carbon sources and/or electron donors. Frank & Gaffron (1941) first reported aromatic metabolism in an anoxygenic phototrophic bacterium, Rhodovibrio parvus (now placed in Rhodopseudomonas as Rhodopseudomonas palustris), in which phenylpropionate was transformed to benzoate during phototrophic metabolism. Later, Scher and co-workers (Scher & Allen, 1960; Scher & Proctor, 1960) demonstrated growth of Rhodopseudomonas palustris at the expense of benzoate. The discovery of benzoate utilization by Rhodospirillum fulvum (Pfennig et al., 1965) [renamed as Phaeospirillum fulvum (Imhoff et al., 1998)] led to the investigation of aromatic hydrocarbon degrading capability among other purple non-sulfur bacteria. Rhodopseudomonas palustris (Dutton & Evans, 1967; Harwood & Gibson, 1988), Phaeospirillum fulvum (Pfennig et al., 1965), Rhodopseudomonas acidophila (Yamanaka et al., 1983) [renamed as Rhodoblastus acidophilus (Imhoff, 2001)], Rhodomicrobium vannielii (Wright & Madigan, 1991), Rhodobacter capsulatus (Blasco & Castillo, 1992; Madigan et al., 2001), Rhodobacter sphaeroides (Rajasekhar et al., 2000), Blastochloris sulfoviridis (Zengler et al., 1999) and the bacteriochlorophyll-containing aerobic phototrophic bacterium Porphyrobacter sanguineus (Hiraishi et al., 2002) are phototrophic alphaproteobacteria that are capable of aromatic hydrocarbon metabolism. Phototrophic betaproteobacteria that utilize benzoate include Rhodocyclus purpureus (Pfennig, 1978) and Rhodoferax ferrireducens (Finneran et al., 2003). Although Rhodocyclus gelatinosus [now Rubrivivax gelatinosus (Willems et al., 1991; Imhoff, 1995)] utilizes phloroglucinol for growth and photobiotransforms some other aromatic compounds (Sasikala & Ramana, 1998), to date it is not known whether it can utilize benzoate as a carbon source or electron donor (Whittle et al., 1976). In this paper a new isolate (strain JA2T) is described, which was obtained through phototrophic enrichments on benzoate. Strain JA2T clusters with members of the genus Rubrivivax of the Betaproteobacteria and metabolizes a wide range of aromatic hydrocarbons. Based on phenotypic characteristics and molecular studies, strain JA2T represents a novel species of the genus Rubrivivax, for which the name of Rubrivivax benzoatilyticus sp. nov. is proposed.
Strain JA2T was isolated from the rhizosphere of a flooded paddy field near Eluru, Andhra Pradesh, India. For isolation and cultivation of the isolate, Pfennig's mineral salts medium (Biebl & Pfennig, 1981) supplemented with benzoate (3 mM) as a carbon source and ammonium chloride (0.12 %) as a nitrogen source was used, with incubation under light (2400 lx) at 30±2 °C. Purification of the isolate was achieved by repeated streaking on agar slants that were sealed with suba seals and flushed with argon and incubated phototrophically (2400 lx) at 30±2 °C. For comparative analysis, Rubrivivax gelatinosus ATCC 17011T was used under identical culture conditions. No distinct variation was observed in the colony morphologies of strain JA2T and Rubrivivax gelatinosus ATCC 17011T. Colonies of strain JA2T grown photoheterotrophically were round, convex, smooth and dark-orangebrown in colour and surrounded by a thin opaque margin. Microscopical observations, such as cell shape, size, cell division and flagella, were performed using a phase-contrast microscope (Olympus-B201) and the internal membrane structures were viewed with a transmission electron microscope after the cells were processed as described by Hanada et al. (2002). Polyhydroxyalkanoates of a culture of strain JA2T grown for 48 h on Pfennig's medium supplemented with 0.3 % (w/v) pyruvate were stained with Sudan black B (2 % in ethanol) (Smibert & Krieg, 1981) or Nile blue (Ostle & Holt, 1982).
Substrate utilization of carbon sources/electron donors was done in Pfennig's mineral salts medium by replacing benzoate with the test aliphatic substrate at a final concentration of 0.3 % (w/v), in a completely filled screw-cap test-tube (10x100 mm). For utilization of aromatic hydrocarbons as carbon sources/electron donors, the test substrates were used at a concentration of 3 mM, with sodium bicarbonate (0.1 %, w/v) in the medium. A culture grown in the absence of any added carbon source was used as the inoculum. Aromatic hydrocarbons such as cyclohexanol, cyclohexanone, catechol and resorcinol were added after membrane filtration to the autoclaved basal medium containing the nitrogen source. Filter-sterilized bicarbonate was added to the medium after autoclaving and the pH of the medium was adjusted with sterile 1 M NaOH or 1 M HCl to pH 6.8. Nitrogen source utilization was tested by replacing ammonium chloride with various nitrogen sources (0.12 %, w/v). Diazotrophy was tested by growing the cells under a N2 atmosphere and also by using assays for acetylene reduction activity (Sasikala et al., 1990). Dry weight was determined from an OD660 versus dry weight graph, ranging from 0.1 to1.0 OD660 prepared for strain JA2T (OD660 of 0.1 was equal to 0.3 mg dry weight per ml). In vivo absorption spectra of cells collected from photoheterotrophic (malate; 0.3 %, w/v) cultures grown for 30 h 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 elution of the cell suspension with acetone through a 10x200 mm column packed with aluminium oxide. Whole cellular components (fatty acids, intracellular and membrane proteins, polysaccharides, photosynthetic pigments and nucleic acids) of strain JA2T were distinguished from Rubrivivax gelatinosus ATCC 17011T based on information obtained from fourier-transform infrared (FT-IR) spectroscopy. For FT-IR spectroscopy, 23 ml exponentially grown culture was lyophilized and processed with KBr to make a pellet. Spectral characteristics were recorded in the transmission mode between wave numbers 4000 and 400 cm1 at a resolution of 4 cm1 and 20 kHz scan speed in a Perkin Elmer FT-IR spectrophotometer (model 128).
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). Cell material for 16S rRNA gene sequencing was taken from 12 ml culture. PCR and 16S rRNA gene sequencing were performed as described previously (Shivaji et al., 2000). Sequences were aligned using the CLUSTAL W program (Thompson et al., 1994). 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). Maximum-likelihood phylogenetic trees were established using the DNAML module of the PHYLIP 3.6 program package. Bootstrap analysis was performed. DNADNA hybridization analysis was performed at the DSMZ (Germany).
Uprooted paddy at booting stage together with the soil was collected in a polyethylene bag and the rhizosphere soil was used after 3 days for phototrophic enrichment on benzoate. Six colonies obtained during purification were designated strains JA2T, JA4, JA5, JA6, JA7 and JA8. Colony morphologies of strains JA2T, JA4 and JA6 were identical: colonies were round, dark-orangebrown in colour with a thin opaque margin and grew on benzoate as a sole source of carbon/electron donor. Strain JA2T was characterized further using a polyphasic approach.
Cells of strain JA2T stained Gram-negative. They were rod-shaped (see Supplementary Fig. S1 in IJSEM Online), 0.71.0 µm in width and 2.06.0 µm in length, motile by a single polar flagellum and formed a rosette-like arrangement. Electron micrographs of ultrathin sections of the cells revealed the absence of internal membrane structures in strain JA2T (see Supplementary Fig. S2 in IJSEM Online). Stationary-phase cells of strain JA2T contained refractive granules. Sudan black and Nile blue staining indicated the presence of polyhydroxyalkanoates. On average, the cells contained 1530 polyhydroxyalkanoate granules per cell, as observed in ultrathin sections (Fig. 1). In addition, growth of the granules at various stages could be observed (using transmission electron microscopy) and, sometimes, almost the whole cell was filled with a single large granule. Eventually, the granules even burst open the cell and were released as small ovoid granules (Fig. 1). The polyhydroxyalkanoate content of the cells reached up to 8085 % of the total cell dry mass. The photosynthetically grown cell suspensions were orangebrown in colour. Whole-cell absorption spectra of strain JA2T had absorption maxima at 378, 488, 520, 590, 802 and 884 nm, confirming the presence of bacteriochlorophyll a (BChl a) (Supplementary Fig. S3 in IJSEM Online) and most probably the carotenoids spirilloxanthin and sphaeroidene.
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Strain JA2T grew photolithoautotrophically [anaerobically in the light (2400 lx), with H2 (20 %, v/v)/thiosulfate (0.1 %, w/v) and NaHCO3 (0.1 %, w/v)], photo-organoheterotrophically [anaerobically in the light (2400 lx) with malate (0.3 %, w/v)] and chemo-organoheterotrophically [microaerobically in the dark with malate (0.3 %, w/v)]. Fermentative growth [anaerobically in the dark with glucose/fructose (0.3 %, w/v)] and chemolithoautotrophy [microaerobically in the dark with thiosulfate (0.1 %, w/v) and NaHCO3 (0.1 % w/v)] could not be demonstrated. The substrates that were utilized as carbon sources and electron donors under photo-organoheterotrophic conditions are given in the species description.
Ammonium chloride and molecular nitrogen were the nitrogen sources utilized by strain JA2T; urea, nitrate or nitrite did not support growth. NaCl was not obligatory for growth of strain JA2T, but up to 5 % (v/v) was tolerated. In addition, the strain did not require any growth factors (vitamins) and there was no enhancement in the biomass yield in their presence. Production of indole from tryptone was not observed; however, replacement of tryptone with tryptophan resulted in indole production. Gelatin liquefaction was observed. Using FT-IR spectroscopic data (Supplementary Fig. S4 in IJSEM Online), strain JA2T showed visible spectral differences from Rubrivivax gelatinosus ATCC 17011T, indicating variations in the composition of aromatic compounds (at 980 cm1), polysaccharides (13001100 cm1) and esters (1726 cm1).
Aromatic hydrocarbons that supported photo-organoheterotrophic growth of strain JA2T included benzoate, 2-aminobenzoate (anthranilate), 4-aminobenzoate, 4-hydroxybenzoate, 2-carboxybenzoate (phthalate), phenylalanine, trans-cinnamate, benzamide, salicylate (2-hydroxybenzoate), cyclohexanone, cyclohexanol and cyclohexane-2-carboxylate (Table 1). Growth and simultaneous utilization of benzoate is shown in Supplementary Fig. S5 in IJSEM Online. The doubling time of strain JA2T was about 35 h on benzoate, compared with 8 h on malate.
Table 1. Growth and biomass yield of strain JA2T on various aromatic hydrocarbons as carbon source/electron donors Values shown are means of experiments performed in duplicate after 7 days of light (2400 lx) anaerobic incubation at 30±2 °C. Phototrophic benzoate-grown cultures were used as the initial inoculum (5 %, v/v). The compounds were utilized at a concentration of 3 mM, in the presence of (0.1 %, w/v). OD660 values: +++, >0.50; ++, 0.30.49; +, 0.210.29; GI, <0.2 (growth inhibition). NA, Not applicable.
The G+C content of strain JA2T was 74.9 mol% (by HPLC). The 16S rRNA gene sequence of strain JA2T formed a cluster with members of the genus Rubrivivax and showed 99 % sequence similarity with Rubrivivax gelatinosus DSM 1709T (=ATCC 17011T). However, DNADNA hybridization of strain JA2T with Rubrivivax gelatinosus ATCC 17011T revealed a relatedness value of only 54.8 %.
The genus Rhodocyclus with three species, Rhodocyclus purpureus, Rhodocyclus tenuis and Rhodocyclus gelatinosus, was described in the first edition of Bergey's Manual of Systematic Bacteriology (Imhoff & Trüper, 1989). Based on 16S rRNA gene sequence analysis, even though all three species were grouped in the class Betaproteobacteria (Stackebrandt et al., 1988), significant phylogenetic differences separated Rhodocyclus gelatinosus from the other two species, thus necessitating the description of a new genus, Rubrivivax, with Rubrivivax gelatinosus as the type species (Willems et al., 1991). Rubrivivax gelatinosus is phylogenetically related to members (genera Tepidimonas, Aquabacterium, Ideonella, Leptothrix, Roseateles, Sphaerotilus, Thiomonas, Xylophilus) of the family incertae sedis (Cole et al., 2003) [Rubrivivax gelatinosus was previously included in the family Comamonadaceae (Willems et al., 1991; Wen et al., 1999)], separating it from the genera Rhodoferax and Rhodocyclus. The genus Rhodoferax, a later addition to the family Comamonadaceae, to date includes two phototrophic species, Rhodoferax fermentans (Hiraishi et al., 1991) and Rhodoferax antarcticus (Madigan et al., 2000), and the non-pigmented Rhodoferax ferrireducens (Finneran et al., 2003). Pairwise sequence comparisons and distance matrix tree analysis showed that the genera Rhodoferax, Rubrivivax and Rhodocyclus formed three separate lineages within the Betaproteobacteria (Hiraishi, 1994). Based on the 16S rRNA gene sequence analysis, strain JA2T clustered closely with members of the family incertae sedis within the genus Rubrivivax (Fig. 2). However, the morphological, physiological and biochemical traits (Table 2) and DNADNA hybridization studies significantly differentiated this strain from the only reported species of Rubrivivax, Rubrivivax gelatinosus, and therefore strain JA2T is described here as a novel species, Rubrivivax benzoatilyticus. FT-IR spectroscopic data, which are considered to be a connecting link between genomic and phenotypic approaches (Amiel et al., 2001), have shown differences between strain JA2T and Rubrivivax gelatinosus ATCC 17011T in a few major cellular components, viz., aromatic compounds, polysaccharides and esters (see Supplementary Fig. S4 in IJSEM Online).
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Table 2. Characteristics that differentiate strain JA2T from related freshwater purple betaproteobacteria Strains: 1, Strain JA2T; 2, Rubrivivax gelatinosus ATCC 17011T; 3, Rhodoferax fermentans JCM 7819T; 4, Rhodocyclus purpureus DSM 168T; 5, Rhodocyclus tenuis DSM 109T. All strains are chemo-organotrophic and utilize acetate, butyrate, succinate and ethanol. +, Positive/substrate utilized; , negative/not utilized; NT, not tested.
Curved cells commonly observed with Rubrivivax gelatinosus (Willems et al., 1991) were not observed with strain JA2T, the cells of which were always straight rods. One significant difference between strain JA2T and the other Rubrivivax species is the absence of internal membrane structures, which form tubular intrusions in Rubrivivax gelatinosus (Willems et al., 1991). In terms of the lack of internal membrane structures, strain JA2T more probably resembles Rhodoferax species (Hiraishi et al., 1991). Preliminary analysis of the carotenoid content of strain JA2T indicated that it probably has both sphaeroidene and spirilloxanthin series of carotenoids, again similar to Rubrivivax gelatinosus. This is in contrast to Rhodobacter capsulatus and Rhodobacter sphaeroides, which only have carotenoids of the sphaeroidene series (Pinta et al., 2003; Harada et al., 2001; Steiger et al., 2000). The other major difference between strain JA2T and many other phototrophic members of the Alphaproteobacteria and Betaproteobacteria is the lack of any vitamin requirement for growth, which is rather rare in this group (Imhoff, 1995). Rhodocyclus tenuis and Roseateles depolymerans (an aerobic BChl a-containing bacterium; Suyama et al., 1999) are the only other species of phototrophic Betaproteobacteria that do not have a requirement for vitamins.
Strain JA2T produced large amounts (up to 85 % of the cell dry weight) of polyhydroxyalkanoates. A similar yield of polyhydroxyalkanoate from Ralstonia (Alcaligenes) eutropha has been reported, with accumulation of up to 96 % of the cellular dry weight (Pedrosalio et al., 1985). This is exploited commercially for biopolyester production (Sasikala & Ramana, 1996).
Strain JA2T did not produce indole directly from tryptone; however, indole production from tryptophan was observed. This suggests that strain JA2T is unable to hydrolyse tryptone, in contrast to the extensive proteolytic capabilities of Rubrivivax gelatinosus (Tanskul et al., 2003). Therefore we recommend the use of tryptophan for the indole production (tryptophanase) test rather than tryptone. Production of indole from tryptophan, catalysed by the enzyme tryptophanase, is a common diagnostic marker for differentiating members of Enterobacteriaceae, including Escherichia coli, Proteus vulgaris, Providencia spp. and Morganella spp. (Sonnenwirth, 1980), which belong to the Gammaproteobacteria.
Anoxygenic phototrophic bacteria capable of photo-organotrophic utilization of aromatic hydrocarbons mainly belong to the Alphaproteobacteria and among the photosynthetic Betaproteobacteria, Rhodocyclus purpureus and strain JA2T are capable of benzoate utilization. In addition, anaerobic aromatic hydrocarbon degradation among the Alphaproteobacteria is restricted to the photosynthetic members, whereas it is common among members of the Betaproteobacteria, including species of Thauera and Azoarcus (Heider & Fuchs, 1997). Strain JA2T degraded a variety of aromatic hydrocarbons (Table 1), adding to the aromatic hydrocarbon degrading diversity of phototrophic bacteria (Sasikala & Ramana, 1998).
Description of Rubrivivax benzoatilyticus sp. nov.
Rubrivivax benzoatilyticus (ben.zo'at.i.ly'ti.cus. N.L. n. benzoas -atis benzoate; Gr. adj. lutikos able to loose, able to dissolve; N.L. adj. lyticus dissolving; N.L. masc. adj. benzoatilyticus dissolving benzoate, named after its utilization of benzoate).
Cells are rod-shaped, 0.71.0 µm wide and 2.06.0 µm long. Motile by means of a single polar flagellum. Colour of the cell suspension is orangebrown and cells multiply by binary fission. Colony morphology on agar slants is round, convex and smooth, with dark orangebrown colour surrounded by thin opaque margin. Internal membranes are absent. Intact cells have absorption maxima at 378, 488, 520, 590, 802 and 884 nm. Photosynthetic pigments are BChl a and probably carotenoids of the alternative spirilloxanthin series with both sphaeroidene and spirilloxanthin. Growth modes are photo- and chemo-organoheterotrophy and photolithoautotrophy. Organic substrates photometabolized include formate, acetate, propionate, butyrate, valerate, succinate, malate, fumarate, oxaloacetate, 2-oxoglutarate, pyruvate, glucose, fructose, tartarate, glutamate, glycerol, ethanol and benzoate. Does not metabolize lactate, citrate, mannitol, methanol or sorbitol. Inorganic electronic donors such as molecular hydrogen and thiosulfate support growth. NaCl and vitamins are not required for growth. The DNA G+C content of the type strain is 74.9 mol% (HPLC).
The type strain is JA2T (=ATCC BAA-35T=JCM 13220T=MTCC 7087T). Habitat is rhizosphere of flooded paddy.
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