Abstract
Abbreviations: ITS, internally transcribed spacer; PHC, poly(hexamethylene carbonate)
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, ITS1, nifH and puf gene sequences determined in this study are AM501443–AM501446, AM501462, AM501479 and AM501480, and AM773545–AM773547, respectively.
Carbon source utilization data (Biolog), DNA–DNA hybridization values and a 16S rRNA gene sequence matrix are available as supplementary tables with the online version of this paper.
The genera Aquabacterium (Kalmbach et al., 1999), Ideonella (Malmqvist et al., 1994), Rubrivivax (Willems et al., 1991), Roseateles (Suyama et al., 1999), Azohydromonas (Xie & Yokota, 2005), Mitsuaria (Amakata et al., 2005) and Pelomonas (Xie & Yokota, 2005; Gomila et al., 2007) are phylogenetically closely related to the Sphaerotilus–Leptothrix group. These genera form the Rubrivivax line of descent, which is associated with the family Comamonadaceae (Wen et al., 1999). Willems & Gillis (2001) consider these genera to be of incertae sedis within the family Comamonadaceae. The relative branching order of most species within the Rubrivivax group is difficult to determine, mainly because of the limited number of variable positions available for estimating phylogenetic distance values (Spring, 2001).
Roseateles depolymerans 61AT (=CCUG 52219T=DSM 11813T), which is closely related to species of the genera Mitsuaria and Pelomonas, was isolated in 1998 from river water and described in 1999 as the first bacteriochlorophyll a-containing, obligate aerobe belonging to the Betaproteobacteria. The strain was isolated originally as a degrader of poly(hexamethylene carbonate) (PHC) and is considered to be an aerobic phototroph (Suyama et al., 1999). Since 2005, three novel strains (CCUG 48205T, CCUG 52220 and CCUG 52222T) have been isolated from different sources, including industrial water, freshwater and soil, and were identified provisionally as Roseateles depolymerans-like organisms. The present study was undertaken to characterize and determine the taxonomic positions of these organisms. The data demonstrated that strains CCUG 48205T and CCUG 52222T should be classified as representing two novel species, with the names Roseateles aquatilis sp. nov. and Roseateles terrae sp. nov., respectively.
The reference strains used in this study included Pelomonas saccharophila CCUG 32988T, Pelomonas aquatica CCUG 52575T, Pelomonas puraquae CCUG 52769T, Mitsuaria chitosanitabida IAM 14711T and Roseateles depolymerans CCUG 52219T, as well as Roseateles depolymerans strain CCUG 52220, Roseateles sp. strain CCUG 52222T and Roseateles depolymerans-like strain CCUG 48205T. All of the strains used in this study were cultivated in R2A medium (Reasoner & Geldreich, 1985), containing (l–1): 0.5 g yeast extract, 0.5 g acid hydrolysate of casein, 0.5 g glucose, 0.5 g starch, 0.3 g K2HPO4, 0.3 g sodium pyruvate, 0.25 g pancreatic digest of casein, 0.25 g peptic digest of animal tissue, 0.0492 g MgSO4 . 7H2O, supplemented with 1.5 % (w/v) agar (Scharlab) for solid medium. The bacteria were incubated for 3–4 days at 30 °C, if not stated otherwise.
Cell size, morphology and flagella insertion were determined using transmission electron microscopy of cells from the exponential growth phase in R2A broth. A Hitachi model H600 electron microscope was used at 75 kV. The samples were negatively stained with phosphotungstic acid (1 % w/v, pH 7.0), as described by Lalucat (1988).
The following phenotypic tests were carried out: API 20NE (identification system for Gram-negative, non-enterobacterial rods) and API 50 CH, according to the instructions of the manufacturer. Biolog tests were also carried out according to the instructions of the manufacturer. Briefly, the procedure was as follows: a homogeneous suspension of cells grown on plates of trypticase soy agar supplemented with sheep blood was made in the GN/GP inoculating fluid and 150 µl of the suspension was dispensed into each well of a GN microplate, which was then incubated for up to 96 h at 30 °C. The absorbance values were recorded periodically over a 4-day period, at 0, 4, 8, 24, 48, 72 and 96 h incubation time, using an automated Microplate reader. Absorbance was measured in dual wavelength mode, at 590 nm (as respiratory reduction will cause the tetrazolium dye to turn purple and absorb light at 590 nm) and at 750 nm as a second reference wavelength.
Conventional phenotypic tests were done according to the methods of Cowan (1974). Growth rates were determined at different temperatures (4, 10, 20, 30, 37 and 45 °C) in R2A medium, and aerobic autotrophic growth with hydrogen was tested in mineral medium, as described by Aragno & Schlegel (1992), at different O2 partial pressures. For the detection of photosynthetic pigments, cells were grown in R2A medium, PHC medium (or PHC medium supplemented with Tween 80) or in medium lacking organic carbon as described by Suyama et al. (1999, 2002). Lipophilic pigments were extracted from fresh wet cells cultured in the four media, under light or in the dark, using acetone [90 % (v/v) with 1 % MgCO3 at 4 °C for 24 h). Absorption spectra were recorded spectrophotometrically (Ultrospec III; Pharmacia LKB) between wavelengths of 360 and 900 nm.
Colonies of strains CCUG 52219T and CCUG 52220 grown on R2A medium for 5 days were 4–5 mm in diameter, transparent, darker in the centre, circular and flat, with an undulate margin, and smooth and translucent. Strain CCUG 52222T could be differentiated by means of a more mucous appearance of the colonies, which were 4–6 mm in diameter, less transparent, circular and convex, with an undulate transparent margin. Colonies of strain CCUG 48205T were 4–6 mm in diameter, clear white, circular and umbonate, with an undulate margin, and translucent. Old colonies showed concentric circles.
Cells were rod-shaped (2 µm in length), Gram-negative and motile, by means of a single polar flagellum. All strains grew at 20 and 30 °C. None of the strains were able to grow at 4 or 45 °C. The range of growth temperatures is indicated in Table 1. Differential phenotypic characteristics and substrates assimilated or utilized as sole carbon and energy sources are listed in Table 1. Carbon sources utilized in Biolog plates are given in Supplementary Table S1, available in IJSEM Online. Differential phenotypic characteristics obtained in the Biolog test are summarized in Table 2. None of the novel isolates were able to produce pigments when incubated for 1 week in the dark or under light in R2A medium or PHC medium, or in PHC medium supplemented with Tween 80, as well as in mineral medium.
Table 1. Differential phenotypic characteristics between species of the genera Mitsuaria, Pelomonas and Roseateles Strains: 1, Roseateles depolymerans CCUG 52219T; 2, Roseateles depolymerans CCUG 52220; 3, strain CCUG 52222T (Roseateles terrae sp. nov.); 4, strain CCUG 48205T (Roseateles aquatilis sp. nov.); 5, M. chitosanitabida IAM 14711T; 6, P. saccharophila CCUG 32988T; 7, P. puraquae CCUG 52769T; 8, P. aquatica CCUG 52575T. All strains are oxidase-positive. +, Positive; –, negative; W, weak; V, variable; ND, not determined.
Table 2. Differential carbon source utilization (Biolog) among the Roseateles strains studied Strains: 1, Roseateles depolymerans CCUG 52219T; 2, Roseateles depolymerans CCUG 52220; 3, strain CCUG 52222T (Roseateles terrae sp. nov.); 4, strain CCUG 48205T (Roseateles aquatilis sp. nov.). +, Positive; –, negative.
Gas chromatography of fatty acid methyl esters was performed at CCUG in a highly standardized way, similar to that of the MIDI Sherlock MIS system. Fatty acids were identified and quantified, and the relative amount of each fatty acid in a strain was expressed as a percentage of the total fatty acids in the profile of that strain. The major cellular fatty acids detected in all Roseateles strains analysed (CCUG 48205T, CCUG 52219T, CCUG 52220 and CCUG 52222T) were cis-9-hexadecanoic and hexadecanoic acids, as indicated in Table 3. Clear differences between Roseateles, Mitsuaria and Pelomonas could be established in the relative amounts of dodecanoic and cis-11-octodecanoic acids. The two novel species proposed in the genus Roseateles can be differentiated chemotaxonomically from Roseateles depolymerans by the relative amount of dodecanoic acid.
Table 3. Cellular fatty acid composition (%) of the bacteria studied and reference strains Strains: 1, P. saccharophila CCUG 32988T; 2, P. puraquae CCUG 52769T; 3, P. aquatica CCUG 52575T; 4, M. chitosanitabida IAM 14711T; 5, Roseateles depolymerans CCUG 52219T; 6, Roseateles depolymerans CCUG 52220; 7, strain CCUG 52222T (Roseateles terrae sp. nov.); 8, strain CCUG 48205T (Roseateles aquatilis sp. nov.). –, Below the limit of detection.
Genomic DNA was isolated according to the method of Marmur (1961). DNA–DNA hybridizations were performed, in duplicate, using a non-radioactive method as described by Ziemke et al. (1998). Reference DNAs of one strain of each group (P. saccharophila CCUG 32988T, P. puraquae CCUG 52769T, P. aquatica CCUG 52575T, M. chitosanitabida IAM 14711T, Roseateles depolymerans CCUG 52219T, Roseateles sp. strain CCUG 52222T and Roseateles depolymerans-like strain CCUG 48205T) were double-labelled with DIG-11-dUTP and biotin-16-dUTP using a Nick Translation kit (Roche). DNA–DNA hybridizations were performed using chromosomal DNA of each type strain as probes against the other strains (see Supplementary Table S2 in IJSEM Online). Strain CCUG 52220 showed 100 % DNA–DNA relatedness to the type strain of Roseateles depolymerans. The levels of DNA–DNA relatedness of strains CCUG 48205T and CCUG 52222T to the other strains were less than 57 %, and was 37.1 % between the two strains. This indicates that three different genomic groups can be delineated in the genus Roseateles.
The 16S rRNA, internally transcribed spacer 1 (ITS1; between the 16S and the 23S genes), nifH (nitrogenase) and Rubisco genes were amplified from genomic DNA by PCR, and sequenced as described previously (Gomila et al., 2007). Gene sequences were aligned with the homologous sequences of the closest relatives retrieved using the BLAST analysis tool and the NCBI nucleotide sequence database. The alignments were done using a hierarchical method for multiple alignments implemented in the program CLUSTAL_X (Thompson et al., 1997). Automatically aligned sequences were checked manually. Sequence similarities and evolutionary distances were calculated with programs implemented in PHYLIP (Phylogeny Inference Package, version 3.5c) (Felsenstein, 1989). The 16S rRNA gene sequence of strain CCUG 52220 was 99.7 % similar to that of Roseateles depolymerans CCUG 52219T (Fig. 1). Strain CCUG 52222T clustered in the same phylogenetic branch of the 16S rRNA gene sequence tree, with a sequence similarity of 99.6 % with that of the type strain, and was similar to the sequences of the species in the genus Pelomonas (between 97.2 and 97.9 %) and to the type strain of M. chitosanitabida (98.1 %). Strain CCUG 48205T also grouped in the Roseateles branch, with a similarity of 98.6 % to the 16S rRNA gene sequence of the type strain of Roseateles depolymerans (see Supplementary Table S3 in IJSEM Online). The type strain of Roseateles depolymerans, as well as strains CCUG 52220 and CCUG 52222T, showed two ITS1 amplicons of different sizes and were not sequenced. Rubisco genes cbbL of green or red form I enzymes and cbbM of the form II enzyme did not amplify in any of the strains. The nifH genes in strains CCUG 52219T, CCUG 52220 and CCUG 52222T were PCR-amplified and sequenced, and showed the same nucleotide sequence.
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Amplification of the partial puf (photosynthetic) gene operon was done for the four Roseateles strains using primers pufLF (5'-CTKTTCGACTTCTGGGTSGG-3') and pufMR (5'-CCCATSGTCCAGCGCCAGAA-3'). The following PCR conditions were employed: an initial cycle at 94 °C for 4 min, 35 cycles at 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1.5 min, and a final cycle at 72 °C for 10 min. A fragment of about 1.5 kb in size was amplified for strains CCUG 52219T, CCUG 52220 and CCUG 52222T. The PCR product was sequenced and compared with databases, confirming that photosynthetic genes are present in these strains. DNA of strain CCUG 48205T did not amplify. A Southern blot analysis was also performed with the four Roseateles strains by digestion of the genomic DNAs with the restriction enzymes ApaI and EcoRI (GE Health Care), followed by hybridization with the amplified puf gene fragment of strain CCUG 52219T as a probe. Strain CCUG 48205T did not hybridize with the probe, but the other three strains gave a positive signal (data not shown).
In addition to the phylogenetic, genomic and chemotaxonomic differences, strain CCUG 48205T could be differentiated from other Roseateles strains by its narrow range of temperatures for growth, by the assimilation of different carbon sources, catalase, β-glucosidase and β-galactosidase reactions and by the absence of nif and puf genes. Strain CCUG 52222T could be differentiated from Roseateles depolymerans by its profile of carbon sources assimilated and by the urease reaction.
Based on these results, we conclude that strain CCUG 52220 belongs to the species Roseateles depolymerans and that strains CCUG 48205T and CCUG 52222T represent two novel species, with the names Roseateles aquatilis sp. nov. and Roseateles terrae sp. nov., respectively.
Emended description of the genus Roseateles Suyama et al. 1999
Roseateles (Ro.se.a.te'les. L. adj. roseus rose-coloured, pink; Gr. adj. ateles defective, incomplete; N.L. masc. n. roseateles the rose-coloured incomplete photosynthetic bacterium).
Cells are motile, Gram-negative straight rods. Cells of some strains are pink in colour under conditions suitable for photosynthetic pigment production. Cis-9-hexadecanoic and hexadecanoic acids are the major cellular fatty acids. Low levels of dodecanoic acid and high levels of cis-11-octadecanoic acid differentiate the genus from other genera in the same phylogenetic branch. Other characteristics of the genus are given by Suyama et al. (1999).
Description of Roseateles aquatilis sp. nov.
Roseateles aquatilis (a.qua.ti'lis. L. masc. adj. aquatilis aquatic, growing in water).
Colonies are 4–6 mm in diameter, clear white, circular and umbonate, with an undulate margin and translucent. Four-day-old colonies show concentric circles. Non-pigmented. Oxidase-positive and catalase-negative. Growth occurs in R2A medium at 20–30 °C, but not at 10 or 45 °C. Cells are rod-shaped (2 µm in length and 0.5 µm in width) with single polar flagella. Nitrate reduction, indole formation, glucose fermentation, arginine dihydrolase, urease, β-galactosidase (PNPG) and aesculin tests are negative. Gelatin hydrolysis is positive. D-Glucose, maltose, gluconate and malic acid are assimilated. L-Arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, capric acid, adipic acid, citrate and phenylacetic acid are not assimilated. Photosynthetic genes (puf) are not detected.
The type strain, CCUG 48205T (=CECT 7248T), was isolated from industrial water in Sweden.
Description of Roseateles terrae sp. nov.
Roseateles terrae (ter'rae. L. gen. n. terrae of the soil).
Colonies are circular, 4–6 mm in diameter and convex, with an undulate transparent margin. Non-pigmented. Oxidase- and catalase-positive. Growth occurs in R2A medium at 10–37 °C, but not at 4 or 45 °C. Cells are rod-shaped (2 µm in length and 0.5 µm in width) with single polar flagella. Nitrate reduction, indole formation, glucose fermentation, arginine dihydrolase and urease tests are negative. β-Galactosidase (PNPG), aesculin and gelatin hydrolysis are positive. D-Glucose, L-arabinose, D-mannose, D-mannitol, maltose, gluconate and capric acid are assimilated. Assimilation of malic acid is extremely weak. N-Acetyl-D-glucosamine, adipic acid, citrate and phenylacetic acid are not assimilated. Photosynthetic genes (puf) are detected, but are not expressed under the experimental conditions used.
The type strain, CCUG 52222T (=CECT 7247T), was isolated from soil in Japan.
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