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1 Área Microbiologia, Departament de Biologia, Universitat de les Illes Balears, and Institut Mediterrani d'Estudis Avançats (CSIC-UIB), 07122 Palma de Mallorca, Illes Balears, Spain
2 Abt. Molekularphysiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, 37077 Göttingen, Germany
3 CCUG, Culture Collection University of Göteborg, Department of Clinical Bacteriology, 41346 Göteborg, Sweden
Correspondence
Jorge Lalucat
jlalucat{at}uib.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the novel strains are: CCUG 52575T (AM51435 and AM501454, for 16S rRNA gene and ITS1, respectively), and for strain CCUG 52769T [AM501439, AM501458, AM501467 and AM501476, for 16S rRNA gene, ITS1, nifH and red-like form I Rubisco genes (cbbL), respectively].
Supplementary data are available with the online version of this paper.
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) and this single strain was subsequently intensively investigated for its carbohydrate metabolism (Doudoroff et al., 1956). Much later Palleroni (1980) described a hydrogen-oxidizing bacterium, Pseudomonas sp. DSM 2583, related to Pseudomonas saccharophila. This new organism shared the basic physiological properties with Pseudomonas saccharophila, but could be easily differentiated by substrate assimilation tests and by DNA–DNA hybridizations. However, it was not proposed as a novel species until other strains of the group were isolated from nature. The nitrogen-fixing ability of the type strain of Pseudomonas saccharophila was later demonstrated by Barraquio et al. (1986). Other strains ascribed to Pelomonas saccharophila have been studied as degraders of aromatic compounds (Stringfellow & Aitken, 1995) and contaminants of pure and ultrapure waters (Kulakov et al., 2002; Gomila et al., 2005). Considering only the type strain, Xie & Yokota (2005) reclassified the type strain of Pseudomonas saccharophila into a new genus, Pelomonas.
In a previous study, haemodialysis water was analysed to monitor its microbiological quality and to identify the bacteria present in this oligotrophic environment (Gomila et al., 2005, 2006). During this study, a group of three strains forming clear brown colonies on R2A agar was isolated from two different samples taken at the same point. The strains were identified by 16S rRNA gene sequences members of the class Betaproteobacteria, closely related to Pelomonas saccharophila (formerly Pseudomonas saccharophila). The present study was undertaken to determine the taxonomic position of nine different strains. Strains CCUG 52769T, CCUG 52770 (=MG64) and CCUG 52771 (=MG63) were isolated on R2A medium (Reasoner & Geldreich, 1985) from haemodialysis water at the Hospital Son Llàtzer (Mallorca, Spain). Five strains deposited at the CCUG as members of the genus Pelomonas were isolated from industrial water in Sweden (CCUG 52428, CCUG 52507, CCUG 52575T, CCUG 52590 and CCUG 52631). The following previously described species were also used in this study: five cultures of the type strain of Pelomonas saccharophila were obtained from the corresponding culture collections (ATCC 15946T, CCUG 32988T, DSM 654T, IAM 14368T and LMG 2256T); strain DSM 2583 (=CCUG 52768), described by Palleroni (1980); the type strain of Mitsuaria chitosanitabida IAM 14711T (=CCUG 52767T); and the type strain of Roseateles depolymerans CCUG 52219T (=DSM 11813T). All strains were cultured in R2A medium 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 and 0.0492 g MgSO4 . 7H2O, supplemented with 1.5 % (w/v) agar for the plates. Bacteria were incubated for 3–4 days at 30 °C unless otherwise stated. Colonies of the haemodialysis water isolates and Pseudomonas sp. DSM 2583 grown on R2A agar for 5 days were 4–5 mm in diameter, pale brown being darker in the centre, circular, raised with entire margin, smooth and translucent. They could easily be removed from the agar. The industrial water isolates formed colonies of a smaller size (1–2 mm) and a different colour (homogeneous yellowish) being darker and opaque, and were more difficult to recultivate after 1 week of incubation, similar to colonies of the Pelomonas saccharophila type strain.
Cell size, morphology and flagella insertion were determined by 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 %, pH 7.0) as described by Lalucat (1988). Cells were rod-shaped (2 µm in length), Gram-negative, and motile by means of a single polarly inserted flagellum (Fig. 1).
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. Growth rate was determined at different temperatures in R2A medium (4, 10, 20, 30, 37 and 45 °C) and aerobic autotrophic growth with hydrogen was tested in mineral medium as described by Aragno & Schlegel (1992) at different O2 partial pressures. All strains grew at 20 and 30 °C. The range of temperatures for heterotrophic growth is indicated in Table 1. The type strain of Pelomonas saccharophila and Pseudomonas sp. DSM 2583 grew autotrophically with hydrogen at 30 °C in mineral medium under an atmosphere of H2/O2/CO2 (80 : 10 : 10, 85 : 5:10 or 88 : 2:10; % by vol.). Strains CCUG 52769T, CCUG 52770 and CCUG 52771 also grew autotrophically with hydrogen but slowly (10 days). None of the five industrial water isolates was able to grow under these conditions, nor were M. chitosanitabida IAM 14711T and R. depolymerans CCUG 52219T. Carbon sources utilized are indicated in the formal description of each novel species. Differential phenotypical characteristics are indicated in Table 1. Several organic substrates were only utilized after prolonged incubation by a small number of cells streaked on agar, probably involving mutational events. Some traits of the original description of Pelomonas saccharophila do not correspond to our observations with strains CCUG 32988T and LMG 2256T. In our hands, Pelomonas saccharophila did not grow at 37 °C and was unable to hydrolyse gelatin or to grow on citrate. Also differences between both strains were detected in the assimilation of D-mannose and malic acid, indicated as +/– in Table 1.
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). The primers 16F945 (5'-GGGCCCGCACAAGCGGTGG-3') and 23R458 (5'-CTTTCCCTCACGGTAC-3') were used for the amplification of ITS1 (Guasp et al., 2000). A fragment of approximately 388 bp of the nifH gene (encoding the dinitrogenase reductase iron protein) was also amplified using primers 19F (5'-GCIWTYTAYGGIAARGGIGG-3') and 407R (5'-AAICCRCCRCAIACIACRTC-3'), where I represents inosine, R represents A or G, W represents A or T and Y represents C or T. The reaction conditions were an initial step of 5 min at 94 °C, followed by 30 s at 94 °C, 1 min at 50 °C and 30 s at 72 °C for 40 cycles, and a final step of 72 °C for 10 min (Ueda et al., 1995). For the amplification of Rubisco genes, three primer sets specific for the form I enzyme (green and red, cbbL) and the form II enzyme were employed (Giri et al., 2004; Selesi et al., 2005). Red-like cbbL genes were amplified with primer pair cbbLR1F (5'-AAGGAYGACGAGAACATC-3') and cbbLR1R (5'-TCGGTCGGSGTGTAGTTGAA-3'). Amplification of green-like cbbL gene primers cbbLG1F (5'-GGCAACGTGTTCGGSTTCAA-3') and cbbLG1R (5'-TTGATCTCTTT CCACGTTTCC-3') were used, where S represents G or C. The cycle conditions for cbbL-specific primers were as follows: initial denaturation at 95 °C for 4 min, followed by 32 cycles of 1 min at 95 °C for denaturation, 1 min of annealing at 62 °C with the red-like and at 60 °C with the green-like cbbL primers, 1 min of elongation at 72 °C, and a final extension step of 10 min at 72 °C. Form II Rubisco genes were amplified with the primer pair cbbMF (5'-ATCATCAARCCSAARCTSGGCCTGCGTCCC-3') and cbbMR (5'-MGAGGTGACSGCRCCGTGRCCRGCMCGRTG-3'), where M represents A or C. The PCR conditions were modified to an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 1 min at 94 °C, 1 min at 65 °C and 1 min at 72 °C, and a final extension at 72 °C for 10 min. Primers hoxF1 (5'-GAYCCNRTNACNMGNATHGARGGNCA-3') and hoxR1 (5'-ACRTGNRYBSVRCANSCVRDVMANGGRTC-3') for the amplification of the hydrogenase gene hoxG were designed from the alignment of different hoxG sequences retrieved from databases, where H represents A, C or T, B is G, T or C, V is G, C or A, D is G, T or A, and N represents G, A, T or C. The touchdown PCR programme used was an initial denaturation step of 5 min at 94 °C followed by 5 cycles of 94 °C for 1 min, 65 °C for 1 min and 72 °C for 105 s, then 20 cycles of 94 °C for 1 min, 65 °C for 1 min (decreasing 0.5 °C per cycle) and 72 °C for 105 s; and 15 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C 105 s; and finally extension at 72 °C for 10 min. PCR amplification was performed with the Personal DNA Thermocycler (Eppendorf). Individual reaction mixtures contained 10 mM Tris/HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 200 µM each of the four deoxynucleoside triphosphates (Amersham Biosciences), 0.5 µl of both forward and reverse primer solutions (10 µM), 1.5 U Taq DNA polymerase (Amersham Biosciences), and 1–3 µl template DNA in a total volume of 50 µl. For the Rubisco, nifH and hoxG genes, the amount of primers was higher: 0.5 µl of both forward and reverse primer solutions (100 µM). A 5 µl sample of the amplified products was analysed by electrophoresis in a 1 % (w/v) agarose gel for 16S rRNA products and in a 2 % (w/v) agarose gel for the other products, and stained with ethidium bromide. For sequencing reactions, the same primers and conditions were used. The amplified products were purified with Microcon centrifugal filter devices (Amicon; Millipore) according to the manufacturer's instructions. Sequencing reactions were carried out using the ABI Prism Big Dye version 3.1 Terminator Cycle sequencing kit and the sequences were read with an automatic sequence analyser (ABI Prism 3730 DNA Sequencer; Applied Biosystems). The 16S rRNA, ITS1, nifH and Rubisco gene sequences were aligned with the closest relatives retrieved from the BLAST nucleotide sequence database. The alignment was done using a hierarchical method for multiple alignments implemented in the CLUSTAL_X program (Thompson et al., 1997). Automatically aligned sequences were checked manually. Similarities and evolutionary distances were calculated with programs contained in PHYLIP (Phylogenetic Inference Package, version 3.5c) (Felsenstein, 1989). Gene distances were calculated from nucleotide sequences by the Jukes–Cantor method (Jukes & Cantor, 1969) and dendrograms were generated by the neighbour-joining, minimum-evolution and maximum-parsimony methods. Bootstrap analysis of 1000 replications was also performed. Topologies of the trees were visualized with the TreeView program (Page, 1996). The dendrograms showed the same topology and only the neighbour-joining tree is given in Fig. 2.
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Gas chromatography of fatty acid methyl esters (FAME) was performed at CCUG in a highly standardized way close 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 were cis-9-hexadecanoic and hexadecanoic acids (Table 2). Clear differences to M. chitosanitabida IAM 14711T and R. depolymerans CCUG 52219T were apparent in the relative amounts of dodecanoic and cis-11-octadecanoic acids. The strains grouped in the three phylogenetic branches according to the 16S rRNA, ITS1, Rubisco and nifH genes could be distinguished chemotaxonomically based on the amounts of 2-hydroxy-dodecanoic and octadecanoic acid.
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. 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 (DSM 654T, CCUG 52769T, CCUG 52219T, IAM 14711T and CCUG 52575T) 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 a probe against the other strains (Supplementary Table S2 available in IJSEM Online). Similarity values between strains of the same phylogenetic branch were higher than 85.0 % and lower than 70.0 % between members of two separate branches, when the mean values were considered. This indicates that three different genomic groups, coinciding with the phylogenetic groups, can be delineated within the genus Pelomonas. Based on the genetic and phenotypic analyses, we conclude that strains CCUG 52769T, CCUG 52770 and CCUG 52771, together with DSM 2583, constitute a novel species and that strains CCUG 52428, CCUG 52507, CCUG 52575T, CCUG 52590 and CCUG 52631 represent another species of the genus Pelomonas. For the first group, the name Pelomonas puraquae, sp. nov. is proposed. The type strain of Pelomonas puraquae is CCUG 52769T. For the second group, the name Pelomonas aquatica sp. nov. is proposed. The type strain of Pelomonas aquatica is CCUG 52575T.
Description of Pelomonas aquatica sp. nov.
Pelomonas aquatica (a.qua'ti.ca. L. fem. adj. aquatica living in water).
The characteristics are the same as those given in the description of the genus, with the following additional traits. Colonies are small (1–2 mm), yellowish, dark and opaque and difficult to recultivate after 1 week of incubation. Cells are rod-shaped (2 µm in length and 0.5 µm in width). Unable to grow autotrophically with hydrogen. Growth occurs at 20–37 °C, with an optimal temperature at about 30 °C; unable to grow at 10 and 45 °C. Positive for nitrate reduction and 
-glucosidase, and negative for urease, gelatin hydrolysis, -galactosidase and glucose fermentation. Unable to grow without combined nitrogen. Unable to grow in D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, gluconate, capric acid, adipic acid, malic acid, citrate and phenylacetate. Able to utilize: 
-cyclodextrin, D-fructose, dextrin, uridine, glycogen, thymidine, Tween 40, DL-lactic acid, L-proline, trehalose, L-asparagine, glycerol, L-glutamic acid, DL--glycerol phosphate, pyruvic acid methyl ester and -hydroxybutyric acid.
The type strain, CCUG 52575T (=CECT 7233T), was isolated from industrial water in Sweden.
Description of Pelomonas puraquae sp. nov.
Pelomonas puraquae (pur.a.quae. L. adj. purus -a -um pure; L. n. aqua water; N.L. gen. n. puraquae of pure water).
The characteristics are the same as those given in the description of the genus, with the following additional traits. Colonies are 4–5 mm in diameter, pale brown being darker in the centre, circular, raised with entire margin, smooth and translucent. Can easily be removed from the agar. Cells are rod-shaped (2 µm in length and 0.5 µm in width). Able to grow autotrophically with hydrogen. Growth occurs at 10–37 °C, with an optimal temperature about 37 °C; unable to grow at 4 and 45 °C. Variable for nitrate reduction and positive for -glucosidase, -galactosidase and gelatin hydrolysis, negative for urease and glucose fermentation. Able to grow in L-arabinose, D-glucose, maltose, sucrose, D-cellobiose, acetate, -ketoglutarate, fumarate, glutamate, lactate, malic acid, pyruvate, L-arginine, L-asparagine, L-histidine, L-serine and L-valine. Variable growth for citrate and succinate. Negative for D-mannose, D-mannitol, N-acetyl-D-glucosamine, gluconate, capric acid, adipic acid and phenylacetate. Able to utilize Tweens 40 and 80, pyruvic acid methyl ester, -hydroxybutyric acid and glycyl L-glutamic acid.
The type strain, CCUG 52769T (=CECT 7234T), was isolated from haemodialysis water in Mallorca, Spain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Barraquio, W. L., Padre, B. C., Jr, Watanabe, I. & Knowles, R. (1986). Nitrogen fixation by Pseudomonas saccharophila Doudoroff ATCC 15946. J Gen Microbiol 132, 237–241.
Cowan, S. T. (1974). Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge University Press.
Doudoroff, M. (1940). The oxidative assimilation of sugars and related substances by Pseudomonas saccharophila with a contribution to the problem of the direct respiration of di- and polysaccharides. Enzymologia 9, 59–72.
Doudoroff, M., Palleroni, N. J., McGee, J. & Ohara, M. (1956). Metabolism of carbohydrates by Pseudomonas saccharophila. J Bacteriol 71, 196–201.
Felsenstein, J. (1989). PHYLIP – phylogeny inference package (version 3.2). Cladistics 5, 164–166.
Giri, B. J., Bano, N. & Hollibaugh, J. T. (2004). Distribution of RuBisCO genotypes along a redox gradient in Mono Lake, California. Appl Environ Microbiol 70, 3443–3448.
Gomila, M., Gascó, J., Busquets, A., Gil, J., Bernabeu, R., Buades, J. M. & Lalucat, J. (2005). Identification of culturable bacteria present in haemodialysis water and fluid. FEMS Microbiol Ecol 52, 101–114.[CrossRef][Medline]
Gomila, M., Gascó, J., Gil, J., Bernabeu, R., Iñigo, V. & Lalucat, J. (2006). A molecular microbial ecology approach to studying hemodialysis water and fluid. Kidney Int 70, 1567–1576.[CrossRef][Medline]
Guasp, C., Moore, E. R. B., Lalucat, J. & Bennasar, A. (2000). Utility of internally-transcribed 16S–23S rDNA spacer regions for the definition of Pseudomonas stutzeri genomovars and other Pseudomonas species. Int J Syst Evol Microbiol 50, 1629–1639.[Abstract]
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kulakov, L. A., McAlister, M. B., Ogden, K. L., Larkin, M. J. & O'Hanlon, J. F. (2002). Analysis of bacteria contaminating ultrapure water in industrial systems. Appl Environ Microbiol 68, 1548–1555.
Lalucat, J. (1988). Analysis of refractile (R) bodies. In Methods in Microbiology – Electron Microscopy in Microbiology, vol. 20, pp. 79–90. Edited by F. Mayer. Academic Press.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Palleroni, N. J. (1980). Isolation and properties of a new hydrogen bacterium related to Pseudomonas saccharophila. J Gen Microbiol 117, 155–161.
Reasoner, D. J. & Geldreich, E. E. (1985). A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol 49, 1–7.
Selesi, D., Schmid, M. & Hartmann, A. (2005). Diversity of green-like and red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils. Appl Environ Microbiol 71, 175–184.
Stringfellow, W. T. & Aitken, M. D. (1995). Competitive metabolism of naphthalene, methylnaphthalenes, and fluorene by phenanthrene-degrading pseudomonads. Appl Environ Microbiol 61, 357–362.[Abstract]
Suyama, T., Shigematsu, T., Takaichi, S., Nodasaka, Y., Fujikawa, S., Hosoya, H., Tokiwa, Y., Kanagawa, T. & Hanada, S. (1999). Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the -subclass of the proteobacteria. Int J Syst Bacteriol 49, 449–457.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Ueda, T., Suga, Y., Yahiro, N. & Matsuguchi, T. (1995). Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177, 1414–1417.
Xie, C. H. & Yokota, A. (2005). Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australiaca sp. nov. and Pelomonas saccharophila gen. nov., comb. nov. respectively. Int J Syst Evol Microbiol 55, 2419–2425.
Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.
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