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1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany
2 BRAIN Aktiengesellschaft, 64673 Zwingenberg, Germany
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 38124 Braunschweig, Germany
4 Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, 28359 Bremen, Germany
Correspondence
Peter Kämpfer
peter.kaempfer{at}agrar.uni-giessen.de
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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Phylogenetic trees and a table showing fatty acid compositions are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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and, at present, comprises five species: Hydrogenophaga flava, Hydrogenophaga pseudoflava, Hydrogenophaga palleronii, Hydrogenophaga taeniospiralis (Willems et al., 1989) and Hydrogenophaga intermedia (Contzen et al., 2000). Members of this genus are chemo-organotrophic or chemolithoautotrophic, using the oxidation of H2 as an energy source and CO2 as a carbon source, these being major differentiating characteristics for distinguishing between them and other genera of the family Comamonadaceae (Wen et al., 1999; Spring et al., 2004).
Numerous studies applying cultivation-independent methods have revealed that members of the 
1-group of the Proteobacteria are abundant in activated sludge from wastewater treatment plants (e.g. Amann et al., 1996b; Snaidr et al., 1997). Since members of this group, although closely related, are physiologically diverse, it is almost impossible to infer the metabolic phenotype of members of this group by 16S rRNA gene sequence comparison studies. To determine their physiological traits, members of this important bacterial group were isolated, by a directed cultivation procedure, from activated sludge of the wastewater treatment plant München I (Großlappen, Germany) as described by Schulze et al. (1999). The isolates obtained were screened by whole-cell hybridization with specific probes directed against signature regions of 16S rRNA sequences. Isolates that hybridized to probe BONE23a (Amann et al., 1996b) directed against members of the 1-group of the Proteobacteria were further grouped using the probes LDI (Wagner et al., 1994) and SNA8b (Amann et al., 1996b). Two strains (BSB 9.5T and BSB 41.8T) that hybridized to probe LDI were also detected by the probe HYD208 directed against members of the genus Hydrogenophaga, which seemed to represent an abundant group of wastewater bacteria (Amann et al., 1996a). Therefore, these two isolates were chosen for further investigation. Both strains were maintained on nutrient agar (Difco). Type strains of all Hydrogenophaga species with validly published names were used for comparison.
Cell morphology was examined by using phase-contrast microscopy (Leitz). Cell dimensions were measured with an ocular (x10) and an objective (x100/1·25). Gram-staining was performed by using Hucker's modification (Gerhardt et al., 1994). Colony morphology was studied using a stereo microscope (model SZ 11; Olympus).
The effects of different temperatures on growth were determined on Bacto nutrient agar (Oxoid) incubated at 5, 10, 28, 37, 45 and 50 °C. Physiological tests in microtitre plates were performed as described previously (Kämpfer et al., 1991). Tests were read after 7 days at 30 °C. Chemolithoautotrophic growth of both strains was tested under the conditions described by the DSMZ. Both strains were grown on medium 81 (Malik & Schlegel, 1981) under an atmosphere of O2/CO2/H2/N2 (approximately 2 : 10 : 60 : 28, by vol.) (Malik & Schlegel, 1981). Nitrate reduction and denitrification were tested in R2A broth (Difco) supplemented with 10 mM nitrate under aerobic and anaerobic conditions. The obligately aerobic heterotrophic strains BSB 9.5T and BSB 41.8T grew as circular, entire, slightly convex, smooth, pale-yellow colonies on R2A agar (Oxoid). The cells were Gram-negative, non-spore-forming, motile, rod-shaped organisms. Both strains grew at 28 °C on nutrient agar, R2A agar and tryptone soy broth agar (Oxoid), but only very weak growth was observed on MacConkey agar (Oxoid). They did not grow on nutrient agar at 5 or 10 °C, but grew well in a temperature range from 20 to 37 °C. Neither strain grew at 40 or 45 °C. Both strains were oxidase- and catalase-positive. Only a few organic compounds could be used as sole sources of carbon (see Table 1 and the species descriptions). Differentiation from all five Hydrogenophaga species is possible on the basis of the results of several tests. Both strains could be differentiated on the basis of the utilization of 2-oxoglutarate and L-histidine. A detailed comparison was made with all previously published data (Willems et al., 1989; Contzen et al., 2000). Table 1 shows only those tests for which identical results were obtained with the method used in this study and the method used by Willems et al. (1989). Only strain BSB 9.5T showed good chemolithotrophic growth. Strain BSB 41.8T was not able to grow chemolithoautotrophically under the conditions described – an additional important feature enabling differentiation between the two strains. The utilization of thiosulfate was tested with all type strains of Hydrogenophaga in R2A medium supplemented with 10 mM Na2S2O3.5H2O, as described by Spring et al. (2004). Only the type strains of H. palleronii (DSM 63T) and H. intermedia (DSM 5680T) were positive and oxidized thiosulfate to sulfate.
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. Phylogenetic analysis was performed using the ARB software package (Ludwig et al., 2004) as well as the software package MEGA version 2.1 (Kumar et al., 2001) after multiple alignment of the data by CLUSTAL_X (Thompson et al., 1997). Calculation of distances (with distance options according to the Kimura-2 model) and clustering with the neighbour-joining method and maximum parsimony was performed. Bootstrap values based on 1000 replications were determined to show support for branching points (results are available as supplementary figures in IJSEM Online). Nearly complete 16S rRNA gene sequences of BSB 9.5T and BSB 41.8T, respectively comprising 1503 and 1527 nucleotide positions, were determined by PCR amplification and sequencing of the PCR-amplified 16S rRNA genes. A comparative analysis and estimation of the phylogenetic relationships demonstrated that the two strains show 99·9 % sequence similarity and cluster within the genus Hydrogenophaga, being most closely related to H. palleronii (98·5 %) and H. taeniospiralis (98·0 %).
The G+C content was determined by reversed-phase HPLC of nucleosides according to Mesbah et al. (1989).
For fatty acid analysis, cells were grown on YPG agar (Contzen et al., 2000). The fatty acid methyl esters were prepared and analysed as described elsewhere (Kämpfer & Kroppenstedt, 1996). When grown on YPG agar, the two strains were very similar with regard to their fatty acid patterns and, as with the type strains of all Hydrogenophaga species, contained the fatty acids 15 : 0, 16 : 0, summed feature 4 (16 : 1
7c and/or 15 : 0 iso 2-OH) and summed feature 7 (18 : 17c, 18 : 19t and/or 18 : 112t) (see the supplementary table available in IJSEM Online). These fatty acids have also been detected in previous studies and are characteristic of all Hydrogenophaga species with validly published names (Willems et al., 1989). The presence of the hydroxy fatty acid 8 : 0 3-OH in all Hydrogenophaga species could be confirmed, although this fatty acid could be detected only in trace amounts in H. pseudoflava LMG 5945T and H. taeniospiralis DSM 2082T. H. intermedia S1T (when grown on YPG agar) contained 8 : 0 3-OH in larger amounts (>2·5 %), a feature shared only by H. palleronii DSM 63T. Additionally, both isolates (BSB 9.5T and BSB 41.8T) and the type strains H. palleronii DSM 63T and H. intermedia S1T produced large amounts of the cyclopropane fatty acid, 17 : 0 cyclo, in agreement with data in the literature (Willems et al., 1989).
DNA–DNA hybridization experiments were performed with both isolates and with the type strains of all Hydrogenophaga species by using the method described by Ziemke et al. (1998) except that, for nick translation, 2 µg DNA was labelled during a 3 h incubation at 15 °C. Strains BSB 9.5T and BSB 41.8T showed a DNA–DNA hybridization value of 51 % (mean value from four experiments, including reciprocal analyses), indicating that they belong to different species, despite their close 16S rRNA gene sequence similarity. The following DNA–DNA hybridization values between BSB 9.5T and other Hydrogenophaga type strains were found: H. flava, 30 %; H. pseudoflava, 26 %; H. palleronii, 29 %; H. taeniospiralis, 13 %; H. intermedia, 12 %. The DNA–DNA hybridization values between BSB 41.8T and other Hydrogenophaga type strains were as follows: H. flava, 32 %; H. pseudoflava, 28 %; H. palleronii, 31 %; H. taeniospiralis, 26 %; H. intermedia, 9 %.
Although both strains showed a very high degree of 16S rRNA gene sequence similarity, they could be differentiated from each other and from other species of the genus Hydrogenophaga by means of DNA–DNA hybridization and a few physiological tests. In conclusion, we propose the names Hydrogenophaga defluvii sp. nov. for strain BSB 9.5T and Hydrogenophaga atypica sp. nov. for strain BSB 41.8T.
Description of Hydrogenophaga defluvii sp. nov.
Hydrogenophaga defluvii (de.flu'vi.i. L. n. defluvium sewage; L. gen. n. defluvii of sewage).
Cells are Gram-negative, rod-shaped, motile and 1·5 µm long by 0·5 µm wide, with rounded ends. Metabolism is oxidative. Oxidase-positive. Able to grow chemolithoautotrophically on H2 under the conditions described. Chemolithoautotrophic growth of H. defluvii (in contrast to that of H. flava) is not inhibited by high levels of oxygen in the atmosphere (5 % O2 or more). Thiosulfate is not oxidized to sulfate. The fatty acid pattern is characterized by the presence of the fatty acids typical of the genus Hydrogenophaga, as well as the hydroxylated fatty acid 8 : 0 3-OH and also small amounts of 9 : 0 3-OH. Phylogenetically, the species is a member of the genus Hydrogenophaga. On YPG agar at 25 °C, colonies are circular, entire, slightly convex, smooth and pale yellow. Growth occurs at 37 °C but not at 10 °C. Only a few organic compounds can be used as sole sources of carbon: gluconate, glutarate, lactate, 3-hydroxybutyrate, pyruvate, suberate, L-alanine, L-leucine, L-aspartate, L-histidine, phenylalanine, L-proline, 3-hydroxybenzoate and 4-hydroxybenzoate. Furthermore, tests for hydrolysis of L-alanine p-nitroanilide (pNA) are positive. Does not utilize N-acetyl-D-glucosamine, L-arabinose, arbutin, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-mannitol, adipate, D-melibiose, L-rhamnose, ribose, sucrose, salicin, trehalose, D-xylose, adonitol, inositol, sorbitol, putrescine, acetate, propionate, cis-aconitate, trans-aconitate, 4-aminobutyrate, azelate, citrate, fumarate, glutarate, itaconate, D-malate, mesaconate, 2-oxoglutarate, -alanine, L-ornithine, L-serine, L-tryptophan or phenylacetate as sole sources of carbon. Tests for hydrolysis of p-nitrophenyl (pNP) 
-D-glucopyranoside, pNP -D-glucopyranoside, pNP -D-galactopyranoside, pNP -D-glucuronide, pNP phenylphosphonate, pNP phosphorylcholine, 2-deoxythymidine-5'-pNP phosphate, glutamate-
-3-carboxy pNP-ester and L-proline pNA are negative. The G+C content of the genomic DNA is 65 mol%. Characteristics used for differentiation from other Hydrogenophaga species are given in Table 1
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The type strain, BSB 9.5T (=DSM 15341T=CIP 108119T), was isolated from activated sludge in Munich, Germany.
Description of Hydrogenophaga atypica sp. nov.
Hydrogenophaga atypica (Gr. pref. a-, an- not; L. adj. typicus, -a, -um from Gr. adj. tupikos typical; N.L. fem. adj. atypica atypical).
Cells are Gram-negative, rod-shaped, motile and 1·5 µm long by 0·5 µm wide, with rounded ends. Oxidative metabolism. Oxidase-positive. Unable to grow chemolithoautotrophically on H2 under the conditions described. Thiosulfate is not oxidized to sulfate. The fatty acid pattern is characterized by the presence of the fatty acids typical of the genus Hydrogenophaga with the hydroxylated fatty acid 8 : 0 3-OH and also 9 : 0 3-OH. Phylogenetically, the species is a member of the genus Hydrogenophaga. On YPG agar at 25 °C, colonies are circular, entire, slightly convex, smooth and pale yellow. Growth occurs at 37 °C but not at 10 °C. Only a few organic compounds can be used as sole sources of carbon: gluconate, glutarate, lactate, 3-hydroxybutyrate, 2-oxoglutarate, pyruvate, phenylalanine, L-proline, 3-hydroxybenzoate and 4-hydroxybenzoate. Furthermore, tests for hydrolysis of L-alanine pNA are positive. Does not utilize N-acetyl-D-glucosamine, L-arabinose, arbutin, D-cellobiose, D-fructose, D-galactose, D-glucose, D-mannose, D-mannitol, adipate, D-melibiose, L-rhamnose, ribose, sucrose, salicin, trehalose, D-xylose, adonitol, inositol, sorbitol, putrescine, acetate, propionate, cis-aconitate, trans-aconitate, suberate, 4-aminobutyrate, azelate, citrate, fumarate, glutarate, itaconate, D-malate, mesaconate, L-alanine, -alanine, L-leucine, L-aspartate, L-histidine, L-ornithine, L-serine, L-tryptophan or phenylacetate as sole sources of carbon. Tests for hydrolysis of pNP -D-glucopyranoside, pNP -D-glucopyranoside, pNP -D-galactopyranoside, pNP -D-glucuronide, pNP phenylphosphonate, pNP phosphorylcholine, 2-deoxythymidine-5'-pNP phosphate, glutamate--3-carboxy pNP-ester and L-proline pNA are negative. The G+C content of the genomic DNA is 64 mol%. Characteristics used for differentiation from the other Hydrogenophaga species are given in Table 1.
The type strain, BSB 41.8T (=DSM 15342T=CIP 108118T), was isolated from activated sludge in Munich, Germany.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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