Publications (22)84.48 Total impact
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Chapter: Molecular Ecology of N2-Fixing Microbes Associated with Gramineous Plants: Hidden Activities of Unknown Bacteria
01/2006: pages 173-198; -
Article: Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.
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ABSTRACT: In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.Applied and Environmental Microbiology 09/2001; 67(8):3655-64. · 3.83 Impact Factor -
Article: Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens.
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ABSTRACT: Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (> or =86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.Systematic and Applied Microbiology 08/2001; 24(2):245-51. · 3.37 Impact Factor -
Article: Role of a ferredoxin gene cotranscribed with the nifHDK operon in N(2) fixation and nitrogenase "switch-off" of Azoarcus sp. strain BH72.
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ABSTRACT: The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of nifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to nifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a sigma(54)-type promoter. Shorter transcripts sequentially missing genes of the 3' part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% +/- 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxN mutant which carried a nifH promoter-driven fdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxN deletion also had an effect on the rapid "switch-off" of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN in trans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.Journal of Bacteriology 07/2001; 183(12):3752-60. · 3.83 Impact Factor -
Article: Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens.
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ABSTRACT: Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.Journal of Bacteriology 05/2001; 183(8):2634-45. · 3.83 Impact Factor -
Article: Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72.
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ABSTRACT: PII-like signal transmitter proteins are involved in the regulation of ammonium assimilation and nitrogen fixation. We report the identification of three PII-like proteins in the diazotrophic, endophytic proteobacterium Azoarcus sp. BH72, encoded by glnB (monocistronically transcribed) or in the glnKamtB and glnYamtY operons. Phylogenetic analysis revealed that glnB, glnK and glnY represent distinct lineages within the Proteobacteria. A combined approach of two-dimensional gel electrophoresis, Western blotting with paralogue-specific antibodies, N-terminal sequencing and marker exchange mutagenesis allowed us to analyse PII protein expression of Azoarcus sp. BH72 in vivo. GlnK and GlnB were present on all nitrogen sources. Knock-out mutant analysis revealed that GlnB was the only detectable PII protein in a glnK- background, whereas GlnY was only present in a glnK/glnB- double mutant. Nitrogen limitation enhanced transcript abundance of glnK strongly, glnY moderately and glnB not at all in wild-type, glnB-/glnK- or glnK- backgrounds respectively. Phenotypic characterization of knock-out mutants revealed that, unlike in other Proteobacteria, neither glnK nor glnB were essential for nitrogen fixation. As the growth of a double mutant was drastically impaired only on minimal media, both proteins are probably involved in the control of ammonium and nitrate assimilation. The PII-like proteins differed from each other in details of N-sensing. They were covalently modified by uridylylation upon nitrogen limitation, as shown by mass spectrometry; however, the modification patterns in relation to the supplied nitrogen source differed. The novel paralogue GlnY was unusual, as it only occurred in the uridylylated state in vivo and thus lacked a deuridylylation response to nitrogen excess.Molecular Microbiology 11/2000; 38(2):276-88. · 5.01 Impact Factor -
Article: Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races.
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ABSTRACT: Several diazotrophic species of Azoarcus spp. occur as endophytes in the pioneer plant Kallar grass. The purpose of this study was to screen Asian wild rice and cultivated Oryza sativa varieties for natural association with these endophytes. Populations of culturable diazotrophs in surface-sterilized roots were characterized by 16S rDNA sequence analysis, and Azoarcus species were identified by genomic fingerprints. A. indigens and Azoarcus sp. group C were detected only rarely, whereas Azoarcus sp. group D occurred frequently in samples of flooded plants: in 75% of wild rice, 80% of land races of O. sativa from Nepal and 33% of modern cultivars from Nepal and Italy. The putatively endophytic populations of diazotrophs differed with the rice genotype. The diversity of cultured diazotrophs was significantly lower in wild rice species than in modern cultivars. In Oryza officinalis (from Nepal) and O. minuta (from the Philippines), Azoarcus sp. group D were the predominant diazotrophic putative endophytes in roots. In contrast, their number was significantly lower in modern cultivars of O. sativa, whereas numbers and diversity of other diazotrophs, such as Azospirillum spp., Klebsiella sp., Sphingomonas paucimobilis, Burkholderia sp. and Azorhizobium caulinodans, were increased. In land races of O. sativa, the diazotrophic diversity was equally high; however, Azoarcus sp. was found in high apparent numbers. Similar differences in populations were also observed in a culture-independent approach comparing a wild rice (O. officinalis) and a modern-type O. sativa plant: in clone libraries of root-associated nitrogenase (nifH) gene fragments, the diazotrophic diversity was lower in the wild rice species. New lineages of nifH genes were detected, e.g. one deeply branching cluster within the anf (iron) nitrogenases. Our studies demonstrate that the natural host range of Azoarcus spp. extends to rice, wild rice species and old varieties being preferred over modern cultivars.Environmental Microbiology 05/2000; 2(2):131-41. · 5.84 Impact Factor -
Article: Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and new species, Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov.
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ABSTRACT: The taxonomic structure of members of the genus Azoarcus sensu lato was reassessed in a polyphasic approach. Two species, Azoarcus communis and Azoarcus indigens, three unnamed species containing diazotrophs associated with Kallar grass roots (groups C, D) and a group of strains (E) isolated from fungi were analysed. They were compared by PAGE analyses of cellular proteins, genomic fingerprints, morphological and nutritional features to new isolates from rice roots. All strains within groups C, D and E containing 5-12 isolates showed group-specific cell and colony morphology and carbon source utilization patterns, with exception of the obligately microaerobic strain BS20-3, a member of group C. All strains, with this exception, also had almost indistinguishable electrophoretic protein patterns and genomic fingerprints generated with tDNA-directed primers, suggesting they belong to the same species. Phylogenetic analyses of almost complete 16S rDNA sequences carried out with three different algorithms (neighbour-joining, maximum-likelihood, parsimony) revealed that Azoarcus sensu lato is not monophyletic. Groups C, D and E formed three distinct lineages located between the Azoarcus/Thauera and the Rhodocyclus clusters. Phylogenetic distances between groups C, D and E were as large as between other genera (93-94% sequence similarity). This suggested they have the rank of three different genera. Since it was possible to differentiate them from each other and other related bacteria by phenotypic features, three new genera with one type species each are proposed: Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov. and Azonexus fungiphilus gen. nov., sp. nov.International journal of systematic and evolutionary microbiology 04/2000; 50 Pt 2:649-59. · 2.27 Impact Factor -
Article: Type IV pili are involved in plant-microbe and fungus-microbe interactions.
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ABSTRACT: Adherence of bacteria to eukaryotic cells is essential for the initiation of infection in many animal and human pathogens, e.g. Neisseria gonorrhoeae and Pseudomonas aeruginosa. Adhesion-mediating type IV pili, filamentous surface appendages formed by pilin subunits, are crucial virulence factors. Here, we report that type IV pilus-dependent adhesion is also involved in plant-bacteria and fungus-bacteria interactions. Nitrogen-fixing, endophytic bacteria, Azoarcus sp., can infect the roots of rice and spread systemically into the shoot without causing symptoms of plant disease. Formation of pili on solid media was dependent on the pilAB locus. PilA encodes an unusually short (6.4 kDa) putative pilin precursor showing 100% homology to the conserved N-terminus of the Pseudomonas aeruginosa type IV pilin. PilB encodes for a 14.2 kDa polypeptide showing similarity to FimF, a component of type I fimbriae of Escherichia coli. It was found to be extruded beyond the cell surface by immunofluorescence studies, and it may, therefore, be part of a pilus assembly complex or the pilus itself. Both genes are involved in the establishment of bacteria on the root surface of rice seedlings, as detected by fluorescence microscopy. Moreover, both genes are necessary for bacterial adhesion to the mycelium of an ascomycete, which was isolated from the same rhizosphere as the bacteria. In co-culture with the fungus, Azoarcus sp. forms complex intracytoplasmic membranes, diazosomes, which are related to efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process, as diazosomes were absent and nitrogen fixation rates were decreased in pilAB mutants in co-culture.Molecular Microbiology 11/1998; 30(1):7-17. · 5.01 Impact Factor -
Article: Life in grasses: diazotrophic endophytes.
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ABSTRACT: N2-fixing bacteria such as Azoarcus spp., Herbaspirillum spp, and Acetobacter diazotrophicus can infect the interior of gramineous plants without causing symptoms of plant disease but do not survive in soil. Like phytopathogens, they can penetrate into central tissues and spread systemically. There is no evidence for an endosymbiosis in living plant cells; however, the bacteria are physiologically active in the plant apoplast.Trends in Microbiology 05/1998; 6(4):139-44. · 7.91 Impact Factor -
Article: Use of green fluorescent protein to detect expression of nif genes of Azoarcus sp. BH72, a grass-associated diazotroph, on rice roots.
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ABSTRACT: A gfp (green fluorescent protein) cassette for transcriptional fusions has been developed to study gene expression in Azoarcus sp. BH72 in association with plant roots. The bacteria expressed nitrogenase genes (nifHDK) in the rhizosphere, on root tips, and in epidermal cells of rice seedlings. Green fluorescent protein fusions also visualized promoter activity of single cells in soil.Molecular Plant-Microbe Interactions 02/1998; 11(1):71-5. · 4.43 Impact Factor -
Article: Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints.
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ABSTRACT: Most species of the diazotrophic Proteobacteria Azoarcus spp. occur in association with grass roots, while A. tolulyticus and A. evansii are soil bacteria not associated with a plant host. To facilitate species identification and strain comparison, we developed a protocol for PCR-generated genomic fingerprints, using an automated sequencer for fragment analysis. Commonly used primers targeted to REP (repetitive extragenic palindromic) and ERIC (enterobacterial repetitive intergenic consensus) sequence elements failed to amplify fragments from the two species tested. In contrast, the BOX-PCR assay (targeted to repetitive intergenic sequence elements of Streptococcus) yielded species-specific genomic fingerprints with some strain-specific differences. PCR profiles of an additional PCR assay using primers targeted to tRNA genes (tDNA-PCR, for tRNA(IIe)) were more discriminative, allowing differentiation at species-specific (for two species) or infraspecies-specific level. Our protocol of several consecutive PCR assays consisted of 16S ribosomal DNA (rDNA)-targeted, genus-specific PCR followed by BOX- and tDNA-PCR; it enabled us to assign new diazotrophic isolates originating from fungal resting stages (sclerotia) to known species of Azoarcus. The assignment was confirmed by phylogenetic analysis of 16S rDNA sequences. Additionally, the phylogenetic distances and the lack of monophyly suggested emendment of the genus Azoarcus: the unnamed species Azoarcus groups C and D and a new group (E) of Azoarcus, which was detected in association with fungi, are likely to have the taxonomic rank of three different genera. According to its small subunit rRNA, the sclerotium-forming basidiomycete was related to the Ustilagomycetes, facultatively biotrophic parasites of plants. Since they occurred in a field which was under cultivation with rice and wheat, these fungi might serve as a niche for survival for Azoarcus in the soil and as a source for reinfection of plants.Applied and Environmental Microbiology 12/1997; 63(11):4331-9. · 3.83 Impact Factor -
Article: Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass.
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ABSTRACT: Nitrogenase is a functionally constant protein catalyzing N2 reduction, which is found in many phylogenetic lineages of Archaea and Bacteria. A phylogenetic analysis of nif genes may provide insights into the evolution of the bacterial genomes. Moreover, it may be used to study diazotrophic communities, when classical isolation techniques may fail to detect all contributing populations. Among six species of the genus Azoarcus, diazotrophic Proteobacteria of the beta subclass, the deduced amino acid sequences of nifH genes of two species were unusually divergent from each other. Nitrogenases of the "authentic" Azoarcus branch formed a monophyletic unit with those of gamma Proteobacteria, thus being in accordance with 16S ribosomal DNA phylogeny. The nitrogenase proteins of the two aberrant strains clustered within the alpha proteobacterial clade with rhizobial nitrogenases. This relationship was supported by bootstrap values of 87 to 98% obtained by various distance and maximum parsimony methods. Phylogenetic distances of NifH proteins indicate a possible lateral gene transfer of nif genes to Azoarcus from a common donor of the alpha subclass at the time of species diversification or several more recent, independent transfers. Application of the phylogenetic analysis to DNA isolated from environmental samples demonstrated novel habitats for Azoarcus: in guts of termites and rice grown in Japan, nifH genes belonging to the authentic Azoarcus branch were detected. This is the first evidence suggesting the occurrence of Azoarcus spp. in a plant other than its originally described host, Kallar grass. Moreover, evidence for expression of nif genes inside grass roots was obtained by in situ hybridization studies with antisense nifH probes.Journal of Bacteriology 08/1997; 179(13):4172-8. · 3.83 Impact Factor -
Article: Terminal oxidases of Azoarcus sp. BH72, a strictly respiratory diazotroph.
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ABSTRACT: The nitrogen-fixing, grass-associated bacterium Azoarcus sp. BH72 was characterized with respect to its terminal oxidases. Inhibitory respiratory analysis revealed the presence of at least one cytochrome c oxidase and one quinol oxidase. The cytochrome c oxidase was preferably used by the cells under aerobic, whereas the quinol oxidase seemed to be dominant under microaerobic, nitrogen-fixing conditions. Differential spectroscopy and heme analysis of the membrane preparations indicated that the cytochrome c oxidase is probably of the cb type.FEBS Letters 04/1997; 404(2-3):143-7. · 3.54 Impact Factor -
Article: Global changes in protein composition of N2-fixing-Azoarcus sp. strain BH72 upon diazosome formation.
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ABSTRACT: The strictly respiratory, diazotrophic bacterium Azoarcus sp. strain BH72 fixes nitrogen under microaerobic conditions. In empirically optimized batch cultures at nanomolar O2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N2 fixation are formed. Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, Kallar grass. To initiate studies on function of diazosomes and regulation of their formation, diazosome-containing bacteria were compared with respect to composition or total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions. In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions. N-terminal amino acid sequence analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P(II), an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism. According to its electrophoretic mobility, it might be uridylylated in diazosome-free cells but not in diazosome-containing cells, or it may represent a second, not identical P(II) protein. Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp. strain BH72 in Southern hybridizations.Journal of Bacteriology 11/1996; 178(19):5748-54. · 3.83 Impact Factor -
Article: Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72.
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ABSTRACT: We report the discovery of novel subcellular structures related to bacterial nitrogen fixation in the strictly respiratory diazotrophic bacterium Azoarcus sp. BH72, which was isolated as an endophyte from Kallar grass. Nitrogenase is derepressed under microaerobic conditions at O2 concentrations in the micromolar range. With increasing O2 deprivation, bacteria can develop into a hyperinduced state, which is characterized by high specific rates of respiration and efficient nitrogen fixation at approximately 30 nM O2. Ultrastructural analysis of cells in the course of hyperinduction revealed that complex intracytoplasmic membrane systems are formed, which consist of stacks of membranes and which are absent under standard nitrogen-fixing conditions. The iron protein of nitrogenase was highly enriched on these membranes, as evidenced by immunohistochemical studies. Membrane deficiency in NifH/K- mutants, a deletion mutant in the nifK gene and the character of NH+4-grown cells suggested, in concert with the membrane localization of nitrogenase, that these structures are specialized membranes related to nitrogen fixation. We propose the term 'diazosomes' for them. Development of intracytoplasmic membranes coincides with the appearance of a high-molecular-mass form of the iron protein of nitrogenase, which was detectable in membrane fractions. Mutational analysis, and determination of the N-terminal amino acid sequence indicate that the nifH gene product is covalently modified by a mechanism probably different from adenosine diphosphoribosylation. Development of diazosomes in nitrogen-fixing cells can be induced in pure cultures and in co-culture with a fungus isolated from the rhizosphere of Kallar grass.Molecular Microbiology 11/1995; 18(2):225-36. · 5.01 Impact Factor -
Article: Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences.
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ABSTRACT: The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)Applied and Environmental Microbiology 07/1995; 61(6):2257-61. · 3.83 Impact Factor -
Article: Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72.
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ABSTRACT: Azoarcus sp. strain BH72 is an aerobic diazotrophic bacterium that was originally found as an endophyte in Kallar grass. Anticipating that these bacteria are exposed to dissolved O2 concentrations (DOCs) in the nanomolar range during their life cycle, we studied the impact of increasing O2 deprivation on N2 fixation and respiration. Bacteria were grown in batch cultures, where they shifted into conditions of low pO2 upon depletion of O2 by respiration. During incubation, specific rates of respiration (qO2) and efficiencies of carbon source utilization for N2 reduction increased greatly, while the growth rate did not change significantly, a phenomenon that we called "hyperinduction." To evaluate this transition from high- to low-cost N2 fixation in terms of respiratory kinetics and nitrogenase activities at nanomolar DOC, bacteria which had shifted to different gas-phase pO2s in batch cultures were subjected to assays using leghemoglobin as the O2 carrier. As O2 deprivation in batch cultures proceeded, respiratory Km (O2) decreased and Vmax increased. Nitrogenase activity at nanomolar DOC increased to a specific rate of 180 nmol of C2H4 min-1 mg of protein-1 at 32 nM O2. Nitrogenase activity was proportional to respiration but not to DOC in the range of 12 to 86 nM O2. Respiration supported N2 fixation more efficiently at high than at low respiratory rates, the respiratory efficiency increasing from 0.14 to 0.47 mol of C2H4 mol of O2 consumed-1. We conclude that (i) during hyperinduction, strain BH72 used an increasing amount of energy generated by respiration for N2 fixation, and (ii) these bacteria have a high respiratory capacity, enabling them to develop ecological niches at very low pO2, in which they may respire actively and fix nitrogen efficiently at comparatively high rates.Journal of Bacteriology 09/1994; 176(15):4726-33. · 3.83 Impact Factor -
Article: Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses.
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ABSTRACT: The invasive properties of Azoarcus sp. strain BH72, an endorhizospheric isolate of Kallar grass, on gnotobiotically grown seedlings of Oryza sativa IR36 and Leptochloa fusca (L.) Kunth were studied. Additionally, Azoarcus spp. were localized in roots of field-grown Kallar grass. To facilitate localization and to assure identity of bacteria, genetically engineered microorganisms expressing beta-glucuronidase were also used as inocula. beta-Glucuronidase staining indicated that the apical region of the root behind the meristem was the most intensively colonized. Light and electron microscopy showed that strain BH72 penetrated the rhizoplane preferentially in the zones of elongation and differentiation and colonized the root interior inter- and intracellularly. In addition to the root cortex, stelar tissue was also colonized; bacteria were found in the xylem. No evidence was obtained that Azoarcus spp. could reside in living plant cells; rather, plant cells were apparently destroyed after bacteria had penetrated the cell wall. A common pathogenicity test on tobacco leaves provided no evidence that representative strains of Azoarcus spp. are phytopathogenic. Compared with the control, inoculation with strain BH72 significantly promoted growth of rice seedlings. This effect was reversed when the plant medium was supplemented with malate (0.2 g/liter). N2 fixation was apparently not involved, because the same response was obtained with a nifK mutant of strain BH72, which has a Nif- phenotype. Also, Western blot (immunoblot) analysis of protein extracts from rice seedlings gave no indication that nitrogenase was present. PCR and Western immunoblotting, using primers specific for eubacteria and antibodies recognizing type-specific antigens, respectively, indicated that strain BH72 could colonize rice plants systemically, probably mediated by longitudinal spreading through vessels.Journal of Bacteriology 05/1994; 176(7):1913-23. · 3.83 Impact Factor -
Article: 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs.
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ABSTRACT: Phylogenetic analyses after reverse transcriptase sequencing of 16S rRNA of nitrogen-fixing, grass-associated Azoarcus strains confirmed their affiliation to the beta subdivision of the Proteobacteria. Strains representing three different species formed a phylogenetically coherent unit related to Rhodocyclus purpureus, with actual percent similarities among the three sequences ranging from 93.1 to 97.3%. Within variable regions V2 and V5, we found stretches of sequences considerably conserved within the genus Azoarcus but differing from most other gram-negative bacteria, with the specificity being enhanced when different regions were combined. Genus-specific primers selected from both regions amplified fragments from all but one Azoarcus species in polymerase chain reactions (PCR) but not from any reference strain tested. Primers of lesser specificity generated fragments from members of all five Azoarcus species as well as from some reference strains. Those unspecific amplifications could be differentiated by oligonucleotide hybridization, detecting only fragments generated from Azoarcus strains except strain 6a3, which represents the same group which could not be detected by genus-specific PCR. Thus we propose the application of PCR amplification with 16S rRNA-targeted, genus-specific primers in combination with hybridization of a 16S rRNA-targeted oligonucleotide to PCR-generated fragments as diagnostic tests; this allows an initial screening for presence of members of the genus Azoarcus.Applied and Environmental Microbiology 12/1993; 59(11):3816-24. · 3.83 Impact Factor
Top co-authors
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Institutions
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2006
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Universität Bremen
Bremen, Bremen, Germany
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1998–2001
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Max Planck Institute for Terrestrial Microbiology
Marburg an der Lahn, Hesse, Germany
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1995–2000
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Max-Planck-Institut für marine Mikrobiologie
- Abteilung Biogeochemie
Bremen, Bremen, Germany
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1993
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Ghent University
Gent, VLG, Belgium
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