Bradyrhizobium kavangense sp. nov., a symbiotic nitrogen-fixing bacterium from root nodules of traditional Namibian pulses.

Eight strains of symbiotic bacteria from root nodules of local races of cowpea (Vigna unguiculata) and Bambara groundnut (Vigna subterranea) grown on subsistence farmers' fields in the Kavango region, Namibia, were previously characterized and identified as a novel group within the genus Bradyrhizobium. To clarify their taxonomic status, these strains were further characterized using a polyphasic approach. In phylogenetic analysis of the 16S rRNA gene sequence the novel group was most closely related to Bradyrhizobium iriomotense EK05T and Bradyrhizobium ingae BR 10250T, and to 'Bradyrhizobium arachidis' CCBAU 051107 in the ITS sequence analysis. Phylogenetic analysis of concatenated glnII-recA-rpoB-dnaK sequences placed the strains in a lineage distinct from named species of the genus Bradyrhizobium. The species status was validated by results of DNA-DNA hybridization. Phylogenetic analysis of nifH and nodC genes placed the novel strains in a group with 'B. arachidis' CCBAU 051107. The combination of phenotypic characteristics from several tests including carbon source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain 14-3T induces effective nodules on Vigna subterranea, Vigna unguiculata, Arachis hypogaea and Lablab purpureus. Based on the data presented, it is concluded that the strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium kavangense sp. nov. is proposed. The type strain is 14-3T [ = DSM 100299T = LMG 28790T = NTCCM 0012T (Windhoek)]. The DNA G+C content of strain 14-3T is 63.8 mol% (Tm).

Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens.

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.

Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.

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.

Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72.

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.

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.

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.

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races.

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.

Type IV pili are involved in plant-microbe and fungus-microbe interactions.

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.

Life in grasses: diazotrophic endophytes.

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.

Use of green fluorescent protein to detect expression of nif genes of Azoarcus sp. BH72, a grass-associated diazotroph, on rice roots.

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.

Identification of N2-fixing plant- and fungus-associated Azoarcus species by PCR-based genomic fingerprints.

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.

Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the beta subclass.

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.

Induction of complex intracytoplasmic membranes related to nitrogen fixation in Azoarcus sp. BH72.

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.

Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences.

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)

Augmented rates of respiration and efficient nitrogen fixation at nanomolar concentrations of dissolved O2 in hyperinduced Azoarcus sp. strain BH72.

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.

Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses.

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.

Cloning, expression in Escherichia coli, and characterization of cellulolytic enzymes of Azoarcus sp., a root-invading diazotroph.

We screened members of a new genus of grass-associated diazotrophs (Azoarcus spp.) for the presence of cellulolytic enzymes. Out of five Azoarcus strains representing different species, only in the endorhizosphere isolate BH72, which is also capable of invading grass roots, was significant endoglucanase activity, in addition to beta-glucosidase and cellobiohydrolase activity, present. Reducing sugars were readily released from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp. Clones from a plasmid library of strain BH72 expressed all three enzymes in Escherichia coli, apparently not from their own promoter. According to restriction endonuclease mapping and subclone analysis, beta-glucosidase and cellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed. Two isoenzymes of endoglucanase probably resulting from proteolytic cleavage had pI values of 6.4 and 6.1 and an apparent molecular mass of approximately 36 kDa. Cellobiohydrolase and beta-glucosidase activity were conferred by one enzyme 41 kDa in size with a pI of 5.4, which we classified as an unspecific exoglycanase (exg) according to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not microcrystalline cellulose. Both enzymes were not excreted but were associated with the surface of Azoarcus cells. Both activities were only slightly influenced by the presence of CMC or D-glucose in the growth medium but were enhanced by ethanol. egl was located on a large transcript approximately 15 kb in size, which was detectable only in cells grown under microaerobic conditions on N2. Surface-bound exo- and endoglucanases with some unusual regulatory features, detected in this study in a strain which is unable to metabolize cellulose or sugars, might assist Azoarcus sp. strain BH72 in infection of grass roots.

16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs.

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.

Cross-reaction of predominant nitrogen-fixing bacteria with enveloped, round bodies in the root interior of kallar grass.

Diazotrophic rods occurring in high numbers (about 10 rods per g [dry weight] of root) in the root interior of Kallar grass were localized by indirect immunofluorescence in cross sections of the same roots. Specifically stained round bodies which were apparently covered by a nonantigenic envelope were found in the aerenchymatic tissue.

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass.

The effect of oxygen on N(2)-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 muM O(2), some of them being carbon limited. All strains needed a minimum amount of oxygen for N(2)-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O(2) concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N(2) fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N(2) fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O(2) concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.