Biomedical subjects

Thomas Hurek

Publications and source records attributed to Thomas Hurek.

10 recordsLinked to original sources

PubMed2006

A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms.

Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.

Azoarcus↗

PubMed2006

Complete genome of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

Azoarcus↗

PubMed2006

An endoglucanase is involved in infection of rice roots by the not-cellulose-metabolizing endophyte Azoarcus sp. strain BH72.

The nitrogen-fixing endophyte Azoarcus sp. strain BH72 infects roots of Kallar grass and rice inter- and intra-cellularly and can spread systemically into shoots without causing symptoms of plant disease. Although cellulose or its breakdown products do not support growth, this strain expresses an endoglucanase, which might be involved in infection. Sequence analysis of eglA places the secreted 34-kDa protein into the glycosyl hydrolases family 5, with highest relatedness (40% identity) to endoglucanases of the phytopathogenic bacteria Xanthomonas campestris and Ralstonia solanacearum. Transcriptional regulation studied by eglA:: gusA fusion was not significantly affected by cellulose or its breakdown products or by microaerobiosis. Strongest induction (threefold) was obtained in bacteria grown in close vicinity to rice roots. Visible sites of expression were the emergence points of lateral roots and root tips, which are the primary regions of ingress into the root. To study the role in endophytic colonization, eglA was inactivated by transposon mutagenesis. Systemic spreading of the eglA mutant and of a pilAB mutant into the rice shoot could no longer be detected by polymerase chain reaction. Microscopic inspection of infection revealed that the intracellular colonization of root epidermis cells was significantly reduced in the eglA- mutant BHE6 compared with the wild type and partially restored in the complementation mutant BHRE2 expressing eglA. This provides evidence that Azoarcus sp. endoglucanase is an important determinant for successful endophytic colonization of rice roots, suggesting an active bacterial colonization process.

Azoarcus↗

PubMed2006

Characterization of culturable bacterial populations associating with Pinus sylvestris--Suillus bovinus mycorrhizospheres.

Bacterial isolations were carried out on Pinus sylvestris--Suillus bovinus mycorrhizospheres obtained directly from boreal pine forest. When samples were taken during dry weather, the numbers of bacterial colony-forming units were significantly higher in uncolonized short roots and external mycelia than in mycorrhizal roots and soil outside the mycorrhizosphere. In contrast, the colony-forming unit counts were similar in all hypogeous samples after rainy weather. Culturable bacteria were absent from most Suillus bovinus sporocarps. The bacteria isolated from all types of mycorr hizo sphere samples, i.e. short roots, mycorrhizal roots, and external mycelia, consisted primarily of Burkholderia spp., whereas most isolates from soil outside the mycorrhizosphere were identified as Paenibacillus spp. This study shows that mycorrhizal external mycelia can expand the habitat favourable for common rhizosphere bacteria into the soil far from the immediate rhizosphere. Some of these bacteria may help the trees with nitrogen acquisition, since potentially diazotrophic bacteria harbouring nitrogenase reductase (nifH) genes were isolated from mycorrhizal root tips.

Bacteria↗

PubMed2005

Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridizations.

A key issue in applications of short oligonucleotide-based microarrays is how to design specific probes with high sensitivity. Some details of the factors affecting microarray hybridization remain unclear, hampering a reliable quantification of target nucleic acids. We have evaluated the effect of the position of the fluorescent label [position of label (POL)] relative to the probe-target duplex on the signal output of oligonucleotide microarrays. End-labelled single-stranded DNA targets of different lengths were used for hybridization with perfect-match oligonucleotide probe sets targeting different positions of the same molecule. Hybridization results illustrated that probes targeting the labelled terminus of the target showed significantly higher signals than probes targeting other regions. This effect was independent of the target gene, the fluorophore and the slide surface chemistry. Comparison of microarray signal patterns of fluorescently end-labelled, fluorescently internally random-labelled and radioactively end-labelled target-DNAs with the same set of oligonucleotide probes identified POL as a critical factor affecting signal intensity rather than binding efficiency. Our observations define a novel determinant for large differences of signal intensities. Application of the POL effect may contribute to better probe design and data interpretation in microarray applications.

DNA, Single-Stranded↗

PubMed2005

Physical map of the Azoarcus sp. strain BH72 genome based on a bacterial artificial chromosome library as a platform for genome sequencing and functional analysis.

Azoarcus sp. strain BH72 is a Gram-negative proteobacterium of the beta subclass; it is a diazotrophic endophyte of graminaceous plants and can provide significant amounts of fixed nitrogen to its host plant Kallar grass. We aimed to obtain a physical map of the Azoarcus sp. strain BH72 chromosome to be directly used in functional analysis and as a part of an Azoarcus sp. BH72 genome project. A bacterial artificial chromosome (BAC) library was constructed and analysed. A representative physical map with a high density of marker genes was developed in which 64 aligned BAC clones covered almost the entire genome.

Azoarcus↗

PubMed2005

Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice.

Cultivation-independent studies suggest that roots of rice (Oryza sativa) are colonized by a diverse community of nitrogen-fixing bacteria. Here we report for the first time mRNA-based profiling of nitrogenase (nifH) genes, to study the impact of lowland-rice genotypes at the cultivar level on the functional diversity of root-associated diazotrophs. Root RNA extracts from all plants contained nifH mRNA at levels detectable by reverse transcription polymerase chain reaction (RT-PCR). Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed only small interplant variations. However, RNA- and DNA-based profiles obtained from the same root extractions differed from each other, suggesting that presence of diazotrophs did not necessarily coincide with active transcription of nif genes. Application of N-fertilizer at planting had a long-term effect on the profile of expressed nitrogenase genes. Phylogenetic analysis of a clone library constructed for nifH fragments expressed in wild species of rice roots indicated that active diazotrophs were not related to cultured strains. The composition of active diazotrophic communities was compared for six related cultivars of O. sativa, wild species Oryza brachyantha, and a genetic cross between it and cv. IR56, grown under identical conditions in rice field soil in the Philippines without N-fertilizer application. Remarkable varietal differences in root associated nifH-gene expressing communities were detected. This underlines the importance of mRNA-based approaches to study functional diversity and eventually identify key diazotrophs in a particular environment.

Bacteria↗

PubMed2003

Azoarcus sp. strain BH72 as a model for nitrogen-fixing grass endophytes.

The availability of nitrogen often limits plant growth in terrestrial ecosystems. The only biological reaction counterbalancing the loss of N from soils or ecosystems is biological nitrogen fixation, the enzymatic reduction of N2 to ammonia. Some gramineous crops such as certain Brazilian sugar cane cultivars or Kallar grass can derive a substantial part of the plant nitrogen from biological nitrogen fixation. Our research on grass-associated diazotrophs focuses on endophytic bacteria, microorganisms that multiply and spread inside plants without causing damage of the host plants or conferring an ecological threat to the plant. This review summarizes the current knowledge on the diazotrophic endophyte Azoarcus sp. BH72, which is capable of colonizing the interior of rice roots, one of the globally most important crops.

Azoarcus↗

PubMed2003

Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice.

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N-fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T-RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10-fold range of template concentrations. These results clearly demonstrated that our T-RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic.

Bacteria↗

PubMed2002

Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state.

The extent to which the N2-fixing bacterial endophyte Azoarcus sp. strain BH72 in the rhizosphere of Kallar grass can provide fixed nitrogen to the plant was assessed by evaluating inoculated plants grown in the greenhouse and uninoculated plants taken from the natural environment. The inoculum consisted of either wild-type bacteria or nifK- mutant strain BHNKD4. In N2-deficient conditions, plants inoculated with strain BH72 (N2-fixing test plants) grew better and accumulated more nitrogen with a lower delta15N signature after 8 months than did plants inoculated with the mutant strain (non-N2-fixing control plants). Polyadenylated or polymerase chain reaction-amplified BH72 nifH transcripts were retrieved from test but not from control plants. BH72 nifH transcripts were abundant. The inocula could not be reisolated. These results indicate that Azoarcus sp. BH72 can contribute combined N2 to the plant in an unculturable state. Abundant BH72 nifH transcripts were detected also in uninoculated plants taken from the natural environment, from which Azoarcus sp. BH72 also could not be isolated. Quantification of nitrogenase gene transcription indicated a high potential of strain BH72 for biological N2 fixation in association with roots. Phylogenetic analysis of nitrogenase sequences predicted that uncultured grass endophytes including Azoarcus spp. are ecologically dominant and play an important role in N2-fixation in natural grass ecosystems.

Azoarcus↗