Transcriptional profiling of nitrogen fixation and the role of NifA in the diazotrophic endophyte Azoarcus sp. strain BH72
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ABSTRACT:
ORGANISM(S): Azoarcus sp. Azoarcus olearius
PROVIDER: GSE49913 | GEO | 2013/08/15
SECONDARY ACCESSION(S): PRJNA215319
REPOSITORIES: GEO
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Project description:BackgroundThe model endophyte Azoarcus sp. strain BH72 is known to contribute fixed nitrogen to its host Kallar grass and also expresses nitrogenase genes endophytically in rice seedlings. Availability of nitrogen is a signal regulating the transcription of nitrogenase genes. Therefore, we analysed global transcription in response to differences in the nitrogen source.Methodology/principal findingsA DNA microarray, comprising 70-mer oligonucleotides representing 3989 open reading frames of the genome of strain BH72, was used for transcriptome studies. Transcription profiles of cells grown microaerobically on N2 versus ammonium were compared. Expression of 7.2% of the genes was significantly up-regulated, and 5.8% down-regulated upon N2 fixation, respectively. A parallel genome-wide prediction of σ(54)-type promoter elements mapped to the upstream region of 38 sequences of which 36 were modulated under the N2 response. In addition to modulation of genes related to N2 fixation, the expressions of gene clusters that might be related to plant-microbe interaction and of several transcription factors were significantly enhanced. While comparing under N2-fixation conditions the transcriptome of wild type with a nifLA(-) insertion mutant, NifA being the essential transcriptional activator for nif genes, 24.5% of the genome was found to be affected in expression. A genome-wide prediction of 29 NifA binding sequences matched to 25 of the target genes whose expression was differential during microarray analysis, some of which were putatively negatively regulated by NifA. For selected genes, differential expression was corroborated by real time RT-PCR studies.Conclusion/significanceOur data suggest that life under conditions of nitrogen fixation is an important part of the lifestyle of strain BH72 in roots, as a wide range of genes far beyond the nif regulon is modulated. Moreover, the NifA regulon in strain BH72 appears to encompass a wider range of cellular functions beyond the regulation of nif genes.
| S-EPMC3916325 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-08-15 | E-GEOD-49913 | biostudies-arrayexpress
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored.
2013-08-01 | GSE49394 | GEO
Project description:Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.
| S-EPMC9769520 | biostudies-literature
Project description: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.
| S-EPMC95253 | biostudies-literature
Project description:BACKGROUND: Bacterial communication is involved in regulation of cellular mechanisms such as metabolic processes, microbe-host interactions or biofilm formation. In the nitrogen-fixing model endophyte of grasses Azoarcus sp. strain BH72, known cell-cell signaling systems have not been identified; however, the pilA gene encoding the structural protein of type IV pili that are essential for plant colonization appears to be regulated in a population density-dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Our data suggest that pilAB expression is affected by population density, independent of autoinducers typical for gram-negative bacteria, likely depending on unknown secreted molecule(s) that can be produced by different bacterial species. We used transcriptomic and proteomic approaches to identify target genes and proteins differentially regulated in conditioned supernatants in comparison to standard growth conditions. Around 8% of the 3992 protein-coding genes of Azoarcus sp. and 18% of the detected proteins were differentially regulated. Regulatory proteins and transcription factors among the regulated proteins indicated a complex hierarchy. Differentially regulated genes and proteins were involved in processes such as type IV pili formation and regulation, metal and nutrient transport, energy metabolism, and unknown functions mediated by hypothetical proteins. Four of the newly discovered target genes were further analyzed and in general they showed regulation patterns similar to pilAB. The expression of one of them was shown to be induced in plant roots. CONCLUSION/SIGNIFICANCE: This study is the first global approach to initiate characterization of cell density-dependent gene regulation mediated by soluble molecule(s) in the model endophyte Azoarcus sp. strain BH72. Our data suggest that the putative signaling molecule(s) are also produced by other Proteobacteria and might thus be used for interspecies communication. This study provides the foundation for the development of robust reporter systems for Azoarcus sp. to analyze mechanisms and molecules involved in the population-dependent gene expression in this endophyte in future.
| S-EPMC3262810 | biostudies-literature
Project description: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.
| S-EPMC205294 | biostudies-other
Project description:Model endophyte Azoarcus sp. BH72 is known to contribute fixed nitrogen to its host Kallar grass by nitrogen fixation and also expresses nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. Therefore, we analysed global transcription in response to differences in the nitrogen source. Transcription profiles of cells grown microaerobically (0.6% oxygen) on minimal medium with nitrogen (N2-fixing) versus ammonium (combined nitrogen) were compared using a genome-wide microarray approach and differences in the gene expression profile were monitored. RNA from cells grown on nitrogen-free synthetic medium under nitrogen fixation (experiment) and combined nitrogen source as ammonium chloride (control) was used for two-color whole-genome microarray approach.
2013-08-01 | E-GEOD-49394 | biostudies-arrayexpress
Project description:Azoarcus sp. BH72 is able to communicate via cell density-dependent gene regulation. Here, the impact of cell-free conditioned culture supernatants, obtained from stationary phase Azoarcus wild type cultures, on gene expression was investigated determining changes in transcript profiles when early exponential aerobic cultures were incubated with cell-free culture supernatants for one and four hours. Bacterial communication via quorum sensing (QS) is involved in the regulation of several cellular mechanisms such as metabolic processes, microbe-host interactions or biofilm formation. The nitrogen-fixing model endophyte of grasses Azoarcus sp. strain BH72 shows density-dependent gene regulation in the absence of common hydrophobic autoinducers for pilA encoding the structural protein of type IV pili that are essential for plant colonization. Here, we used a transcriptomic approach to identify target genes differentially regulated under QS conditions in conditioned supernatants in comparison to standard growth conditions.
2011-12-01 | GSE25939 | GEO
Project description:Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we investigated whether root exudates of the host might play a role in initiating the endophyte-rice interaction. The whole genome microarray approach was used to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O. sativa cv. Nipponbare in order to identify differentially regulated genes. Azoarcus sp. strain BH72 was grown in the presence or absence of root exudates of Oryza sativa cv. Nipponbare for two different time points, and differences in the gene expression profile were monitored.
2012-11-30 | GSE35187 | GEO