Environmental factors affecting the expression of pilAB as well as the proteome and transcriptome of the grass endophyte Azoarcus sp. strain BH72.
ABSTRACT: 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.
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. Overall design: Analysis used RNA from the early exponential growth phase as control samples for comparison to the quorum-sensing condition samples taken at one hour and four hours after incubation with cell-free culture supernatants.
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. Analysis used RNA from the early exponential growth phase as control samples for comparison to the quorum-sensing condition samples taken at one hour and four hours after incubation with cell-free culture supernatants.
Project description:Research on the interaction between the non-nodule-forming bacterial endophytes and their host plants is still in its infancy. Especially the understanding of plant control mechanisms which govern endophytic colonization is very limited. The current study sets out to determine which hormonal signaling pathway controls endophytic colonization in rice, and whether the mechanisms deviate for a pathogen. The endophyte Azoarcus olearius BH72-rice model was used to investigate root responses to endophytes in comparison to the recently established pathosystem of rice blight Xanthomonas oryzae pv. oryzae PXO99 (Xoo) in flooded roots. In the rice root transcriptome, 523 or 664 genes were found to be differentially expressed in response to Azoarcus or Xoo colonization, respectively; however, the response was drastically different, with only 6% of the differentially expressed genes (DEGs) overlapping. Overall, Xoo infection induced a much stronger defense reaction than Azoarcus colonization, with the latter leading to down-regulation of many defense related DEGs. Endophyte-induced DEGs encoded several enzymes involved in phytoalexin biosynthesis, ROS (reactive oxygen species) production, or pathogenesis-related (PR) proteins. Among putative plant markers related to signal transduction pathways modulated exclusively during Azoarcus colonization, none overlapped with previously published DEGs identified for another rice endophyte, Azospirillum sp. B510. This suggests a large variation in responses of individual genotypic combinations. Interestingly, the DEGs related to jasmonate (JA) signaling pathway were found to be consistently activated by both beneficial endophytes. In contrast, the salicylate (SA) pathway was activated only in roots infected by the pathogen. To determine the impact of SA and JA production on root colonization by the endophyte and the pathogen, rice mutants with altered hormonal responses were employed: mutant cpm2 deficient in jasmonate synthesis, and RNA interference (RNAi) knockdown lines of NPR1 decreased in salicylic acid-mediated defense responses (NPR1-kd). Only in cpm2, endophytic colonization of Azoarcus was significantly increased, while Xoo colonization was not affected. Surprisingly, NPR1-kd lines showed slightly decreased colonization by Xoo, contrary to published results for leaves. These outcomes suggest that JA but not SA signaling is involved in controlling the Azoarcus endophyte density in roots and can restrict internal root colonization, thereby shaping the beneficial root microbiome.
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. RNA from cells grown on synthetic medium for 1 and 4 hours respectively in presence (experiment) and absence (control) of exudates was used for two color whole genome microarray approach.
Project description:BACKGROUND: The 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 FINDINGS: A 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 ?(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/SIGNIFICANCE: Our 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.
Project description:Low oxygen tensions are often encountered in flooded soils of rice fields by root-associated, strictly respiratory, beta proteobacterium, Azoarcus sp. BH72 which fixes nitrogen only under microaerobic condition. In this study, genome wide oligonucleotide microarrays were used compare the global transcription profile of Azoarcus sp. BH72 under microaerobic condition with cells grown under aerobic condition, both with ammonia as sole nitrogen source. The outcome of this study will provide a better insight about the establishment of this endophyte in the microaerobic environment, probably prevailing inside of the rice root niche . RNA from cells grown under microaerobic condition with 0.3% oxygen (experiment) and aerobic condition with 21% oxygen (control), respectively was used for two color whole genome microarray approach.
Project description:DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.
Project description:P(II)-like signal transmitter proteins, found in Bacteria, Archaea, and plants, are known to mediate control of carbon and nitrogen assimilation. They indirectly regulate the activity of key metabolic enzymes and transcription factors by protein-protein interactions with signal transduction proteins. Many Proteobacteria harbor two paralogous P(II)-like proteins, GlnB and GlnK, whereas a novel third P(II) paralogue (GlnY) was recently identified in Azoarcus sp. strain BH72, a diazotrophic endophyte of grasses. In the present study, evidence was obtained that the P(II)-like proteins have distinct roles in mediating nitrogen and oxygen control of nif gene transcription and nitrogenase activity. Full repression of nif gene transcription in the presence of a combined nitrogen source or high oxygen concentrations was observed in wild-type and glnB and glnK knockout mutants, revealing that GlnB and GlnK can complement each other in mediating the repression. In contrast, in a glnBK double mutant strain in the presence of only GlnY, nif gene transcription was still detectable, albeit at a lower level, on nitrate or 20% oxygen. As another level of control, nitrogenase activity was regulated by at least three types of mechanisms in strain BH72: covalent modification of dinitrogenase reductase (NifH), probably by ADP-ribosylation, and two other, unknown means. Functional inactivation upon ammonium addition (switch-off) required the putative high-affinity ammonium transporter AmtB and GlnK, but not GlnB or GlnY. Functional inactivation in response to anaerobiosis did not depend on AmtB, GlnK, or GlnB. In contrast, covalent modification of NifH required both GlnB and GlnK and AmtB as response to ammonium addition, whereas it required either GlnB or GlnK and not AmtB when cells were shifted to anaerobiosis. In a glnBK double mutant expressing only GlnY, NifH modification was completely abolished, further revealing functional differences between the three P(II) paralogues.
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.
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.