Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571.
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ABSTRACT:
SUBMITTER: Liu X
PROVIDER: S-EPMC7750401 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. RESULTS: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. CONCLUSION: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.
| S-EPMC2443382 | biostudies-literature
Project description:Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells.
| S-EPMC3416630 | biostudies-literature
Project description:Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.
| S-EPMC7141136 | biostudies-literature
Project description:Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.
| S-EPMC205018 | biostudies-other
Project description:The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.
| S-EPMC2725507 | biostudies-literature
Project description:The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.
| S-EPMC2075074 | biostudies-literature
Project description:The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37?kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ?cheY1 mutant exhibited decreased frequency of direction reversal, whereas the ?cheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ?cheY1 and ?cheY2 mutants was lower than that in the WT, but the ?cheY2 mutant had more obvious EPS defects that were similar to those of the ?cheY1 ?cheY2 and ?eps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ?cheY1 and ?cheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ?cheY2 mutant was severely impaired compared to that of the ?cheY1 mutant. Taken together, the ?cheY2 phenotypes are more severe than the ?cheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ?cheY2 single-mutant phenotype. These defects of ?cheY1 and ?cheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCE Azorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.
| S-EPMC7376556 | biostudies-literature
Project description:Bacterial signal transduction pathways are important for a variety of adaptive responses to environment, such as two-component systems (TCSs). In this paper, we reported the characterization of a transcriptional regulator in Azorhizobium caulinodans ORS571, ActR, with an N-terminal receiver domain and one C-terminal OmpR/PhoB-type DNA binding domain. Sequence analysis showed that ActR shared a high similarity with FtcR regulator of Brucella melitensis 16M known to be involved in flagellar regulation. The structural gene of this regulator was largely distributed in Alphaproteobacteria, in particular in Rhizobiales and Rhodobacterales, and was located within clusters of genes related to motility functions. Furthermore, we studied the biological function of ActR in A. caulinodans grown at the free-living state or in association with Sesbania rostrata by constructing actR gene deletion mutant. In the free-living state, the bacterial flagellum and motility ability were entirely deleted, the expression of flagellar genes was downregulated; and the exopolysaccharide production, biofilm formation, and cell flocculation decreased significantly compared with those of the wild-type strain. In the symbiotic state, ΔactR mutant strain showed weakly competitive colonization and nodulation on the host plant. These results illustrated that FtcR-like regulator in A. caulinodans is involved in flagellar biosynthesis and provide bacteria with an effective competitive nodulation for symbiosis. These findings improved our knowledge of FtcR-like transcriptional regulator in A. caulinodans.
| S-EPMC8639532 | biostudies-literature
Project description:This study focuses on the function of the gene praR that encodes a putative transcription factor in Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata. The praR gene is a homolog of the phrR gene of Sinorhizobium medicae WSM419, and the praR and phrR homologs are distributed throughout the class Alphaproteobacteria. The growth and nitrogen fixation activity of an A. caulinodans praR deletion mutant in the free-living state were not significantly different from those of the wild-type strain. However, the stem nodules formed by the praR mutant showed lower nitrogen fixation activity than the wild-type stem nodules. Microscopy revealed that infected host cells with an oval or elongated shape were observed at early stages in the nodules formed by the praR mutant, but these infected cells gradually fell into two types. One maintained an oval or elongated shape, but the vacuoles in these cells gradually enlarged and the bacteria gradually disappeared. The other cells were shrunken with bacteria remaining inside. Microarrays revealed that genes homologous to the reb genes of Caedibacter taeniospiralis were highly expressed in the praR mutant. Furthermore, the stem nodules formed by an A. caulinodans mutant with a deletion of praR and reb-homologous genes showed high nitrogen fixation activity, comparable to that of the wild-type stem nodules, and were filled with oval or elongated host cells. These results suggest that PraR controls the expression of the reb-homologous genes and that high expression of reb-homologous genes causes aberrance in A. caulinodans-S. rostrata symbiosis.
| S-EPMC2876453 | biostudies-literature
Project description:BackgroundSwitchgrass (Panicum virgatum L.) is an important biofuel crop that may contribute to replacing petroleum fuels. However, slow seedling growth and soil salinization affect the growth and development of switchgrass. An increasing number of studies have shown that beneficial microorganisms promote plant growth and increase tolerance to salinity stress. However, the feasibility of inoculating switchgrass with Azorhizobium caulinodans ORS571 to enhance the growth and salt tolerance of its seedlings is unclear. Our previous study showed that A. caulinodans ORS571 could colonize wheat (Triticum aestivum L.) and thereby promote its growth and development and regulate the gene expression levels of microRNAs (miRNAs).ResultsIn this study, we systematically studied the impact of A. caulinodans ORS571 on switchgrass growth and development and the response to salinity stress; we also studied the underlying mechanisms during these biological processes. Inoculation with A. caulinodans ORS571 significantly alleviated the effect of salt stress on seedling growth. Under normal conditions, A. caulinodans ORS571 significantly increased fresh plant weight, chlorophyll a content, protein content, and peroxidase (POD) activity in switchgrass seedlings. Under salt stress, the fresh weight, dry weight, shoot and root lengths, and chlorophyll contents were all significantly increased, and some of these parameters even recovered to normal levels after inoculation with A. caulinodans ORS571. Soluble sugar and protein contents and POD and superoxide dismutase (SOD) activities were also significantly increased, contrary to the results for proline. Additionally, A. caulinodans ORS571 may alleviate salt stress by regulating miRNAs. Twelve selected miRNAs were all upregulated to different degrees under salt stress in switchgrass seedlings. However, the levels of miR169, miR171, miR319, miR393, miR535, and miR854 were decreased significantly after inoculation with A. caulinodans ORS571 under salt stress, in contrast to the expression level of miR399.ConclusionThis study revealed that A. caulinodans ORS571 increased the salt tolerance of switchgrass seedlings by increasing their water content, photosynthetic efficiency, osmotic pressure maintenance, and reactive oxygen species (ROS) scavenging abilities and regulating miRNA expression. This work provides a new, creative idea for improving the salt tolerance of switchgrass seedlings.
| S-EPMC9983177 | biostudies-literature