The volatile organic compound acetoin enhances the colonization of Azorhizobium caulinodans ORS571 on Sesbania rostrata.

Chemoreceptors play a crucial role in assisting bacterial sensing and response to environmental stimuli. Genome analysis of Azorhizobium caulinodans ORS571 revealed the presence of 43 putative chemoreceptors, but their biological functions remain largely unknown. In this study, we identified the chemoreceptor AmaP (methyl-accepting protein of A. caulinodans), characterized by the presence of the CHASE3 domain and exhibited a notable response to acetoin. Thus, we investigated the effect of acetoin sensing on its symbiotic association with the host. Our findings uncovered a compelling role for acetoin as a key player in enhancing various facets of A. caulinodans ORS571's performance including biofilm formation, colonization, and nodulation abilities. Notably, acetoin bolstered A. caulinodans ORS571's efficacy in promoting the growth of S. rostrata, even under moderate salt stress conditions. This study not only broadens our understanding of the AmaP protein with its distinctive CHASE3 domain but also highlights the promising potential of acetoin in fortifying the symbiotic relationship between A. caulinodans and Sesbania rostrata.

Expression and Characterization of a New Self-Sufficient P450 Monooxygenase (P450 AZC1) from Azorhizobium caulinodans.

Oxyfunctionalization of non-activated carbon bonds by P450 monooxygenases has drawn great industrial attraction. Self-sufficient P450s containing catalytic heme and reductase domains in a single polypeptide chain offer many advantages since they do not require external electron transfer partners. Here, we report the first P450 enzyme identified and expressed from Azorhizobium caulinodans. Firstly, expression conditions of P450 AZC1 were optimized for enhanced expression in E.coli. The highest P450 content was obtained in E.coli Rosetta DE3 plysS when it was incubated in TB media supplemented with 0.75 mM IPTG, 0.5 mM ALA, and 0.75 mM FeCl3 at 25 °C for 24 hours. Subsequently, the purified enzyme showed a broad substrate spectrum including fatty acids, linear and cyclic alkanes, aromatics, and pharmaceuticals. Finally, P450 AZC1 showed optimal activity at pH 6.0 and 40 °C and a broad pH and temperature profile, making it a promising candidate for industrial applications.

Systematic Analysis of Lysine Acetylation Reveals Diverse Functions in Azorhizobium caulinodans Strain ORS571.

Protein acetylation can quickly modify the physiology of bacteria to respond to changes in environmental or nutritional conditions, but little information on these modifications is available in rhizobia. In this study, we report the lysine acetylome of Azorhizobium caulinodans strain ORS571, a model rhizobium isolated from stem nodules of the tropical legume Sesbania rostrata that is capable of fixing nitrogen in the free-living state and during symbiosis. Antibody enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used to characterize the acetylome. There are 2,302 acetylation sites from 982 proteins, accounting for 20.8% of the total proteins. Analysis of the acetylated motifs showed the preferences for the amino acid residues around acetylated lysines. The response regulator CheY1, previously characterized to be involved in chemotaxis in strain ORS571, was identified as an acetylated protein, and a mutation of the acetylated site of CheY1 significantly impaired the strain's motility. In addition, a Zn+-dependent deacetylase (AZC_0414) was characterized, and the construction of a deletion mutant strain showed that it played a role in chemotaxis. Our study provides the first global analysis of lysine acetylation in ORS571, suggesting that acetylation plays a role in various physiological processes. In addition, we demonstrate its involvement in the chemotaxis process. The acetylome of ORS571 provides insights to investigate the regulation mechanism of rhizobial physiology. IMPORTANCE Acetylation is an important modification that regulates protein function and has been found to regulate physiological processes in various bacteria. The physiology of rhizobium A. caulinodans ORS571 is regulated by multiple mechanisms both when free living and in symbiosis with the host; however, the regulatory role of acetylation is not yet known. Here, we took an acetylome-wide approach to identify acetylated proteins in A. caulinodans ORS571 and performed clustering analyses. Acetylation of chemotaxis proteins was preliminarily investigated, and the upstream acetylation-regulating enzyme involved in chemotaxis was characterized. These findings provide new insights to explore the physiological mechanisms of rhizobia.

A Novel Module Promotes Horizontal Gene Transfer in Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICEAc) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICEAc, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICEAc and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host's flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICEAc by 18.5%. We propose the feasibility of constructing a minimal ICEAc element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.

Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.

Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH3) assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we tested two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation and NH3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses" where N2 fixation and NH3 excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.

Engineered plant control of associative nitrogen fixation.

Engineering N2-fixing symbioses between cereals and diazotrophic bacteria represents a promising strategy to sustainably deliver biologically fixed nitrogen (N) in agriculture. We previously developed novel transkingdom signaling between plants and bacteria, through plant production of the bacterial signal rhizopine, allowing control of bacterial gene expression in association with the plant. Here, we have developed both a homozygous rhizopine producing (RhiP) barley line and a hybrid rhizopine uptake system that conveys upon our model bacterium Azorhizobium caulinodans ORS571 (Ac) 103-fold improved sensitivity for rhizopine perception. Using this improved genetic circuitry, we established tight rhizopine-dependent transcriptional control of the nitrogenase master regulator nifA and the N metabolism σ-factor rpoN, which drove nitrogenase expression and activity in vitro and in situ by bacteria colonizing RhiP barley roots. Although in situ nitrogenase activity was suboptimally effective relative to the wild-type strain, activation was specific to RhiP barley and was not observed on the roots of wild-type plants. This work represents a key milestone toward the development of a synthetic plant-controlled symbiosis in which the bacteria fix N2 only when in contact with the desired host plant and are prevented from interaction with nontarget plant species.

Azorhizobium caulinodans Chemotaxis Is Controlled by an Unusual Phosphorelay Network.

Azorhizobium caulinodans is a nitrogen-fixing bacterium that forms root nodules on its host legume, Sesbania rostrata. This agriculturally significant symbiotic relationship is important in lowland rice cultivation and allows nitrogen fixation under flood conditions. Chemotaxis plays an important role in bacterial colonization of the rhizosphere. Plant roots release chemical compounds that are sensed by bacteria, triggering chemotaxis along a concentration gradient toward the roots. This gives motile bacteria a significant competitive advantage during root surface colonization. Although plant-associated bacterial genomes often encode multiple chemotaxis systems, A. caulinodans appears to encode only one. The che cluster on the A. caulinodans genome contains cheA, cheW, cheY2, cheB, and cheR. Two other chemotaxis genes, cheY1 and cheZ, are located independently from the che operon. Both CheY1 and CheY2 are involved in chemotaxis, with CheY1 being the predominant signaling protein. A. caulinodans CheA contains an unusual set of C-terminal domains: a CheW-like/receiver pair (termed W2-Rec) follows the more common single CheW-like domain. W2-Rec impacts both chemotaxis and CheA function. We found a preference for transfer of phosphoryl groups from CheA to CheY2, rather than to W2-Rec or CheY1, which appears to be involved in flagellar motor binding. Furthermore, we observed increased phosphoryl group stabilities on CheY1 compared to CheY2 and W2-Rec. Finally, CheZ enhanced dephosphorylation of CheY2 substantially more than CheY1 but had no effect on the dephosphorylation rate of W2-Rec. This network of phosphotransfer reactions highlights a previously uncharacterized scheme for regulation of chemotactic responses. IMPORTANCE Chemotaxis allows bacteria to move toward nutrients and away from toxins in their environment. Chemotactic movement provides a competitive advantage over nonspecific motion. CheY is an essential mediator of the chemotactic response, with phosphorylated and unphosphorylated forms of CheY differentially interacting with the flagellar motor to change swimming behavior. Previously established schemes of CheY dephosphorylation include action of a phosphatase and/or transfer of the phosphoryl group to another receiver domain that acts as a sink. Here, we propose that A. caulinodans uses a concerted mechanism in which the Hpt domain of CheA, CheY2, and CheZ function together as a dual sink system to rapidly reset chemotactic signaling. To the best of our knowledge, this mechanism is unlike any that have previously been evaluated. Chemotaxis systems that utilize both receiver and Hpt domains as phosphate sinks likely occur in other bacterial species.

Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans.

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant.

BACKGROUND: A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. RESULTS: In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. CONCLUSIONS: Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

Glutaredoxins Play an Important Role in the Redox Homeostasis and Symbiotic Capacity of Azorhizobium caulinodans ORS571.

Glutaredoxin (GRX) plays an essential role in the control of the cellular redox state and related pathways in many organisms. There is limited information on GRXs from the model nitrogen (N2)-fixing bacterium Azorhizobium caulinodans. In the present work, we identified and performed functional analyses of monothiol and dithiol GRXs in A. caulinodans in the free-living state and during symbiosis with Sesbania rostrata. Our data show that monothiol GRXs may be very important for bacterial growth under normal conditions and in response to oxidative stress due to imbalance of the redox state in grx mutants of A. caulinodans. Functional redundancies were also observed within monothiol and dithiol GRXs in terms of different physiological functions. The changes in catalase activity and iron content in grx mutants were assumed to favor the maintenance of bacterial resistance against oxidants, nodulation, and N2 fixation efficiency in this bacterium. Furthermore, the monothiol GRX12 and dithiol GRX34 play a collective role in symbiotic associations between A. caulinodans and Sesbania rostrata. Our study provided systematic evidence that further investigations are required to understand the importance of glutaredoxins in A. caulinodans and other rhizobia.

CRISPR/Cas9 genome editing shows the important role of AZC_2928 gene in nitrogen-fixing bacteria of plants.

AZC_2928 gene (GenBank accession no. BAF88926.1) of Azorhizobium caulinodans ORS571 has sequence homology to 2,3-aminomutases. However, its function is unknown. In this study, we are for the first time to knock out the gene completely in A. caulinodans ORS571 using the current advanced genome editing tool, CRISPR/Cas9. Our results show that the editing efficiency is 34% and AZC_2928 plays an extremely important role in regulating the formation of chemotaxis and biofilm. CRISPR/Cas9 knockout of AZC_2928 (△AZC_2928) significantly enhanced chemotaxis and biofilm formation. Both chemotaxis and biofilm formation play an important role in nitrogen-fixing bacteria and their interaction with their host plants. Interestingly, AZC_2928 did not affect the motility of A. caulinodans ORS571 and the nodulation formation in their natural host plant, Sesbania rostrata. Due to rhizobia needing to form bacteroids for symbiotic nitrogen fixation in mature nodules, AZC_2928 might have a direct influence on nitrogen fixation efficiency rather than the number of nodulations.

CheY1 and CheY2 of Azorhizobium caulinodans ORS571 Regulate Chemotaxis and Competitive Colonization with the Host Plant.

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 ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 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.IMPORTANCEAzorhizobium 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.

Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571.

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.

Azorhizobium caulinodans c-di-GMP phosphodiesterase Chp1 involved in motility, EPS production, and nodulation of the host plant.

Establishment of the rhizobia-legume symbiosis is usually accompanied by hydrogen peroxide (H2O2) production by the legume host at the site of infection, a process detrimental to rhizobia. In Azorhizobium caulinodans ORS571, deletion of chp1, a gene encoding c-di-GMP phosphodiesterase, led to increased resistance against H2O2 and to elevated nodulation efficiency on its legume host Sesbania rostrata. Three domains were identified in the Chp1: a PAS domain, a degenerate GGDEF domain, and an EAL domain. An in vitro enzymatic activity assay showed that the degenerate GGDEF domain of Chp1 did not have diguanylate cyclase activity. The phosphodiesterase activity of Chp1 was attributed to its EAL domain which could hydrolyse c-di-GMP into pGpG. The PAS domain functioned as a regulatory domain by sensing oxygen. Deletion of Chp1 resulted in increased intracellular c-di-GMP level, decreased motility, increased aggregation, and increased EPS (extracellular polysaccharide) production. H2O2-sensitivity assay showed that increased EPS production could provide ORS571 with resistance against H2O2. Thus, the elevated nodulation efficiency of the ∆chp1 mutant could be correlated with a protective role of EPS in the nodulation process. These data suggest that c-di-GMP may modulate the A. caulinodans-S. rostrata nodulation process by regulating the production of EPS which could protect rhizobia against H2O2.

Control of nitrogen fixation in bacteria that associate with cereals.

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

LuxR-Type Regulator AclR1 of Azorhizobium caulinodans Regulates Cyclic di-GMP and Numerous Phenotypes in Free-Living and Symbiotic States.

LuxR-type regulators play important roles in transcriptional regulation in bacteria and control various biological processes. A genome sequence analysis showed the existence of seven LuxR-type regulators in Azorhizobium caulinodans ORS571, an important nitrogen-fixing bacterium in both its free-living state and in symbiosis with its host, Sesbania rostrata. However, the functional mechanisms of these regulators remain unclear. In this study, we identified a LuxR-type regulator that contains a cheY-homologous receiver (REC) domain in its N terminus and designated it AclR1. Interestingly, phylogenetic analysis revealed that AclR1 exhibited relatively close evolutionary relationships with MalT/GerE/FixJ/NarL family proteins. Functional analysis of an aclR1 deletion mutant (ΔaclR1) in the free-living state showed that AclR1 positively regulated cell motility and flocculation but negatively regulated exopolysaccharide production, biofilm formation, and second messenger cyclic diguanylate (c-di-GMP)-related gene expression. In the symbiotic state, the ΔaclR1 mutant was defective in competitive colonization and nodulation on host plants. These results suggested that AclR1 could provide bacteria with the ability to compete effectively for symbiotic nodulation. Overall, our results show that the REC-LuxR-type regulator AclR1 regulates numerous phenotypes both in the free-living state and during host plant symbiosis.

Inactivation of the Phosphatase CheZ Alters Cell-Surface Properties of Azorhizobium caulinodans ORS571 and Symbiotic Association with Sesbania rostrata.

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.

Bacterioferritin comigratory protein is important in hydrogen peroxide resistance, nodulation, and nitrogen fixation in Azorhizobium caulinodans.

Reactive oxygen species are not only harmful for rhizobia but also required for the establishment of symbiotic interactions between rhizobia and their legume hosts. In this work, we first investigated the preliminary role of the bacterioferritin comigratory protein (BCP), a member of the peroxiredoxin family, in the nitrogen-fixing bacterium Azorhizobium caulinodans. Our data revealed that the bcp-deficient strain of A. caulinodans displayed an increased sensitivity to inorganic hydrogen peroxide (H2O2) but not to two organic peroxides in a growth-phase-dependent manner. Meanwhile, BCP was found to be involved in catalase activity under relatively low H2O2 conditions. Furthermore, nodulation and N2 fixation were significantly impaired by mutation of the bcp gene in A. caulinodans. Our work initially documented the importance of BCP in the bacterial defence against H2O2 in the free-living stage of rhizobia and during their symbiotic interactions with legumes. Molecular signalling in vivo is required to decipher the holistic functions of BCP in A. caulinodans as well as in other rhizobia.

A Dual Role of Amino Acids from Sesbania rostrata Seed Exudates in the Chemotaxis Response of Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans ORS571 can induce nodule formation on the roots and the stems of its host legume, Sesbania rostrata. Plant exudates are essential in the dialogue between microbes and their host plant and, in particular, amino acids can play an important role in the chemotactic response of bacteria. Histidine, arginine, and aspartate, which are the three most abundant amino acids present in S. rostrata seed exudates, behave as chemoattractants toward A. caulinodans. A position-specific-iterated BLAST analysis of the methyl-accepting chemotaxis proteins (MCPs) (chemoreceptors) in the genome of A. caulinodans was performed. Among the 43 MCP homologs, two MCPs harboring a dCache domain were selected as possible cognate amino acid MCPs. After analysis of relative gene expression levels and construction of a gene-deleted mutant strain, one of them, AZC_0821 designed as TlpH, was confirmed to be responsible for the chemotactic response to the three amino acids. In addition, it was found that these three amino acids can also influence chemotaxis of A. caulinodans independently of the chemosensory receptors, by being involved in the increase of the expression level of several che and fla genes involved in the chemotaxis pathway and flagella synthesis. Thus, the contribution of amino acids present in seed exudates is directly related to the role as chemoattractants and indirectly related to the role in the regulation of expression of key genes involved in chemotaxis and motility. This "dual role" is likely to influence the formation of biofilms by A. caulinodans and the host root colonization properties of this bacterium.