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.

The effect of Azorhizobium caulinodans ORS571 and γ-aminobutyric acid on salt tolerance of Sesbania rostrata.

Salt stress seriously affects plant growth and crop yield, and has become an important factor that threatens the soil quality worldwide. In recent years, the cultivation of salt-tolerant plants such as Sesbania rostrata has a positive effect on improving coastal saline-alkali land. Microbial inoculation and GABA addition have been shown to enhance the plant tolerance in response to the abiotic stresses, but studies in green manure crops and the revelation of related mechanisms are not clear. In this study, the effects of inoculation with Azorhizobium caulinodans ORS571 and exogenous addition of γ-Aminobutyric Acid (GABA; 200 mg·L-1) on the growth and development of S. rostrata under salt stress were investigated using potting experiments of vermiculite. The results showed that inoculation with ORS571 significantly increased the plant height, biomass, chlorophyll content, proline content (PRO), catalase (CAT) activity, and superoxide dismutase (SOD) activity of S. rostrata and reduced the malondialdehyde (MDA) level of leaves. The exogenous addition of GABA also increased the height, biomass, and CAT activity and reduced the MDA and PRO level of leaves. In addition, exogenous addition of GABA still had a certain improvement on the CAT activity and chlorophyll content of the ORS571-S. rostrata symbiotic system. In conclusion, ORS571 inoculation and GABA application have a positive effect on improving the salt stress tolerance in S. rostrata, which are closely associated with increasing chlorophyll synthesis and antioxidant enzyme activity and changing the amino acid content. Therefore, it can be used as a potential biological measure to improve the saline-alkali land.

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.

The FtcR-Like Protein ActR in Azorhizobium caulinodans ORS571 Is Involved in Bacterial Motility and Symbiosis With the Host Plant.

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.

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.

The Hypoxia-Associated Localization of Chemotaxis Protein CheZ in Azorhizorbium caulinodans.

Spatial organization of chemotactic proteins is important for cooperative response to external stimuli. However, factors affecting the localization dynamics of chemotaxis proteins are less studied. According to some reports, the polar localization of chemotaxis system I is induced by hypoxia and starvation in Vibrio cholerae. However, in V. cholerae, the chemotaxis system I is not involved in flagellum-mediated chemotaxis, and it may play other alternative cellular functions. In this study, we found that the polar localization of CheZ, a phosphatase regulating chemotactic movement in Azorhizobium caulinodans ORS571, can also be affected by hypoxia and cellular energy-status. The conserved phosphatase active site D165 and the C-terminus of CheZ are essential for the energy-related localization, indicating a cross link between hypoxia-related localization changes and phosphatase activity of CheZ. Furthermore, three of five Aer-like chemoreceptors containing PAS domains participate in the cellular localization of CheZ. In contrast to carbon starvation, free-living nitrogen fixation can alleviate the role of nitrogen limitation and hypoxia on polar localization of CheZ. These results showed that the localization changes induced by hypoxia might be a strategy for bacteria to adapt to complex environment.

Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571.

Chemotaxis is essential for the competitiveness of motile bacteria in complex and harsh environments. The localization of chemotactic proteins in the cell is critical for coordinating a maximal response to chemotactic signals. One chemotaxis protein with a well-defined subcellular localization is the phosphatase CheZ. CheZ localizes to cell poles by binding with CheA in Escherichia coli and other enteric bacteria, or binding with a poorly understood protein called ChePep in epsilon-Proteobacteria. In alpha-Proteobacteria, CheZ lacks CheA-binding sites, and its cellular localization remains unknown. We therefore determined the localization of CheZ in the alpha-Proteobacteria Azorhizobium caulinodans ORS571. A. caulinodans CheZ, also termed as CheZAC, was found to be located to cell poles independently of CheA, and we suspect that either the N-terminal helix or the four-helix bundle of CheZAC is sufficient to locate to cell poles. We also found a novel motif, AXXFQ, which is adjacent to the phosphatase active motif DXXXQ, which effects the monopolar localization of CheZAC. This novel motif consisting of AXXFQ is conserved in CheZ and widely distributed among Proteobacteria. Finally, we found that the substitution of phosphatase active site affects the polar localization of CheZAC. In total, this work characterized the localization pattern of CheZ containing a novel motif, and we mapped the regions of CheZAC that are critical for its polar localization.

Effects of Calcium and Signal Sensing Systems on Azorhizobium caulinodans Biofilm Formation and Host Colonization.

Biofilm formation is important for establishing plants-microbe associations. The role of calcium on biofilm formation has been studied in many bacteria except rhizobia. In this study, we investigated the role of calcium for biofilm formation in Azorhizobium caulindans, which forms nodules in the stem and root of its host plant Sesbania rostrata. We found that calcium is essential for A. caulindans biofilm formation, in addition to the presence of extracellular matrix components, eDNA and proteins. Also, calcium-mediated biofilm formation was tested with chemotaxis, motility, cyclic di-GMP synthesis, and quorum sensing mutants. Finally, calcium was found to promote S. rostrata root colonization of A. caulinodans. In total, these results show that calcium is essential for A. caulindans biofilm formation, and it affects the interaction between A. caulinodans and host plant.

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.

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.

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.

Localization of the reb operon expression is inconsistent with that of the R-body production in the stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR.

Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.

Alkyl hydroperoxide reductase is important for oxidative stress resistance and symbiosis in Azorhizobium caulinodans.

Reactive oxygen species (ROS) are not only toxic products of oxygen from aerobic metabolism or stress but also signalling molecules involved in the development of the legume-Rhizobium symbiosis. To assess the importance of alkyl hydroperoxide reductase (AhpCD) in the nitrogen-fixating bacterium Azorhizobium caulinodans, we investigated the phenotypes of the ∆ahpCD strain with regards to ROS resistance and symbiotic interactions with Sesbania rostrata. The ∆ahpCD strain was notably more sensitive than its parent strain to hydrogen peroxide (H2O2) but not to two organic peroxides, in the early log phase. The expression of ahpCD was not controlled by a LysR-type transcriptional activator either in vitro or in vivo. The catalase activity of the ∆ahpCD strain was affected at a relatively low level of H2O2 stress. Furthermore, the ∆ahpCD strain induced a reduced number of stem nodules in S. rostrata with lowering of nitrogenase activity. These data suggest that A. caulinodans AhpCD is not only important for H2O2 detoxification in vitro but also critical for symbiosis with S. rostrata. Functional analysis of AhpCD is worth investigating in other rhizobia to gain a comprehensive view of its contributions to ROS defence and symbiotic association with legumes.