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.

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.

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.

Functional Exploration of the Bacterial Type VI Secretion System in Mutualism: Azorhizobium caulinodans ORS571-Sesbania rostrata as a Research Model.

The bacterial type VI secretion system (T6SS) has been considered the armed force of bacteria because it can deliver toxin effectors to prokaryotic or eukaryotic cells for survival and fitness. Although many legume symbiotic rhizobacteria encode T6SS in their genome, the biological function of T6SS in these bacteria is still unclear. To elucidate this issue, we used Azorhizobium caulinodans ORS571 and its symbiotic host Sesbania rostrata as our research model. By using T6SS gene deletion mutants, we found that T6SS provides A. caulinodans with better symbiotic competitiveness when coinfected with a T6SS-lacking strain, as demonstrated by two independent T6SS-deficient mutants. Meanwhile, the symbiotic effectiveness was not affected by T6SS because the nodule phenotype, nodule size, and nodule nitrogen-fixation ability did not differ between the T6SS mutants and the wild type when infected alone. Our data also suggest that under several lab culture conditions tested, A. caulinodans showed no T6SS-dependent interbacterial competition activity. Therefore, instead of being an antihost or antibacterial weapon of the bacterium, the T6SS in A. caulinodans ORS571 seems to participate specifically in symbiosis by increasing its symbiotic competitiveness.

A cheZ-Like Gene in Azorhizobium caulinodans Is a Key Gene in the Control of Chemotaxis and Colonization of the Host Plant.

Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY∼P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium-plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant.IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY∼P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY∼P remains poorly characterized among Alphaproteobacteria, except for Sinorhizobium meliloti, which does not contain CheZ but which controls CheY∼P dephosphorylation through a phosphate sink mechanism. Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata, has an orphan cheZ gene besides two cheY genes similar to those in S. meliloti In addition to controlling the chemotaxis response, the CheZ-like protein in strain ORS571 is playing a role by decreasing bacterial adhesion to the host plant, in contrast to the general situation where chemotaxis-associated proteins promote adhesion. In this study, we identified a CheZ-like protein among Alphaproteobacteria functioning in chemotaxis and the A. caulinodans-S. rostrata symbiosis.

A Novel Regulatory Pathway for K+ Uptake in the Legume Symbiont Azorhizobium caulinodans in Which TrkJ Represses the kdpFABC Operon at High Extracellular K+ Concentrations.

Bacteria have multiple K+ uptake systems. Escherichia coli, for example, has three types of K+ uptake systems, which include the low-K+-inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K+ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix- nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix+ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix+ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K+ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K+ conditions. The cytoplasmic K+ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdpFABC under high-K+ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K+ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K+ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K+ uptake systems to control the cytoplasmic K+ levels. In many bacteria, the K+ uptake system KdpFABC is expressed under low-K+ conditions. For years, many researchers have argued over how bacteria sense K+ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K+ concentrations, the detailed mechanism of K+ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K+ concentration and that high extracellular K+ concentrations repress the expression of KdpFABC via TrkJ.

Blue light does not inhibit nodulation in Sesbania rostrata.

Earlier, we reported that root nodulation was inhibited by blue light irradiation of Lotus japonicus. Because some legumes do not establish nodules exclusively on underground roots, we investigated whether nodule formation in Sesbania rostrata, which forms both root and "stem" nodules following inoculation with Azorhizobium caulinodans, is inhibited by blue light as are L. japonicus nodules. We found that neither S. rostrata nodulation nor nitrogen fixation was inhibited by blue light exposure. Moreover, although A. caulinodans proliferation was not affected by blue light irradiation, bacterial survival was decreased. Therefore, blue light appears to impose different responses depending on the legume-rhizobial symbiosis.

Migration of endophytic diazotroph Azorhizobium caulinodans ORS571 inside wheat (Triticum aestivum L) and its effect on microRNAs.

Azorhizobium caulinodans ORS571, a novel rhizobium, forms endosymbionts with its nature host Sesbania rostrata, a semi-aquatic leguminous tree. Recent studies showed that A. caulinodans ORS571, as endophytic rhizobium, disseminated and colonized inside of cereal plants. However, how this rhizobium infects monocot plants and the regulatory mechanism remains unknown. MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression at the post-transcriptional levels. In this study, we employed laser scanning confocal microscope to monitor the pathway that rhizobium invade wheat; we also investigated the potential role of miRNAs during A. caulinodans ORS571 infecting wheat. Our results showed that gfp-labeled A. caulinodans ORS571 infected wheat root hairs and emerged lateral roots, then disseminated and colonized within roots and migrated to other plant tissues, such as stems and leaves. Endophytic rhizobium induced the aberrant expression of miRNAs in wheat with a tissue- and time-dependent manner with a peak at 12-24 h after rhizobium infection. Some miRNAs, such as miR167 and miR393 responded more in roots than that in shoots. In contrast, miR171 responded higher in shoots than that in roots. These results suggested that miRNAs could be responsive to A. caulinodans ORS571 infection and played important role in plant growth, nutrient metabolisms, and wheat-rhizobium interactions.

OxyR-regulated catalase activity is critical for oxidative stress resistance, nodulation and nitrogen fixation in Azorhizobium caulinodans.

The legume-rhizobial interaction results in the formation of symbiotic nodules in which rhizobia fix nitrogen. During the process of symbiosis, reactive oxygen species (ROS) are generated. Thus, the response of rhizobia to ROS is important for successful nodulation and nitrogen fixation. In this study, we investigated how Azorhizobium caulinodans, a rhizobium that forms both root and stem nodules on its host plant, regulates ROS resistance. We found that in-frame deletions of a gene encoding the putative catalase-peroxidase katG or a gene encoding a LysR-family regulatory protein, oxyR, exhibited increased sensitivity to H2O2 We then showed that OxyR positively regulated katG expression in an H2O2-independent fashion. Furthermore, we found that deletion of katG or oxyR led to significant reduction in the number of stem nodules and decrease of nitrogen fixation capacities in symbiosis. Our results revealed that KatG and OxyR are not only critical for antioxidant defense in vitro, but also important for nodule formation and nitrogen fixation during interaction with plant hosts.

A Chemotaxis Receptor Modulates Nodulation during the Azorhizobium caulinodans-Sesbania rostrata Symbiosis.

UNLABELLED: Azorhizobium caulinodans ORS571 is a free-living nitrogen-fixing bacterium which can induce nitrogen-fixing nodules both on the root and the stem of its legume host Sesbania rostrata This bacterium, which is an obligate aerobe that moves by means of a polar flagellum, possesses a single chemotaxis signal transduction pathway. The objective of this work was to examine the role that chemotaxis and aerotaxis play in the lifestyle of the bacterium in free-living and symbiotic conditions. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. Here, we characterized a chemoreceptor of A. caulinodans containing an N-terminal PAS domain, named IcpB. IcpB is a soluble heme-binding protein that localized at the cell poles. An icpB mutant strain was impaired in sensing oxygen gradients and in chemotaxis response to organic acids. Compared to the wild-type strain, the icpB mutant strain was also affected in the production of extracellular polysaccharides and impaired in flocculation. When inoculated alone, the icpB mutant induced nodules on S. rostrata, but the nodules formed were smaller and had reduced N2-fixing activity. The icpB mutant failed to nodulate its host when inoculated competitively with the wild-type strain. Together, the results identify chemotaxis and sensing of oxygen by IcpB as key regulators of the A. caulinodans-S. rostrata symbiosis. IMPORTANCE: Bacterial chemotaxis has been implicated in the establishment of various plant-microbe associations, including that of rhizobial symbionts with their legume host. The exact signal(s) detected by the motile bacteria that guide them to their plant hosts remain poorly characterized. Azorhizobium caulinodans ORS571 is a diazotroph that is a motile and chemotactic rhizobial symbiont of Sesbania rostrata, where it forms nitrogen-fixing nodules on both the roots and the stems of the legume host. We identify here a chemotaxis receptor sensing oxygen in A. caulinodans that is critical for nodulation and nitrogen fixation on the stems and roots of S. rostrata These results identify oxygen sensing and chemotaxis as key regulators of the A. caulinodans-S. rostrata symbiosis.

Comparative genomic and protein sequence analyses of the chemotaxis system of Azorhizobium caulinodans.

Objective: Azorhizobium caulinodans ORS571 can fix nitrogen not only as a free-living organism and an associative-symbiotic bacterium by colonizing the root surface of non-leguminous plants, but also as a symbiotic bacterium by interacting with leguminous plant Sesbania rostrata. Due to its ability to grow and fix nitrogen under three conditions, A. caulinodans uses sophisticated chemotaxis signal transduction systems to transform environmental cues into corresponding behavioral responses. Chemotaxis appears crucial for the growth of A. caulinodansin complicated environment and the construction of associative relationship with the plant. However, little is known about the chemotactic pathway of A. caulinodans. Thus, our study aimed to compare the chemotaxis-like genes of A. caulinodans with those of well-studied species. Methods: NCBI protein BLAST was used for searching sequence similarity with default parameter values against the genomes of A. caulinodans. HMMER3, based on Pfam database, was used for comparative analyses of methyl-accepting chemotaxis protein (MCP). Results: There was a major chemotaxis cluster in A. caulinodans and the CheR methylated MCPs independently of pentapeptide motif. There were 43 MCP homologs containing diverse signal-sensing architectures in A. caulinodans. In addition, cytoplasmic domains of these MCPs were all composed of 38 heptad repeats. Conclusion: Despite the extremely high homology presented between the chemotactic system of A. caulinodans and those of well-studied species, A. caulinodans shows its own unique characteristics. The classification of these chemotactic pathways by comparative genomics enables us to better understand how A. caulinodansresponds to changes in environment via exquisite signal transductions in chemotaxis system.

Effects of alteration in LPS structure in Azorhizobium caulinodans on nodule development.

The lipopolysaccharide (LPS) of Azorhizobium caulinodans ORS571, which forms N2-fixing nodules on the stems and roots of Sesbania rostrata, is known to be a positive signal required for the progression of nodule formation. In this study, four A. caulinodans mutants producing a variety of defective LPSs were compared. The LPSs of the mutants having Tn5 insertion in the rfaF, rfaD, and rfaE genes were more truncated than the modified LPSs of the oac2 mutants. However, the nodule formation by the rfaF, rfaD, and rfaE mutants was more advanced than that of the oac2 mutant, suggesting that invasion ability depends on the LPS structure. Our hypothesis is that not only the wild-type LPSs but also the altered LPSs of the oac2 mutant may be recognized as signal molecules by plants. The altered LPSs may act as negative signals that halt the symbiotic process, whereas the wild-type LPSs may prevent the halt of the symbiotic process. The more truncated LPSs of the rfaF, rfaD, and rfaE mutants perhaps no longer function as negative signals inducing discontinuation of the symbiotic process, and thus these strains form more advanced nodules than ORS571-oac2.

Lon protease of Azorhizobium caulinodans ORS571 is required for suppression of reb gene expression.

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.

Structure of lipid A from a stem-nodulating bacterium Azorhizobium caulinodans.

The structure of the lipid A of the lipopolysaccharide (LPS) from Azorhizobium caulinodans, a symbiont of the tropical legume Sesbania rostrata, was investigated by chemical compositional analyses, mass spectrometry, as well as 1D and 2D NMR spectroscopy techniques. The lipid A backbone was composed of a ß-(1→6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (GlcpN3N) disaccharide and α-D-glucuronic acid (GlcpA). Nuclear magnetic resonance spectroscopy revealed that the GlcpA was connected to the reducing end of the diaminosugar disaccharide via an α-(1→1) glycosidic bond. The lipid A was deprived of phosphate residues. ESI-MS analysis showed that the lipid A preparation was a mixture of molecules due to the occurrence of different acylation patterns. The GlcpN3N disaccharide backbone was N-acylated at the C-2, C-3, C-2' and C-3' positions with 3-OH-18:0, 3-OH-14:0, 3-OH-20:1 and 3-OH-14:0 fatty acids, respectively. Nonpolar fatty acids as well as 3-OH-18:0 were found to be ester-linked. They were attached to hydroxyl groups of primary 3-OH fatty acids giving three acyloxyacyl moieties. Thus, the complete lipid A from A. caulinodans comprised seven acyl residues. Part of the lipid A molecules was esterified by 3-methoxybutyric acid. Azorhizobium caulinodans did not incorporate ω-1 hydroxylated very long chain fatty acids (e.g., 27-OH-28:0) into the lipid A, which makes this variant of endotoxin unusual among rhizobial lipids A.

NAD(P)+-malic enzyme mutants of Sinorhizobium sp. strain NGR234, but not Azorhizobium caulinodans ORS571, maintain symbiotic N2 fixation capabilities.

C(4)-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N(2)-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD(+)-malic enzyme (DME) is required for N(2) fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N(2) fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N(2) at reduced rates, a pckA dme double mutant had no N(2)-fixing activity (Fix(-)). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix(-) phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix(-) nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)(+)-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N(2) fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

Variations in outer-membrane characteristics of two stem-nodulating bacteria of Sesbania rostrata and its role in tolerance towards diverse stress.

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane-protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane-protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

phrR-like gene praR of Azorhizobium caulinodans ORS571 is essential for symbiosis with Sesbania rostrata and is involved in expression of reb genes.

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.