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.

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.

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.

Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island.

Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICEAc) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICEAc-located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.

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.

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.

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).

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.

Bathy phytochromes in rhizobial soil bacteria.

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.

[Identification and functional characterization of genes induced by seed exudates in Azorhizobium caulinodans ORS571].

OBJECTIVE: To identify genes induced by plant seed exudates in Azorhizobium caulinodans ORS571. METHODS: Using promoterless kanamycin resistance gene (Km(r)) on transposon as reporter gene and seed exudates as inducers, we screened genes of interest from transposon insertion mutants libraries. We streaked mutants on TY solid medium with Km, and another with Km and seed exudates correspondingly. If Km(r) is inserted into a gene that can be induced by plant signals, Km(r) will possibly express at the same time. Thus, mutants were selected that can grow on medium with Km and exudates, rather than on medium with Km. RESULTS: We identified a lysE family gene named asiE in strain Azc0 that can be induced by seed exudates and further analysis indicated that the inducing substance is canavanine (CAN). lacZ transcriptional fusion of asiE confirmed that its expression increased by ten-fold or so under the induction of CAN. Besides, lysE gene in four different species of Rhizobia can be induced by CAN. lysE mutants are all sensitive to CAN treatment whereas wild type are resistant. CONCLUSION: The existence of LysE can make rhizobia better survived in the rhizosphere and may play an important role in early stage of interaction between rhizobia and host plant.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571.

BACKGROUND: Biological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem. RESULTS: The 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor. CONCLUSION: The genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

An outer membrane autotransporter, AoaA, of Azorhizobium caulinodans is required for sustaining high N2-fixing activity of stem nodules.

In this study, we investigated the function of a putative high-molecular-weight outer membrane protein, azorhizobial outer membrane autotransporter A (AoaA), of Azorhizobium caulinodans ORS571. Sequence analysis revealed that AoaA was an autotransporter protein belonging to the type V protein secretion system. Azorhizobium caulinodans forms N(2)-fixing nodules on the stems and roots of Sesbania rostrata. The sizes of stem nodules formed by an aoaA mutant having transposon insertion within this ORF were as large as those in the wild-type strain, but the N(2)-fixing activity of the nodules by the aoaA mutant was lower than that of wild-type nodules. cDNA-amplified fragment length polymorphism and reverse transcriptase-PCR analysis revealed that the expressions of several pathogen-related genes of host plants were induced in the aoaA mutant nodules. Furthermore, exopolysaccharide production was defective in the aoaA mutant under free-living conditions. These results indicate that AoaA may have an important role in sustaining the symbiosis by suppressing plant defense responses. The exopolysaccharide production controlled by AoaA might mediate this suppression mechanism.

Responses of Azorhizobium caulinodans to cadmium stress.

The symbiosis of Azorhizobium caulinodans and an annul legume Sesbania rostrata was recently found to be tolerant to cadmium pollution by an unknown mechanism. In this study, A. caulinodans ORS571 and ZY-20 showed much stronger tolerance to cadmium than a mutant ORS571-X15 and a common Rhizobium sp., with minimum inhibitory concentration values as high as 4 and 5 mM (versus 1 and 0.1 mM) on yeast extract mannitol agar medium, respectively. Although Cd uptake by all three strains of A. caulinodans were mostly from absorption rather than binding (both loosely or tightly) on cell surface, in resistant strains a higher portion of extractable Cd was bound on the cell surface vs. absorbed (about 1:2.5 ratio) compared to the sensitive mutant (about 1:35.1 ratio). These results suggest that certain level of metal exclusion by a permeability barrier was involved in the mechanism of resistance to Cd by A. caulinodans ORS571 and ZY-20. Over the 12-h period of cultivation in yeast extract mannitol agar medium with Cd addition, the Cd concentrations in the outer membrane and periplasm and spheroplast were the highest at the first 3 h, and declined steadily over time. The fact that Cd concentrations in spheroplast of all three strains were many folds higher than those in outer membrane and periplasm, suggests that extracellular sequestration was not the only mechanism of Cd tolerance in A. caulinodans. The decline of Cd concentrations was significantly faster and started earlier in strains ORS571 and ZY-20 than in ORS571-X15. This suggests a second, probably more substantial, mechanism involves active transport of the metal from the cell, e.g., some efflux system for maintaining homeostasis under cadmium stress.

Lipopolysaccharides as a communication signal for progression of legume endosymbiosis.

Establishment of a successful symbiosis between rhizobia and legumes results from an elaborate molecular dialogue between both partners. Bacterial nodulation (Nod) factors are indispensable for initiating plant responses, whereas bacterial surface polysaccharides are important for infection progression and nodule development. The mutant ORS571-oac2 of Azorhizobium caulinodans, affected in its surface polysaccharides, provokes a defective interaction with its host Sesbania rostrata. ORS571-oac2 induced structures with retarded development and continued generation of infection centers and organ primordia, leading to multilobed ineffective nodules. Bacterial development throughout the interaction occurred without major defects. A functional bidirectional complementation was obtained upon coinfection of ORS571-oac2 and a Nod factor-deficient mutant, indicating that the Fix- phenotype of ORS571-oac2-induced nodules resulted from the absence of a positive signal from ORS571-oac2. Indeed, the Fix- phenotype could be complemented by coinoculation of ORS571-oac2 with lipopolysaccharides (LPSs) purified from A. caulinodans. Our data show that Nod factors and LPSs are consecutive signals in symbiosis. Nod factors act first to trigger the onset of the nodulation and invasion program; LPSs inform the plant to proceed with the symbiotic interaction and to develop a functional fixation zone.

Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N2 fixation.

Azorhizobium caulinodans thermolabile point mutants unable to fix N2 at 42 degrees C were isolated and mapped to three, unlinked loci; from complementation tests, several mutants were assigned to the fixABCX locus. Of these, two independent fixB mutants carried missense substitutions in the product electron-transferring flavoprotein N (ETFN) alpha-subunit. Both thermolabile missense variants Y238H and D229G mapped to the ETFNalpha interdomain linker. Unlinked thermostable suppressors of these two fixB missense mutants were identified and mapped to the lpdA gene, encoding dihydrolipoamide dehydrogenase (LpDH), immediately distal to the pdhABC genes, which collectively encode the pyruvate dehydrogenase (PDH) complex. These two suppressor alleles encoded LpDH NAD-binding domain missense mutants G187S and E210G. Crude cell extracts of these fixB lpdA double mutants showed 60-70% of the wild-type PDH activity; neither fixB lpdA double mutant strain exhibited any growth phenotype at the restrictive or the permissive temperature. The genetic interaction between two combinations of lpdA and fixB missense alleles implies a physical interaction of their respective products, LpDH and ETFN. Presumably, this interaction electrochemically couples LpDH as the electron donor to ETFN as the electron acceptor, allowing PDH complex activity (pyruvate oxidation) to drive soluble electron transport via ETFN to N2, which acts as the terminal electron acceptor. If so, then, the A. caulinodans PDH complex activity sustains N2 fixation both as the driving force for oxidative phosphorylation and as the metabolic electron donor.

Influence of cell wall degrading enzymes on colonization of N2 fixing bacterium, Azorhizobium caulinodans in rice.

In rice, nodule like structures were formed by inoculation of A. caulinodans combined with growth regulators and enzymes. Among the treatments, combination of cell wall degrading enzyme mixture and NAA with A. caulinodans induced more number of paranodules in rice. Total nitrogen content also increased in treated plants compared to uninoculated control.

Nod factor requirements for efficient stem and root nodulation of the tropical legume Sesbania rostrata.

Azorhizobium caulinodans ORS571 synthesizes mainly pentameric Nod factors with a household fatty acid, an N-methyl, and a 6-O-carbamoyl group at the nonreducing-terminal residue and with a d-arabinosyl, an l-fucosyl group, or both at the reducing-terminal residue. Nodulation on Sesbania rostrata was carried out with a set of bacterial mutants that produce well characterized Nod factor populations. Purified Nod factors were tested for their capacity to induce root hair formation and for their stability in an in vitro degradation assay with extracts of uninfected adventitious rootlets. The glycosylations increased synergistically the nodulation efficiency and the capacity to induce root hairs, and they protected the Nod factor against degradation. The d-arabinosyl group was more important than the l-fucosyl group for nodulation efficiency. Replacement of the 6-O-l-fucosyl group by a 6-O-sulfate ester did not affect Nod factor stability, but reduced nodulation efficiency, indicating that the l-fucosyl group may play a role in recognition. The 6-O-carbamoyl group contributes to nodulation efficiency, biological activity, and protection, but could be replaced by a 6-O-acetyl group for root nodulation. The results demonstrate that none of the studied substitutions is strictly required for triggering normal nodule formation. However, the nodulation efficiency was greatly determined by the synergistic presence of substitutions. Within the range tested, fluctuations of Nod factor amounts had little impact on the symbiotic phenotype.

The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification.

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5-induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD-dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.