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.

CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch.

Regulation and Characterization of Mutants of in .

Symbiosis between and requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of , which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of (P), as well as an additional basal constitutive promoter driving background expression of (P).

Conditional sanctioning in a legume- mutualism.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma.

Global control of bacterial nitrogen and carbon metabolism by a PTS-regulated switch.

The nitrogen-related phosphotransferase system (PTS) of bv. 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival.

NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula.

To overcome nitrogen deficiencies in the soil, legumes enter symbioses with rhizobial bacteria that convert atmospheric nitrogen into ammonium. Rhizobia are accommodated as endosymbionts within lateral root organs called nodules that initiate from the inner layers of Medicago truncatula roots in response to rhizobial perception. In contrast, lateral roots emerge from predefined founder cells as an adaptive response to environmental stimuli, including water and nutrient availability.

A protein complex required for polar growth of rhizobial infection threads.

During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure.

Bacterial Biosensors for in Vivo Spatiotemporal Mapping of Root Secretion.

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea () rhizosphere, a suite of bioreporters has been developed in bv strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo.

Glutathione affects the transport activity of Rhizobium leguminosarum 3841 and is essential for efficient nodulation.

As glutathione (GSH) plays an essential role in growth and symbiotic capacity of rhizobia, a glutathione synthetase (gshB) mutant of Rhizobium leguminosarum biovar viciae 3841 (Rlv3841) was characterised. It fails to efficiently utilise various compounds as a sole carbon source, including glucose, succinate, glutamine and histidine, and shows 60%-69% reduction in uptake rates of glucose, succinate and the non-metabolisable substrate α-amino isobutyric acid. The defect in glucose uptake can be overcome by addition of exogenous GSH, indicating GSH, but not its bacterial synthesis, is required for efficient transport.

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids.

Unlabelled: Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2 Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [(13)C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R.

Multiplicity of Sulfate and Molybdate Transporters and Their Role in Nitrogen Fixation in Rhizobium leguminosarum bv. viciae Rlv3841.

Rhizobium leguminosarum Rlv3841 contains at least three sulfate transporters, i.e., SulABCD, SulP1 and SulP2, and a single molybdate transporter, ModABC.

MgtE From Rhizobium leguminosarum Is a Mg²⁺ Channel Essential for Growth at Low pH and N2 Fixation on Specific Plants.

MgtE is predicted to be a Rhizobium leguminosarum channel and is essential for growth when both Mg²⁺ is limiting and the pH is low. N₂was only fixed at 8% of the rate of wild type when the crop legume Pisum sativum was inoculated with an mgtE mutant of R. leguminosarum and, although bacteroids were present, they were few in number and not fully developed.

Arabinose and protocatechuate catabolism genes are important for growth of biovar in the pea rhizosphere.

Background And Aims: To form nitrogen-fixing nodules on pea roots, biovar must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness.

Methods: Signature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres.

Characterisation of SalRAB a salicylic acid inducible positively regulated efflux system of Rhizobium leguminosarum bv viciae 3841.

Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB.

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor.

Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants.

Plant-microbe interactions in the rhizosphere have important roles in biogeochemical cycling, and maintenance of plant health and productivity, yet remain poorly understood. Using RNA-based metatranscriptomics, the global active microbiomes were analysed in soil and rhizospheres of wheat, oat, pea and an oat mutant (sad1) deficient in production of anti-fungal avenacins. Rhizosphere microbiomes differed from bulk soil and between plant species.

ABC transport is inactivated by the PTS(Ntr) under potassium limitation in Rhizobium leguminosarum 3841.

PTS(Ntr) is a regulatory phosphotransferase system in many bacteria. Mutation of the PTS(Ntr) enzymes causes pleiotropic growth phenotypes, dry colony morphology and a posttranslational inactivation of ABC transporters in Rhizobium leguminosarum 3841. The PTS(Ntr) proteins EI(Ntr) and 2 copies of EIIA(Ntr) have been described previously.

Malonate catabolism does not drive N2 fixation in legume nodules.

Malonyl-coenzyme A (CoA) decarboxylase, malonyl-CoA synthetase, and malonate transporter mutants of Rhizobium leguminosarum bv. viciae and trifolii fixed N2 at wild-type rates on pea and clover, respectively. Thus, malonate does not drive N2 fixation in legume nodules.

Regulatable vectors for environmental gene expression in Alphaproteobacteria.

Two expression vectors utilizing the inducible taurine promoter (tauAp) were developed. Plasmid pLMB51 is a stable low-copy vector enabling expression in the environment and in planta. The higher copy number pLMB509 enables BD restriction-independent cloning, expression, and purification of polyhistidine-tagged proteins.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics.

Background: The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume).

BacA is essential for bacteroid development in nodules of galegoid, but not phaseoloid, legumes.

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7(1-35) and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv.

Pathway of gamma-aminobutyrate metabolism in Rhizobium leguminosarum 3841 and its role in symbiosis.

Pea plants incubated in 15N2 rapidly accumulated labeled gamma-aminobutyrate (GABA) in the plant cytosol and in bacteroids of Rhizobium leguminosarum bv. viciae 3841. Two pathways of GABA metabolism were identified in R.

Characterization of the quaternary amine transporters of Rhizobium leguminosarum bv. viciae 3841.

Rhizobium leguminosarum bv. viciae 3841 contains six putative quaternary ammonium transporters (Qat), of the ABC family. Qat6 was strongly induced by hyperosmosis although the solute transported was not identified.

In vivo restriction endonuclease activity of the Anabaena PCC 7120 XisA protein in Escherichia coli.

Anabaena PCC 7120 genome contains three elements, which get excised out during late stages of heterocyst differentiation by a site-specific recombination process. The XisA protein, which excises the nifD element, shows sequence homology with the integrase family of tyrosine recombinase. The 11 bp target site of XisA CGGAGTAATCC contains a 3 bp inverted repeat.