Elizabeth M. Vanderlinde

Identification of a novel ABC transporter required for desiccation tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841

Rhizobium leguminosarum is a soil bacterium with the ability to form nitrogen-fixing nodules on the roots of leguminous plants. Soil-dwelling, free-living R. leguminosarum often encounters desiccation stress, which impacts its survival within the soil. The mechanisms by which soil bacteria resist the effects of desiccation stress have been described. However, the role of the cell envelope in the desiccation tolerance mechanisms of rhizobia is relatively uncharacterized. Using a transposon mutagenesis approach, a mutant of R. leguminosarum bv. viciae was isolated that was highly sensitive to desiccation. The mutation is located in the ATP-binding protein of an uncharacterized ATP-binding cassette transporter operon (RL2975-RL2977). Exopolysaccharide accumulation was significantly lower in the mutant and the decrease in desiccation tolerance was attributed to the decreased accumulation of exopolysaccharide. In addition to desiccation sensitivity, the mutant was severely impaired in biofilm formation, an important adaptation used by soil bacteria for survival. This work has identified a novel transporter required for physiological traits that are important for the survival of R. leguminosarum in the rhizosphere environment.

Mutation of the Sensor Kinase chvG in Rhizobium leguminosarum Negatively Impacts Cellular Metabolism, Outer Membrane Stability, and Symbiosis

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum.

Characterization of a Gene Family of Outer Membrane Proteins (ropB) in Rhizobium leguminosarum bv. viciae VF39SM and the Role of the Sensor Kinase ChvG in Their Regulation

The outer membrane of Gram-negative bacteria represents the interface between the bacterium and its external environment. It has a critical role as a protective barrier against harmful substances and is also important in host-bacteria interactions representing the initial physical point of contact between the host cell and bacterial cell. RopB is a previously identified outer membrane protein from Rhizobium leguminosarum bv. viciae that is present in free-living cells but absent in bacteroids (H. P. Roest, I. H. Mulders, C. A. Wijffelman, and B. J. Lugtenberg, Mol. Plant Microbe Interact. 8:576-583, 1995). The functions of RopB and the molecular mechanisms of ropB gene regulation have remained unknown. We identified and cloned ropB and two homologs (ropB2 and ropB3) from the R. leguminosarum VF39SM genome. Reporter gene fusions indicated that the expression of ropB was 8-fold higher when cells were grown in complex media than when they were grown in minimal media, while ropB3 expression was constitutively expressed at low levels in both complex and minimal media. Expression of ropB2 was negligible under all conditions tested. The use of minimal media supplemented with various sources of peptides resulted in a 5-fold increase in ropB expression. An increase in ropB expression in the presence of peptides was not observed in a chvG mutant background, indicating a role for the sensor kinase in regulating ropB expression. Each member of the ropB gene family was mutated using insertional mutagenesis, and the mutants were assayed for susceptibility to antimicrobial agents and symbiotic phenotypes. All mutants formed effective nodules on pea plants, and gene expression for each rop gene in bacteroids was negligible. The functions of ropB2 and ropB3 remain cryptic, while the ropB mutant had an increased sensitivity to detergents, hydrophobic antibiotics, and weak organic acids, suggesting a role for RopB in outer membrane stability.

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA(+) ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella.

Atomic force microscopy of a ctpA mutant in Rhizobium leguminosarum reveals surface defects linking CtpA function to biofilm formation

Atomic force microscopy was used to investigate the surface ultrastructure, adhesive properties and biofilm formation of Rhizobium leguminosarum and a ctpA mutant strain. The surface ultrastructure of wild-type R. leguminosarum consists of tightly packed surface subunits, whereas the ctpA mutant has much larger subunits with loose lateral packing. The ctpA mutant strain is not capable of developing fully mature biofilms, consistent with its altered surface ultrastructure, greater roughness and stronger adhesion to hydrophilic surfaces. For both strains, surface roughness and adhesive forces increased as a function of calcium ion concentration, and for each, biofilms were thicker at higher calcium concentrations.

Legume seed exudates and Physcomitrella patens extracts influence swarming behavior in Rhizobium leguminosarum

Plants are known to secrete chemical compounds that can change the behavior of rhizosphere-inhabiting bacteria. We investigated the effects of extracts from legume host plants on the swarming behavior of Rhizobium leguminosarum bv. viciae. We also investigated the effects on swarming when Rhizobium is exposed to extracts from an ancestor to vascular plants, the model bryophyte Physcomitrella patens. Lentil and faba bean seed exudates enhanced and inhibited swarming motility, respectively, whereas pea seed exudates had no observable effect on swarming. Swarming was also enhanced by the moss extracts. Exposure to lentil seed exudates and the moss extract increased flaA expression 2-fold, while faba bean seed exudates exposure decreased expression 3-fold, suggesting that the swarming effect could, in part, be due to regulation of flagellin gene expression. However, the exudates and extracts did not significantly affect flaA gene expression in planktonic motile cells, indicating that the response to flagellar regulation is specific to a physiology unique to the swarming cell. Transmission electron microscopy demonstrated that addition of the lentil seed exudate and the moss extract results in earlier differentiation into swarmer cells, which could contribute to the development of a larger swarming surface area. To gain further mechanistic insight into the effect of the moss extract on swarming, a moss strigolactone-deficient mutant (Ppccd8Δ) was tested. A reduction in the promotive effect was observed, suggesting that the plant hormone strigolactone may be a signalling molecule activating swarming motility in R. leguminosarum.

Homoserine catabolism by Rhizobium leguminosarum bv. viciae 3841 requires a plasmid-borne gene cluster that also affects competitiveness for nodulation.

Homoserine represents a substantial component of pea root exudate that may be important for plant-microbe interactions in the rhizosphere. We identified a gene cluster on plasmid pRL8JI that is required for homoserine utilization by Rhizobium leguminosarum bv. viciae. The genes are arranged as two divergently expressed predicted operons that were induced by L-homoserine, pea root exudate, and were expressed on pea roots. A mutation in gene pRL80083 that prevented utilization of homoserine as a sole carbon and energy source affected the mutants ability to nodulate peas and lentils competitively. The homoserine gene cluster was present in approximately 47% of natural R. leguminosarum isolates (n = 59) and was strongly correlated with homoserine utilization. Conjugation of pRL8JI to R. leguminosarum 4292 or Agrobacterium tumefaciens UBAPF2 was sufficient for homoserine utilization. The presence of L-homoserine increased conjugation efficiency of pRL8JI from R. leguminosarum to a pRL8JI-cured derivative of R. leguminosarum 1062 and to A. tumefaciens UBAPF2, and induced expression of the plasmid transfer gene trbB; however, there was no difference in conjugation efficiency or trbB expression with A. tumefaciens UBAPF2pRL8-Gm as the donor suggesting that other genes in R. leguminosarum may contribute to regulating conjugation of pRL8 in the presence of homoserine.

Metabolic Adaptation of a C-Terminal Protease A-Deficient Rhizobium leguminosarum in Response to Loss of Nutrient Transport

Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in Rhizobium leguminosarum bv. viciae 3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and ctpA (RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the ctpA mutant, which was validated by measured amino acid levels.