Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Site-directed mutagenesis of Bradyrhizobium diazoefficiens USDA 110 aroA improves bacterial growth and competitiveness for soybean nodulation in the presence of glyphosate.

The in vitro growth of Bradyrhizobium diazoefficiens USDA 110 strain is inhibited by glyphosate. The herbicide affects 5-enolpyruvylshikimic acid-3-phosphate synthase, a key enzyme for aromatic aminoacid synthesis. In this study, site-directed mutagenesis was used to change only two nucleotides of the coding region of phosphoenolpyruvate binding site. This change improved the in vitro growth of B. diazoefficiens USDA 110 in the presence of glyphosate, without affecting its normal growth in the absence of the herbicide. Plant co-inoculation experiments demonstrated a better competitiveness of the glyphosate-resistant strain for soybean nodulation in the presence of glyphosate.

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System.

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

Phylogenomic Analyses of Bradyrhizobium Reveal Uneven Distribution of the Lateral and Subpolar Flagellar Systems, Which Extends to Rhizobiales.

Dual flagellar systems have been described in several bacterial genera, but the extent of their prevalence has not been fully explored. Bradyrhizobium diazoefficiens USDA 110T possesses two flagellar systems, the subpolar and the lateral flagella. The lateral flagellum of Bradyrhizobium displays no obvious role, since its performance is explained by cooperation with the subpolar flagellum. In contrast, the lateral flagellum is the only type of flagella present in the related Rhizobiaceae family. In this work, we have analyzed the phylogeny of the Bradyrhizobium genus by means of Genome-to-Genome Blast Distance Phylogeny (GBDP) and Average Nucleotide Identity (ANI) comparisons of 128 genomes and divided it into 13 phylogenomic groups. While all the Bradyrhizobium genomes encode the subpolar flagellum, none of them encodes only the lateral flagellum. The simultaneous presence of both flagella is exclusive of the B. japonicum phylogenomic group. Additionally, 292 Rhizobiales order genomes were analyzed and both flagellar systems are present together in only nine genera. Phylogenetic analysis of 150 representative Rhizobiales genomes revealed an uneven distribution of these flagellar systems. While genomes within and close to the Rhizobiaceae family only possess the lateral flagellum, the subpolar flagellum is exclusive of more early-diverging families, where certain genera also present both flagella.

The tight-adhesion proteins TadGEF of Bradyrhizobium diazoefficiens USDA 110 are involved in cell adhesion and infectivity on soybean roots.

Adhesion of symbiotic bacteria to host plants is an essential early step of the infection process that leads to the beneficial interaction. In the Bradyrhizobium diazoefficiens-soybean symbiosis few molecular determinants of adhesion are known. Here we identified the tight-adhesion gene products TadGEF in the open-reading frames blr3941-blr3943 of the B. diazoefficiens USDA 110 complete genomic sequence. Predicted structure of TadG indicates a transmembrane domain and two extracytosolic domains, from which the C-terminal has an integrin fold. TadE and TadF are also predicted as bearing transmembrane segments. Mutants in tadG or the small cluster tadGEF were impaired in adhesion to soybean roots, and the root infection was delayed. However, nodule histology was not compromised by the mutations, indicating that these effects were restricted to the earliest contact of the B. diazoefficiens and root surfaces. Knowledge of preinfection determinants is important for development of inoculants that are applied to soybean crops worldwide.