Two Functionally Deviating Type 6 Secretion Systems Occur in the Nitrogen-Fixing Endophyte Azoarcus olearius BH72.

Type VI protein secretion systems (T6SSs) have been identified in many plant-associated bacteria. However, despite the fact that effector proteins may modulate host responses or interbacterial competition, only a few have been functionally dissected in detail. We dissected the T6SS in Azoarcus olearius strain BH72, a nitrogen-fixing model endophyte of grasses. The genome harbors two gene clusters encoding putative T6SSs, tss-1 and tss-2, of which only T6SS-2 shared genetic organization and functional homology with the H1-T6SS of Pseudomonas aeruginosa. While tss-2 genes were constitutively expressed, tss-1 genes were strongly up-regulated under conditions of nitrogen fixation. A comparative analysis of the wild type and mutants lacking either functional tss-1 or tss-2 allowed to differentiate the functions of both secretion systems. Abundance of Hcp in the culture supernatant as an indication for T6SS activity revealed that only T6SS-2 was active, either under aerobic or nitrogen-fixing conditions. Our data show that T6SS-2 but not T6SS-1 is post-translationally regulated by phosphorylation mediated by TagE/TagG (PpkA/PppA), and by the phosphorylation-independent inhibitory protein TagF, similar to published work in Pseudomonas. Therefore, T6SS-1 appears to be post-translationally regulated by yet unknown mechanisms. Thus, both T6SS systems appear to perform different functions in Azoarcus, one of them specifically adapted to the nitrogen-fixing lifestyle.

Transcriptomic analysis of responses to exudates reveal genes required for rhizosphere competence of the endophyte Azoarcus sp. strain BH72.

Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we used the whole genome microarray approach to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O. sativa cv. Nipponbare in order to identify differentially regulated genes. On exposure to exudates, an overall expression of 4.4% of the 3992 protein coding genes of Azoarcus sp. strain BH72 was altered, out of which 2.4% was upregulated and 2.0% was downregulated. Genes with modulated expression included a few whose involvement in plant-microbe interaction had already been established, whereas a large fraction comprised of genes encoding proteins with putative or unknown functions. Mutational analysis of several differentially regulated genes like those encoding a minor pilin PilX, signal transduction proteins containing GGDEF domains and a serine-threonine kinase as a putative component of the type IV secretion system (T6SS), revealed their role in host colonization. Our data suggest that strain BH72 may be primed for the endophytic lifestyle by exudates, as the expression of bacterial genes relevant for endophytic colonization of roots is induced by root exudates.

Role for the RecBCD recombination pathway for pilE gene variation in repair-proficient Neisseria gonorrhoeae.

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec(+) bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.