Pathogenic bacteria and mutualistic rhizobia are able to invade and establish chronic infections within their host plants. The success of these plant-bacteria interactions requires evasion of the plant innate immunity by either avoiding recognition or by suppressing host defences. The primary plant innate immunity is triggered upon recognition of common microbe-associated molecular patterns. Different studies reveal striking similarities between the molecular bases underlying the perception of rhizobial nodulation factors and microbe-associated molecular patterns from plant pathogens. However, in contrast to general elicitors, nodulation factors can control plant defences when recognized by their cognate legumes. Nevertheless, in response to rhizobial infection, legumes show transient or local defence-like responses suggesting that Rhizobium is perceived as an intruder although the plant immunity is controlled. Whether these responses are involved in limiting the number of infections or whether they are required for the progression of the interaction is not yet clear. Further similarities in both plant-pathogen and Rhizobium-legume associations are factors such as surface polysaccharides, quorum sensing signals and secreted proteins, which play important roles in modulating plant defence responses and determining the outcome of the interactions.

How does the recipient cell contribute to bacterial conjugation? To answer this question we systematically analyzed the individual contribution of each Escherichia coli gene in matings using plasmid R388 as a conjugative plasmid. We used an automated conjugation assay and two sets of E. coli mutant collections: the Keio collection (3,908 E. coli single-gene deletion mutants) and a collection of 20,000 random mini-Tn10::Km insertion mutants in E. coli strain DH5α. The combined use of both collections assured that we screened > 99% of the E. coli non-essential genes in our survey.

Significance: We report a novel linear mixed-linkage (1→3)(1→4)-β-glucan produced by the bacterium Sinorhizobium meliloti upon raising cyclic diguanylate (c-di-GMP) intracellular levels. This unique bacterial polysaccharide resembles (1→3)(1→4)-β-glucans found in cereals and certain lichens but has a distinctive primary structure. Genes, proteins, and regulatory pathways for producing this new polymer are described. Our findings open the possibility of using bacteria to produce (1→3)(1→4)-β-glucans, which are receiving increasing interest as bioactive compounds, and provide new elements to disclose molecular mechanisms of c-di-GMP regulation as well as for investigating the evolution, activity, and specificity of glycosyl transferases.

Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli , has revealed a novel gene, rctA , as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB , previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales , where they are invariably located adjacent to and divergently transcribed from putative virB -like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA . Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales .

Summary: Cyclic diguanylate (c‐di‐GMP) is a second messenger controlling many important bacterial processes. The phytopathogen Pectobacterium atrosepticum SCRI1043 (Pba1043) possesses a Type I secretion system (T1SS) essential for the secretion of a proteinaceous multi‐repeat adhesin (MRP) required for binding to the host plant. The genes encoding the MRP and the T1SS are tightly linked to genes encoding several putative c‐di‐GMP regulatory components. We show that c‐di‐GMP regulates secreted MRP levels in Pba1043 through the action of two genes encoding predicted diguanylate cyclase (DGC) and phosphodiesterase proteins (ECA3270 and ECA3271). Phenotypic analyses and quantification of c‐di‐GMP levels demonstrated that ECA3270 and ECA3271 regulate secreted MRP levels by increasing and decreasing, respectively, the intracellular levels of c‐di‐GMP. Moreover, ECA3270 represents the first active DGC reported to have an alternative active‐site motif from the ‘canonical’ GG[D/E]EF. ECA3270 has an A‐site motif of SGDEF and analysis of single amino acid replacements demonstrated that the first position of this motif can tolerate functional substitution. Serine in position one of the A‐site is also observed in many other DGCs. Finally, another T1SS‐linked regulator (ECA3265) also plays an important role in regulating secreted MRP, with an altered localization of MRP observed in an ECA3265 mutant background. Mutants defective in these three T1SS‐linked regulators exhibit a reduction in root binding and virulence, confirming that this complex, finely tuned regulation system is crucial in the interaction with host plants.