RESUMEN

The ChvG-ChvI two-component system is conserved among multiple Alphaproteobacteria. ChvG is a canonical two-component system sensor kinase with a single large periplasmic loop. Active ChvG directs phosphotransfer to its cognate response regulator ChvI, which controls transcription of target genes. In many alphaproteobacteria, ChvG is regulated by a third component, a periplasmic protein called ExoR, that maintains ChvG in an inactive state through direct interaction. Acidic pH stimulates proteolysis of ExoR, unfettering ChvG-ChvI to control its regulatory targets. Activated ChvI among different alphaproteobacteria controls a broad range of cellular processes, including symbiosis and virulence, exopolysaccharide production, biofilm formation, motility, type VI secretion, cellular metabolism, envelope composition, and growth. Low pH is a virulence signal in Agrobacterium tumefaciens, but in other systems, conditions that cause envelope stress may also generally activate ChvG-ChvI. There is mounting evidence that these regulators influence diverse aspects of bacterial physiology, including but not limited to host interactions.

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RESUMEN

Many bacteria can migrate from a free-living, planktonic state to an attached, biofilm existence. One factor regulating this transition in the facultative plant pathogen Agrobacterium tumefaciens is the ExoR-ChvG-ChvI system. Periplasmic ExoR regulates the activity of the ChvG-ChvI two-component system in response to environmental stress, most notably low pH. ChvI impacts hundreds of genes, including those required for type VI secretion, virulence, biofilm formation, and flagellar motility. Previous studies revealed that activated ChvG-ChvI represses expression of most of class II and class III flagellar biogenesis genes, but not the master motility regulator genes visN, visR, and rem. In this study, we characterized the integration of the ExoR-ChvG-ChvI and VisNR-Rem pathways. We isolated motile suppressors of the non-motile ΔexoR mutant and thereby identified the previously unannotated mirA gene encoding a 76 amino acid protein. We report that the MirA protein interacts directly with the Rem DNA-binding domain, sequestering Rem and preventing motility gene activation. The ChvG-ChvI pathway activates mirA expression and elevated mirA is sufficient to block motility. This study reveals how the ExoR-ChvG-ChvI pathway prevents flagellar motility in A. tumefaciens. MirA is also conserved among other members of the Rhizobiales suggesting similar mechanisms of motility regulation.

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RESUMEN

Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression.

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RESUMEN

For many pathogenic bacteria surface attachment is a required first step during host interactions. Attachment can proceed to invasion of host tissue or cells or to establishment of a multicellular bacterial community known as a biofilm. The transition from a unicellular, often motile, state to a sessile, multicellular, biofilm-associated state is one of the most important developmental decisions for bacteria. Agrobacterium tumefaciens genetically transforms plant cells by transfer and integration of a segment of plasmid-encoded transferred DNA (T-DNA) into the host genome, and has also been a valuable tool for plant geneticists. A. tumefaciens attaches to and forms a complex biofilm on a variety of biotic and abiotic substrates in vitro. Although rarely studied in situ, it is hypothesized that the biofilm state plays an important functional role in the ecology of this organism. Surface attachment, motility, and cell division are coordinated through a complex regulatory network that imparts an unexpected asymmetry to the A. tumefaciens life cycle. In this review, we describe the mechanisms by which A. tumefaciens associates with surfaces, and regulation of this process. We focus on the transition between flagellar-based motility and surface attachment, and on the composition, production, and secretion of multiple extracellular components that contribute to the biofilm matrix. Biofilm formation by A. tumefaciens is linked with virulence both mechanistically and through shared regulatory molecules. We detail our current understanding of these and other regulatory schemes, as well as the internal and external (environmental) cues mediating development of the biofilm state, including the second messenger cyclic-di-GMP, nutrient levels, and the role of the plant host in influencing attachment and biofilm formation. A. tumefaciens is an important model system contributing to our understanding of developmental transitions, bacterial cell biology, and biofilm formation.