Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens

Abstract

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.

Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis, Horizontal Gene Transfer, and Virulence Gene Expression

“…These include 16 exo genes, responsible for succinoglycan (SCG) biosynthesis (higher expression in ⌬exoR mutants), 51 annotated-and 10 unannotated-motility and chemotaxis genes (lower expression in ⌬exoR mutants), and 22 imp or hcp genes, associated with the type VI secretion system (overexpressed in ⌬exoR mutants). These trends agree with the established hypermucoid and nonmotile phenotypes of ⌬exoR mutants (7,17,18,44) and with the role that ExoR plays in regulating the T6SS presented by Wu et al (22). Noticeably absent from the motility gene category are the class I flagellar synthesis regulators visN, visR (class Ia), and rem (class Ib).…”

“…ChvG-ChvI becomes active under low-pH conditions to elicit their cellular responses. ExoR represses ChvG under neutral conditions, but when A. tumefaciens cells encounter acid, ExoR is proteolyzed to allow the two-component system to become active (21,22). Known targets of regulation by ChvG-ChvI include motility and chemotaxis genes, SCG biosynthesis genes, a type VI secretion system (T6SS), and various virulence genes (22,23).…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…The unmarked genome should help reduce public concerns about the potential toxicity of the current GM crops because of whole T-DNA integration. Apart from T4SS, A. tumefaciens possesses the T6SS which carries antibacterial activity for interbacterial competition; its secretion activity was shown to be suppressed when virulence proteins including T4SS were massively expressed (Wu et al, 2012). Some bacterial T6SS effectors are able to manipulate host immunity in animal systems, but there is no solid evidence to suggest that A. tumefaciens or other plant pathogens can use their T6SS effectors to manipulate host immunity in plants.…”

“…In addition, the affinity of the ChvE protein for sugar acids is increased at low pH, suggesting a link between the sugar and acidity response in A. tumefaciens (Hu et al, 2012). A. tumefaciens utilizes the ChvG/ChvI two-component system to activate the transcription of virG and induces expression of several other bacterial genes (Mantis and Winans, 1992;Chang and Winans, 1996), including genes involved in chemotaxis and motility, several chromosome-encoded virulence genes, succinoglycan biosynthesis genes, and the gene cluster encoding the type VI secretion system (T6SS) (Yuan et al, 2007a;Wu et al, 2008;Wu et al, 2012). Recent studies further identified ExoR as a periplasmic negative regulator acting upstream of the ChvG sensor kinase to repress acid-inducible gene expression (Wu et al, 2012;Heckel et al, 2014) and uncovered a role for the T6SS in interbacterial competition activity during the plant colonization process (Ma et al, 2014).…”

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

“…Agrobacterium tumefaceins C58 harbors one T6SS that is activated transcriptionally by an ExoR-ChvG/ChvI signaling cascade and posttranslationally via threonine phosphorylation when sensing acidity (34)(35)(36). Three type VI effectors, namely type VI amidase effector (Tae) and type VI DNase effectors (Tde1 and Tde2), confer antibacterial activity of this bacterium.…”