Sensor Kinases RetS and LadS RegulatePseudomonas syringaeType VI Secretion and Virulence Factors

Abstract

Pseudomonas syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This study demonstrates that a 29.9-kb cluster of genes in the B728a genome shares homology to the novel type VI secretion system (T6SS) locus recently described for other Gram-negative bacteria. Western blot analyses showed that B728a secretes Hcp, a T6SS protein, in culture and that this secretion is dependent on clpV, a gene that likely encodes an AAA ؉ ATPase. In addition, we have identified two B728a sensor kinases that have homology to the P. aeruginosa proteins RetS and LadS. We demonstrate that B728a RetS and LadS reciprocally regulate the T6SS and collectively modulate several virulence-related activities. Quantitative PCR analyses indicated that RetS and LadS regulate genes associated with the type III secretion system and that LadS controls the expression of genes involved in the production of the exopolysaccharides alginate and levan. These analyses also revealed that LadS and the hybrid sensor kinase GacS positively regulate the expression of a putative novel exopolysaccharide called Psl. Plate assays demonstrated that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affected B728a population levels on the surfaces of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…As is the case for many other plant and animal pathogens, P. syringae secretes a variety of products, including quorum-sensing compounds, phytotoxins, antibiotics, exoproteases, fluorescent pigments, and alginate, which influence infection and disease processes (363). These secreted products as well as biofilm formation, host colonization, and lesion formation are all regulated by the GacS/GacA TCS (364)(365)(366)(367)(368)(369)(370)(371)(372)(373)(374)(375)(376)(377)(378)(379). Furthermore, genetic analyses of various P. syringae pathovars have identified homologs of the RsmA-inhibitory sRNAs RsmX, RsmY, and RsmZ of P. fluorescens, as well as RsmB of P. carotovorum (55).…”

Nooks and Crannies in Type VI Secretion Regulation

“…A probably similar regulatory pathway controls the expression of the P. syringae pv. syringae and P. fluorescens Pf-5 T6SS gene clusters (48,91). Interestingly, an orphan sensor of the RetS family, AtsR, has been reported to repress the expression of the B. cenocepacia T6SS gene cluster (5).…”

Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

“…Cues from within the host intracellular environment likely play a role in the regulation of the S. Typhimurium T6SS, as it has been determined that transcription of sciS increases approximately 16 h after the invasion of macrophages, and transcript levels are negatively correlated with the activity of the two-component response regulator of the SPI-2 T3SS, SsrB (38). Two-component signaling plays a major role in the regulation of virulence in S. Typhimurium, and these systems may regulate T6SS activity in concert with noncore T6SS genes, as is the case for the sensor kinases LadS and RetS in Pseudomonas syringae (44). A hallmark of T6SS activity has been the stable expression of phage-like proteins Hcp and VgrG, which form the contractile sheath and tailspike, respectively (with the ortholog of the latter annotated as VrgS in S. Typhimurium).…”

“…The expression of these proteins in some organisms is absent under experimental conditions, likely due to the absence of inducing stimuli. This can be circumvented by the use of strains with constitutive T6SS activity, as in the case of a retS mutant of P. syringae or the V52 V. cholerae strain (43,44). Whether or not stable expression and secretion of the Hcp and VrgS proteins occur under in vitro or in vivo conditions in S. Typhimurium is unknown.…”