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Coordinated control of the type IV pili and c-di-GMP-dependent antifungal antibiotic production in Lysobacter by the response regulator PilR.

ABSTRACT: In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)-mediated twitching motility as well as synthesis of the heat-stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c-di-GMP; however, the mechanism remained unknown. Here, we describe how PilR, c-di-GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c-di-GMP synthases) and found that PilR binds to the promoter region of lchD and down-regulates its transcription. The DNA-binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c-di-GMP-bound and c-di-GMP-free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS-PilR two-component system activates T4P-motility while simultaneously decreasing c-di-GMP levels and promoting HSAF production via the highly specific LchD-c-di-GMP-Clp pathway. Coordinated increase in motility and secretion of the "long-distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili-based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS-PilR system, and highlighted exquisite specificity in c-di-GMP-mediated circuits.

SUBMITTER: Xu K

PROVIDER: S-EPMC8035640 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenesIMPORTANCE PilR is a widespread response regulator of the two-component system known for regulating type IV pilus synthesis in proteobacteria. Here we report that, in the soil bacterium Lysobacter enzymogenes, PilR regulates pilus synthesis and twitching motility, as expected. Unexpectedly, PilR was also found to control intracellular levels of the second messenger c-di-GMP, which in turn inhibits production of the antifungal antibiotic HSAF. The coordinated production of type IV pili and antifungal antibiotics has not been observed previously.

Project description:Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. A large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs), and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components employ various methods to avoid undesired cross talk to maintain signaling specificity. The synthesis of the antibiotic HSAF (heat-stable antifungal factor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE, LchP, and a c-di-GMP effector, Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, that participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP display local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. IMPORTANCE Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases [DGCs]), c-di-GMP-degrading enzymes (phosphodiesterases [PDEs]), and c-di-GMP effectors. These components deploy various methods to avoid undesired cross talk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes. We provide direct evidence to show that the DGC and PDE form a complex and also indirect evidence to argue that they may balance a local c-di-GMP pool to control antibiotic production. These results represent an important finding regarding the mechanism of a DGC and PDE pair to control the expression of specific c-di-GMP signaling pathways.

Project description:Most bacteria respond to surfaces by biogenesis of intracellular c-di-GMP, which inhibits motility and induces secretion of biofilm-promoting adherence factors. Bacterial cellulose is a widespread biofilm component whose secretion in Gram-negative species requires an inner membrane, c-di-GMP-dependent synthase tandem (BcsAB), an outer membrane porin (BcsC), and various accessory subunits that regulate synthase assembly and function as well as the exopolysaccharide's chemical composition and mechanical properties. We recently showed that in Escherichia coli, most Bcs proteins form a megadalton-sized secretory nanomachine, but the role and structure of individual regulatory components remained enigmatic. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner BcsF. Crystallographic and solution-based data show that BcsE's predicted GIL domain is a degenerate receiver-GGDEF domain tandem (BcsEREC*-GGDEF*), where the divergent diguanylate cyclase module binds both dimeric c-di-GMP and BcsQ through mutually independent interfaces. In addition, we reveal that a third N-terminal domain (BcsENTD) determines the protein's homooligomerization and targeting of BcsERQ to the membrane as well as previously unreported interactions with transcription antitermination complex components. Together, the data suggest that BcsE acts on multiple levels to fine-tune bacterial cellulose secretion, from the early stages of secretion system assembly to the maintenance of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation.IMPORTANCE Bacterial cellulose is a widespread biofilm component that can modulate microbial fitness and virulence both in the environment and infected hosts. Whereas its secretion generally involves an inner membrane c-di-GMP-dependent synthase tandem (BcsAB) across the bacterial domain of life, enterobacteria feature sophisticated Escherichia coli-like Bcs secretion systems, where multiple additional subunits are either required for secretion or contribute to the maximal production of the polysaccharide in vivo. Here, we demonstrate that essential-for-secretion BcsR and BcsQ regulate each other's folding and stability and are recruited to the inner membrane via c-di-GMP-sensing BcsE and its intraoperon partner, BcsF. Crystallographic and functional data reveal that BcsE features unexpected domain architecture and likely acts on multiple levels to fine-tune bacterial cellulose production, from the early stages of secretion system assembly to the maintenence of a membrane-proximal pool of dimeric c-di-GMP for processive synthase activation.

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Coordinated control of the type IV pili and c-di-GMP-dependent antifungal antibiotic production in Lysobacter by the response regulator PilR.

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