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A Master Regulator BrpR Coordinates the Expression of Multiple Loci for Robust Biofilm and Rugose Colony Development in Vibrio vulnificus.

ABSTRACT: Vibrio vulnificus, a fulminating human pathogen, forms biofilms to enhance its survival in nature and pathogenicity during host infection. BrpR is the transcriptional regulator governing robust biofilm and rugose colony formation in V. vulnificus, but little is known about both the direct regulon of BrpR and the role of BrpR in regulation of downstream genes. In this study, transcript analyses revealed that BrpR is highly expressed and thus strongly regulates the downstream gene in the stationary and elevated cyclic di-GMP conditions. Transcriptome analyses discovered the genes, whose expression is affected by BrpR but not by the downstream regulator BrpT. Two unnamed adjacent genes (VV2_1626-1627) were newly identified among the BrpR regulon and designated as brpL and brpG in this study. Genetic analyses showed that the deletion of brpL and brpG impairs the biofilm and rugose colony formation, indicating that brpLG plays a crucial role in the development of BrpR-regulated biofilm phenotypes. Comparison of the colony morphology and exopolysaccharide (EPS) production suggested that although the genetic location and regulation of brpLG are distinct from the brp locus, brpABCDFHIJK (VV2_1574-1582), brpLG is also responsible for the robust EPS production together with the brp locus genes. Electrophoretic mobility shift assays and DNase I protection assays demonstrated that BrpR regulates the expression of downstream genes in distinct loci by directly binding to their upstream regions, revealing a palindromic binding sequence. Altogether, this study suggests that BrpR is a master regulator coordinating the expression of multiple loci responsible for EPS production and thus, contributing to the robust biofilm and rugose colony formation of V. vulnificus.

SUBMITTER: Hwang SH

PROVIDER: S-EPMC8268162 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vibrio vulnificus is a potent opportunistic human pathogen that contaminates the human food chain by asymptomatically colonizing seafood. The expression of the 9-gene brp exopolysaccharide locus mediates surface adherence and is controlled by the secondary signaling molecule c-di-GMP and the regulator BrpT. Here, we show that c-di-GMP and BrpT also regulate the expression of an adjacent 5-gene cluster that includes the cabABC operon, brpT, and another VpsT-like transcriptional regulator gene, brpS The expression of the 14 genes spanning the region increased with elevated intracellular c-di-GMP levels in a BrpT-dependent manner, save for brpS, which was positively regulated by c-di-GMP and repressed by BrpT. BrpS repressed brpA expression and was required for rugose colony development. The mutation of its consensus WFSA c-di-GMP binding motif blocked these activities, suggesting that BrpS function is dependent on binding c-di-GMP. BrpT specifically bound the cabA, brpT, and brpS promoters, and binding sites homologous to the Vibrio cholerae VpsT binding site were identified upstream of brpA and brpT Transcription was initiated distal to brpA, and a conserved RfaH-recruiting ops element and a potential Rho utilization (rut) terminator site were identified within the 100-bp leader region, suggesting the integration of early termination and operon polarity suppression into the regulation of brp transcription. The GC content and codon usage of the 16-kb brp region was 5.5% lower relative to that of the flanking DNA, suggesting its recent assimilation via horizontal transfer. Thus, architecturally, the brp region can be considered an acquired biofilm and rugosity island that is subject to complex regulation.IMPORTANCE Biofilm and rugose colony formation are developmental programs that underpin the evolution of Vibrio vulnificus as a potent opportunistic human pathogen and successful environmental organism. A better understanding of the regulatory pathways governing theses phenotypes promotes the development and implementation of strategies to mitigate food chain contamination by this pathogen. c-di-GMP signaling is central to both pathways. We show that the molecule orchestrates the expression of 14 genes clustered in a 16-kb segment of the genome that governs biofilm and rugose colony development. This region exhibits the hallmarks of horizontal transfer, suggesting complex regulatory control of a recently assimilated genetic island governing the colonization response of V. vulnificus.

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A Master Regulator BrpR Coordinates the Expression of Multiple Loci for Robust Biofilm and Rugose Colony Development in Vibrio vulnificus.

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