OpaR Controls the Metabolism of c-di-GMP in Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, has a strong ability to form biofilms on surfaces. Quorum sensing (QS) is a process widely used by bacteria to communicate with each other and control gene expression via the secretion and detection of autoinducers. OpaR is the master QS regulator of V. parahaemolyticus operating under high cell density (HCD). OpaR regulation of V. parahaemolyticus biofilm formation has been reported, but the regulatory mechanisms are still not fully understood. bis-(3′-5′)-cyclic di-GMP (c-di-GMP) is an omnipresent intracellular second messenger that regulates diverse behaviors of bacteria including activation of biofilm formation. In this work, we showed that OpaR repressed biofilm formation and decreased the intracellular concentration of c-di-GMP in V. parahaemolyticus RIMD2210633. The OpaR box-like sequences were detected within the regulatory DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979, encoding a group of GGDEF and/or EAL-type proteins. The results of qPCR, LacZ fusion, EMSA, and DNase I footprinting assays demonstrated that OpaR bound to the upstream DNA regions of scrA, VP0117, VPA0198, VPA1176, and VP0699 to repress their transcription, whereas it positively and directly regulated the transcription of scrG and VP2979. Thus, transcriptional regulation of these genes by OpaR led directly to changes in the intracellular concentration of c-di-GMP. The direct association between QS and c-di-GMP metabolism in V. parahaemolyticus RIMD2210633 would be conducive to precise control of gene transcription and bacterial behaviors such as biofilm formation.

Biological and transcriptional studies reveal VmeL is involved in motility, biofilm formation and virulence in Vibrio parahaemolyticus

“…Crystal violet staining assays were tested as previously described ( Zhang et al, 2021a ) with some modifications. Overnight cultures were diluted at 1:100 in fresh MLB medium, 200 μL aliquots of the test organism suspension were cultured in 96-well microtiter dishes (Costar, 42592), and then incubated for 48 h at 30 °C without shaking.…”

“…Both assays were performed as described formerly ( Enos-Berlage et al, 2005 ; Zhang et al, 2021a ). For morphology analysis, overnight cultures were diluted at 1:100 in fresh MLB medium, then, the logarithmic bacterial culture was homogenized, and 2 μL aliquots were spotted on heart infusion medium (HI) (ELITE Biotech, Shanghai, China) plates containing 2% NaCl and 2% agar, then incubated at 37°C for at least 48 h. For the CR binding assay, the mixed bacterial culture was spotted on CR plates [HI plates added 80 ug/ml Congo red (Yuanye Bio-Technology, Shanghai, China)], and then incubated at 30°C for 8 d. Images of each strain were recorded every two days.…”

Characterization of Vibrio parahaemolyticus isolated from stool specimens of diarrhea patients in Nantong, Jiangsu, China during 2018–2020

“…CV staining was performed as previously described [30]. Briefly, the overnight cultures were diluted 50-fold into 5 mL HI broth and cultured at 37˚C with shaking at 200 rpm to OD 600 equals to 1.4.…”

H-NOX proteins in the virulence of pathogenic bacteria

“…These transcription factors reciprocally inhibit each other's transcription and thus comprise a master switch for gene expression between low and high cell density states. Among numerous other functions [61], OpaR represses biofilm formation at high cell densities [62] although this property may be strain dependent. AphA is the predominant regulator at low cell densities, and in V. cholerae it positively regulates genes involved in biofilm formation [63] and virulence [64].…”