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.

Keywords: Vibrio vulnificus; biofilm; colony morphology; exopolysaccharide; transcriptional regulator.

FIGURE 1

BrpR and BrpT regulate the expression of the brp locus and cabABC in a sequential cascade. In V. vulnificus, BrpR induces the brpT expression, and BrpT in turn activates the expression of the brp locus and cabABC in a sequential manner. The brp locus and cabABC are responsible for the production of the brp-EPS and the matrix protein CabA, respectively, and together contribute to the development of a structured biofilm matrix. The genome of V. vulnificus contains three distinct loci, the rbd, brp, and EPS-III loci, responsible for EPS production. Among them, expression of only the brp locus is activated by BrpR and BrpT as indicated.

FIGURE 2

BrpR activates brpT in a growth phase and c-di-GMP-dependent manner. (A) Total RNAs were isolated from the parent strain grown to an A₆₀₀ of 0.5 (exponential phase) or 2.0 (stationary phase) with or without 0.01% arabinose. The brpR expression was determined by qRT-PCR analysis, and the brpR expression in the exponential phase without arabinose was set at 1. (B) Total RNAs were isolated from the parent and ΔbrpR strains grown to an A₆₀₀ of 0.5 (exponential phase) or 2.0 (stationary phase) with 0.01% arabinose. The brpT expression was determined by qRT-PCR analysis, and the brpT expression of the parent strain in the exponential phase was set at 1. Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005; *, P < 0.05; ns, not significant).

FIGURE 3

Transcriptome analyses for the genes differentially expressed by BrpR. Transcriptome analyses plotted the genes down-regulated (A) or up-regulated (B) by the brpR deletion as dots. The red dots (A) or blue dots (B) represent the differentially expressed genes with annotated gene products, and the black dots indicate the genes encoding hypothetical proteins. The gray dashed lines indicate the cutoffs for differential expression of fold change > 2 and P value < 0.01.

FIGURE 4

Effects of BrpR and BrpT on the expression of downstream genes. Total RNAs were isolated from the parent and mutant strains grown to an A₆₀₀ of 2.0 with 0.01% arabinose. The brpR, brpT, brpA, brpL, brpG, and VV1_2302 expression was determined by qRT-PCR analysis, and the expression of each gene in the parent strain was set at 1. ND, not detected. Parent, parent strain; ΔbrpR, ΔbrpR mutant; ΔbrpT, ΔbrpT mutant; ΔbrpRΔbrpT, ΔbrpRΔbrpT double mutant. Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005; *, P < 0.05; ns, not significant).

FIGURE 5

Biofilm formation impaired by the deletion of brpLG. (A) Biofilms of the parent and mutant strains were grown in VFMG supplemented with 0.01% arabinose for 24 h, and then stained with 1% crystal violet. The crystal violet was eluted and its absorbance at 570 nm (A₅₇₀) was determined to quantify the biofilms. (B) Biofilms of the parent and mutant strains were grown in VFMG supplemented with 0.01% arabinose and 100 μg/ml kanamycin for 24 h for complementation experiments. The biofilm formation was quantified in the same manner as described above. Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005; ns, not significant).

FIGURE 6

Colony rugosity impaired by the deletion of brpLG. (A) The parent and mutant strains were spotted onto VFMG agar supplemented with 0.02% arabinose and incubated for 24 h. (B) The parent and mutant strains were spotted onto VFMG agar supplemented with 0.02% arabinose and 100 μg/ml kanamycin and incubated for 24 h for complementation experiments. Each colony that represented the mean rugosity from at least three independent experiments was visualized using a stereomicroscope. All images are shown at the same scale, and 1-mm scale bars are shown on the images of the parent strain.

FIGURE 7

Comparison of colony morphology resulting from the deletion of brpLG and the brp locus genes. The parent and mutant strains were spotted onto VFMG agar supplemented with 0.02% arabinose and grown for 24 h. Each colony that represented the mean rugosity from at least three independent experiments was visualized using a stereomicroscope. All images are shown at the same scale, and a 1-mm scale bar is shown on the image of the parent strain. The genes deleted in each strain are shown in top and left panels.

FIGURE 8

EPS production impaired by the deletion of brpLG and the brp locus genes. (A) EPS extracts were prepared from the parent and mutant strains and resolved on a 4% polyacrylamide gel by SDS-PAGE. The gel was stained with Stains-All and photographed. (B) EPS production was quantified from the intensity of each lane, and the production of the parent strain was set at 100% in each experiment. Average and SD of the intensity values from three independent experiments were represented. Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005 relative to the parent strain).

FIGURE 9

Direct binding of BrpR-His6 to upstream regions of the BrpR regulon. The 6-FAM-labeled DNA fragments (5 nM) for the upstream regions of brpT (A), VV1_2302 (B), and brpL (C) were incubated with increasing amounts of BrpR-His6 as indicated in the presence of c-di-GMP (50 μM). For competition analysis, the same but unlabeled DNA fragments were used as self-competitors. Various amounts of self-competitors were added as indicated to the reaction mixtures before the addition of BrpR-His6. B, bound DNA; F, free DNA.

FIGURE 10

Similar binding affinity of BrpR-His6 in the presence or absence of c-di-GMP. The 6-FAM-labeled DNA fragments (5 nM) for the upstream regions of brpT (A), VV1_2302 (B), and brpL (C) were incubated with increasing amounts of BrpR-His6 as indicated in the presence or absence of c-di-GMP (50 μM). B, bound DNA; F, free DNA.

FIGURE 11

Specific and conserved binding sequences of BrpR-His6. (A–C) The 6-FAM-labeled DNA fragments (40 nM) for the upstream regions of brpT (A), VV1_2302 (B), and brpL (C) were incubated with or without BrpR (1 μM) in the presence or absence of c-di-GMP (50 μM), and then digested with DNase I. The regions protected from DNase I cleavage by BrpR are expanded on the right panel, and the peaks in the BrpR binding sites are filled black. Nucleotides are numbered relative to the first base of each ORF. (D) The sequences of the BrpR-His6 binding sites are aligned. The palindromic sequence for the BrpR-His6 binding is shown in red below, and the bases that match the palindromic sequence are shown in bold.

FIGURE 12

Effects of the mutation in the palindromic sequence on the BrpR-His6 binding. (A) The sequences of 38-bp DNA oligonucleotides which contain the wild-type (BRPBwt) or mutated (BRPBmt1, BRPBmt2, and BRPBmt3) palindromic sequences for the BrpR-His6 binding are shown. The palindromic sequences are shown in bold, and the mutated bases are shown in red. (B) The 6-FAM-labeled DNA oligonucleotides were incubated with increasing amounts of BrpR-His6 as indicated in the presence of c-di-GMP (50 μM). B, bound DNA; F, free DNA.

FIGURE 13

BrpR-coordinated regulation of multiple loci for robust biofilm and rugose colony development in V. vulnificus. The stationary and elevated intracellular c-di-GMP conditions activate the brpR expression in V. vulnificus. BrpR subsequently represses the EPS-III locus but activates brpLG and brpT by directly binding to specific sequences in their upstream regions. BrpT then activates the cabABC operon and the brp locus. The proteins expressed from brpLG and the brp locus comprise the EPS biosynthesis machinery responsible for the brp-EPS production. The brp-EPS and the matrix protein CabA are secreted to the extracellular milieu and contribute to robust biofilm and rugose colony development of V. vulnificus.