The <i>cabABC</i> Operon Essential for Biofilm and Rugose Colony Development in <i>Vibrio vulnificus</i>

<div><p>A transcriptome analysis identified <i>Vibrio vulnificus cabABC</i> genes which were preferentially expressed in biofilms. The <i>cabABC</i> genes were transcribed as a single operon. The <i>cabA</i> gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the <i>cabA</i> mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the <i>cabA</i> mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the <i>cabA</i> mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render <i>V</i>. <i>vulnificus</i> to survive in hostile environments and reach a concentrated infective dose.</p></div

Calcium-induced conformational change and multimerization of CabA.

<p>(A) Typical far-UV CD spectra of CabA in the absence (black dotted line) and presence (blue line) of calcium. MRE, mean residue ellipticity. (B) Calcium-dependent multimerization of CabA was assessed using size-exclusion chromatography. CabA was eluted in the absence (yellow dotted line) and presence of calcium (20 mM, orange line; 35 mM, blue line; 50 mM, green line). The molecular weight standards (gray dotted line), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa) are indicated. Elution peaks corresponding to monomeric and multimeric CabA are indicated by a black dashed line and arrows, respectively.</p

Structure of biofilms formed on oyster shells.

<p>Biofilms of JN111 and its isogenic mutant (A) or CMCP6 and its isogenic mutant (B) were grown on oyster shells for 24 h in 24-well microtiter plates containing VFMG-CF supplemented with or without 0.01% arabinose and with 10 mM CaCl₂. The biofilms were fixed, dehydrated, coated with platinum, and visualized using SEM (Supra 55VP, Zeiss) at a 20000× magnification. Bars, 1 μm; JN111, parent strain; YM112D, <i>cabA</i> mutant (of JN111); CMCP6, wild type; YM112, <i>cabA</i> mutant (of CMCP6).</p

Effects of the <i>cabA</i> mutation on colony morphology.

<p>(A) The strains were spotted onto VFMG-CF agar plates supplemented with or without 0.02% of arabinose and 10 mM of CaCl₂. Then, the plates were incubated at 30°C for 3 d. Ampicillin and kanamycin (200 μg/ml for each) were used to maintain plasmids in the strains. Each colony representing the mean rugosity of colony morphology from at least three independent experiments was visualized using a stereomicroscope (Stemi DV4, Zeiss) at an 8× magnification. Bars, 1 mm; JN111 (pJK1113), parent strain; <i>cabA</i> (pJK1113), <i>cabA</i> mutant; <i>cabA</i> (pYM1109), complemented strain.</p

Distribution of CabA in biofilms.

<p>Biofilms of the strains were grown on the nickel grids containing VFMG-CF supplemented with 0.01% of arabinose and 10 mM of CaCl₂. CabA in the biofilms was detected with the rabbit anti-CabA antibody, labeled with the secondary antibody conjugated to 10-nm gold particles, and then visualized using TEM (JEM1010, JEOL) at a 40000× magnification. Black dots represent the CabA protein labeled with the antibody conjugated to gold particles. Bars, 200 nm. JN111 (pJK1113), parent strain; <i>cabA</i> (pJK1113), <i>cabA</i> mutant; <i>cabA</i> (pYM1109), complemented strain.</p

Effects of purified CabA protein on biofilm formation and colony morphology.

<p>(A) Biofilms of the strains were grown with VFMG-CF supplemented with 0.01% arabinose, and various amounts of exogenously-provided calcium-free CabA in the absence or presence of 10 mM CaCl₂. After 24 h incubation, biofilms were quantified using CV staining. Error bars represent the SD. **, <i>P</i><0.005 relative to the JN111 biofilm grown with 10 mM CaCl₂. (B) The strains were spotted onto VFMG-CF agar plates supplemented with 0.02% of arabinose and with or without 10 mM CaCl₂. After being grown for 12 h at 30°C, calcium-free CabA were added exogenously onto the growing colonies as indicated and then further incubated for 2.5 d. Each colony representing the mean rugosity of colony morphology from at least three independent experiments was visualized using a stereomicroscope (Stemi DV4, Zeiss) at an 8× magnification. Bars, 1 mm; JN111, parent strain; <i>cabA</i>, <i>cabA</i> mutant.</p

CabA in the cell and matrix fraction of biofilms.

<p>Biofilms of the strains were grown in the flask containing VFMG-CF supplemented with 0.01% of arabinose and 10 mM CaCl₂. (A and B, upper panels) Cell and matrix fractions of the biofilms were prepared and the resulting fractions, equivalent to 10 μg of total proteins, were resolved on SDS-PAGE and immunoblotted using the rabbit anti-CabA antibody. (A and B, lower panels) In order to demonstrate that cells were not lysed during the fractionation procedures, an intracellular regulator IscR was detected in each fraction using the rabbit anti-IscR antibody prepared previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005192#ppat.1005192.ref031" target="_blank">31</a>]. The protein size markers (Precision Plus Protein Standards; Bio-Rad Laboratories) and CabA (<i>arrows</i>) are shown in kDa. JN111 (pJK1113), parent strain; <i>cabA</i> (pJK1113), <i>cabA</i> mutant; <i>cabA</i> (pYM1109), complemented strain; <i>cabB</i> (pJK1113), <i>cabB</i> mutant; <i>cabB</i> (pJN1502), complemented strain; <i>cabC</i> (pJK1113), <i>cabC</i> mutant; <i>cabC</i> (pJN1403), complemented strain; Cell, cell fraction; Matrix, matrix fraction.</p

Effects of the <i>cabA</i> mutation on biofilm formation.

<p>(A and C) Biofilms of the strains were grown for 24 h on 96-well microtiter plate wells containing VFMG-CF supplemented with or without 0.01% arabinose and with various levels of CaCl₂, and quantified using CV staining. (B) Biofilms of the strains were grown with VFMG-CF containing 0.01% arabinose and 10 mM CaCl₂, and quantified as described above. Ampicillin and kanamycin (100 μg/ml for each) were used to maintain plasmids in the strains. JN111 (pJK1113), parent strain; <i>cabA</i> (pJK1113), <i>cabA</i> mutant; <i>cabA</i> (pYM1109), complemented strain. **, <i>P</i><0.005 relative to the JN111 biofilm. Error bars represent the SD. (D) EPS extracts were prepared from the strains grown on the LBS agar plates supplemented with 0.02% arabinose and resolved on a 4% stacking SDS-PAGE. JN111, parent strain; <i>cabA</i>, <i>cabA</i> mutant.</p

Effects of the <i>cabA</i> mutation on biofilm structure.

<p>Biofilms of the strains were grown for 3 d in the flow cells containing VFMG-CF supplemented with 0.01% arabinose and 10 mM CaCl₂. (A) Biofilms were stained by LIVE/DEAD <i>Bac</i>Light Viability Kit (Invitrogen), and CSLM (LSM710, Zeiss) images were acquired at a 100× magnification. The depth of the Z-stack is indicated below the images in μm. Images of flow cell chambers were presented on the upper-right corner of each CSLM image. (B) Biofilms of the strains were fixed, dehydrated, coated with platinum, and visualized using SEM (Supra 55VP, Zeiss) at a 30000× magnification. Bars, 100 μm (A) and 1 μm (B); JN111, parent strain; <i>cabA</i>, <i>cabA</i> mutant.</p

Genetic organization of the <i>cabABC</i> operon and amino acid sequence of CabA.

<p>(A) The physical map of the <i>cabABC</i> operon located between <i>brpT</i> and <i>brpABCDFHIJK</i> gene cluster. The <i>open arrows</i> represent the coding regions and transcriptional directions of the genes. The figure was derived using the <i>V</i>. <i>vulnificus</i> CMCP6 genome sequences. The gene identifications are shown above each coding region. The size of the <i>brpABCDFHIJK</i> operon is reduced as indicated. The <i>grey boxes</i> represent the deleted regions of the <i>cabA</i>, <i>cabB</i>, <i>cabC</i>, and <i>brpA</i> mutants, respectively. The <i>nptI</i> insertion site is indicated as a <i>triangle</i>. The primers RTcabA and RTcabC2, used for the RT-PCR analysis, are depicted by <i>solid bars</i>. (B) Analysis of the <i>cabABC</i> transcript by RT-PCR. Total RNA was isolated from JN111 grown with LBS containing 0.01% arabinose and used for the synthesis of cDNA by reverse transcription. The cDNA (lane 1), DNase I-treated RNA (negative control; lane 2), and genomic DNA (positive control; lane 3) were used as templates for PCR. Molecular size markers (1 kb plus ladder; Invitrogen) and a PCR product are indicated. (C) The amino acid sequence was deduced from the nucleotide sequence of the <i>cabA</i> coding region (VV2_1571) and the four putative calcium-binding motifs (GGXG(N/D)DX(L/I/F)X) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005192#ppat.1005192.ref028" target="_blank">28</a>] are indicated with <i>asterisks</i>.</p