Effects of Cyclic di-GMP and Biofilm Development on Transcriptome Profile of Vibrio vulnificus

Abstract

Biofilm formation is regulated by many factors including cyclic diguanylic acid (c-di-GMP) and calcium. Overexpression of diguanylate cyclase increased intracellular c-di-GMP level and promoted biofilm formation and rugose colony development of Vibrio vulnificus. Microarray analysis revealed that intracellular c-di-GMP influences the expression of over 5% of the V. vulnificus genome. Among the c-di-GMP regulon, the genes from several functional categories were identified to affect biofilm formation. From these combined results, it was confirmed that c-di-GMP regulates diverse cellular processes associated with the adherence to surfaces and the biofilm formation.

V. vulnificus exhibited distinct stages of biofilm development, including initial attachment, maturation, and dispersion. Transcriptomic comparison between cells in biofilm and planktonic cells were performed using RNA sequencing technique. The sequencing data revealed that, comparing to the transcriptome in planktonic cells, the expression of 10% of the V. vulnificus genome was influenced in biofilm cells regardless of the stages of biofilm development. The expression levels of the two gene clusters, brp locus and the loci encoding calcium-binding protein and type 1 secretion system components, were changed dynamically with the development stages. Against my expectation, biofilm dispersion factor, vvpE, showed highly increased expression in the transition from the mature stage to the dispersion stage in biofilm development.

As the locus encoding calcium-binding protein shows notable expression levels when biofilm forms, it is inevitable to identify the relation between calcium and biofilm formation of V. vulnifius. In this study, a putative calcium binding protein, CabA, induced under high c-di-GMP level was identified from V. vulnificus. The coding regions of cabA and its adjacent gene cluster cabBC encoding type 1 secretion system (T1SS) components are located between a polysaccharide gene cluster, brp, and a gene encoding putative regulator of brp, BrpT. Reverse transcription-PCR results indicated that cabA is co-transcribed with cabB and cabC. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) and isothermal titration calorimetry (ITC) analyses demonstrated that CabA binds to calcium. To examine the role of CabA under high and low levels of c-di-GMP, we constructed the modified V. vulnificus strain in which the expression of dcpA encoding diguanylate cyclase is under the arabinose-inducible promoter. When high levels of c-di-GMP were present, a considerable decrease in the biofilm formation of the mutant was observed with increasing calcium concentrations which was not shown under low levels of c-di-GMP. Confocal laser scanning microscope examination of flow-cell-grown biofilms revealed that the wild type forms well-structured and mushroom-shaped biofilms, whereas the cabA mutant makes unstructured and loose biofilms. c-di-GMP-induced colony rugosity of V. vulnificus was also found to be mediated by CabA. Western blot analysis demonstrated that CabA was secreted into biofilm matrix by the T1SS. The exogenously added CabA restored the biofilm formation activity and colony rugosity of the cabA mutant only in the calcium-containing media. It was additionally confirmed that the expression of cabABC operon was activated by BrpT. Consequently, the combined results indicated that CabA, a c-di-GMP-regulated calcium binding protein, is the major protein component of V. vulnificus biofilm matrix and plays crucial roles in biofilm formation.