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

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 최상호.-
dc.description.abstractBiofilm 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.
dc.description.tableofcontentsAbstract i
Contents iv
List of Figures ix
List of Tablesxi
Chapter 1. Introduction 1
1-1. Vibrio vulnificus 2
1-1-1. Disease and pathogenesis of V. vulnificus 2
1-1-2. Virulence factors of V. vulnificus 3
1-2. Biofilm 7
1-2-1. Biofilm matrix . 7
1-2-2. Development stages of biofilm .. 8
1-2-3. Cyclic di-GMP . 9
1-3. Objective of this study. 11
Chapter 2. Transcriptome Analysis of V. vulnificus in Response to Increased Cyclic di-GMP Levels 13
2-1. Introduction 14
2-2. Materials and Method 16
2-2-1. Strains, plasmids, and culture media 16
2-2-2. Manipulation of intracellular level of c-di-GMP 16
2-2-3. Biofilm formation 17
2-2-4. Colony morphology assay 17
2-2-5. Transcriptome analysis 18
2-2-6. Quantitative real-time PCR (qRT PCR) 18
2-2-7. Generation of the c-di-GMP regulon mutants 19
2-2-8. Microarray data accession number 20
2-3. Results 25
2-3-1. Intracellular c-di-GMP concentrations of V. vulnificus strains 25
2-3-2 The effects of c-di-GMP on biofilm and rugose colony formation of V. vulnificus 27
2-3-3. Identification of the c-di-GMP regulon 29
2-3-4. Verification of the genes up-regulated biofilm cells compared to plaktonic cells 41
2-3-5. Biofilm-forming ability of wild type and various mutant 44
2-4. Discussion 45
Chapter 3. The Analysis of V. vulnificus Transcriptome in Planktonic and Biofilm Cells at the Different Stages of Biofilm Development Using RNA Sequencing 49
3-1. Introduction 50
3-2. Materials and Methods 52
3-2-1. Culture conditions and biolm formation 52
3-2-2. RNA extraction, sequencing, and data analysis 53
3-3. Results and discussion 55
3-3-1. Development stage of V. vulnificus biofilms 55
3-3-2. RNA-seq analysis of the transcriptome of biofilm and planktonic cells from the different growth phases 57
3-3-3. Increase of the expression levels of biofilm-associated genes in mature biofilm cells 139
3-3-4. Dynamic changes of the expression levels of biofilm-associated genes with the biofilm development stages 142
Chapter 4. CabA, a c-di-GMP-Regulated Calcium Binding Protein, Is an Essential Component of Biofilm Matrix of V. vulnificus 144
4-1. Introduction 145
4-2. Materials and Methods 148
4-2-1. Strains, plasmids, and culture media 148
4-2-2. Construction of JN111 for manipulating c-di-GMP level 148
4-2-3. RNA purification and transcript analysis 148
4-2-4. Purification of CabA, metal contents analysis, and isothermal titration calorimetry (ITC) 149
4-2-5. Generation of the cabA and cabBC mutant 150
4-2-6. Biofilm formation 153
4-2-7. Colony morphology assay 153
4-2-8. Biofilm fractionation and Western blot analysis 153
4-2-9. E. coli dual plasmid system 154
4-3. Results 155
4-3-1. Identification of a cabABC operon 155
4-3-2. Calcium-binding ability of CabA 158
4-3-3. Manipulation of intracellular c-di-GMP levels 160
4-3-4. Effect of cabA mutation on static biofilm formation 163
4-3-5. Effect of cabA mutation on biofilm structure 165
4-3-6. Effect of cabA mutation on rugose colony morphology 167
4-3-7. CabA, secreted by type 1 secretion system (T1SS), exists in biofilm matrix 169
4-3-8. Purified CabA restored biofilm formation activity and colony rugosity of the cabA mutant 171
4-3-9. Effect of BrpT on cabABC expression 174
4-4. Discussion 176
Chapter 5. Conclusion 181
References 183
초 록 198
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dc.publisher서울대학교 대학원-
dc.subjectVibrio vulnificus-
dc.titleEffects of Cyclic di-GMP and Biofilm Development on Transcriptome Profile of Vibrio vulnificus-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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