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Quorum sensing-dependent proteomics and anticipation of stationary-phase stress in Burkholderia glumae : Burkholderia glumae 균의 quorum sensing 의존 단백질체 분석과 생장 정지기 스트레스에 대한 예측

DC Field Value Language
dc.contributor.advisor황인규-
dc.contributor.author구은혜-
dc.date.accessioned2017-07-13T08:18:08Z-
dc.date.available2017-07-13T08:18:08Z-
dc.date.issued2013-02-
dc.identifier.other000000008511-
dc.identifier.urihttps://hdl.handle.net/10371/119425-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2013. 2. 황인규.-
dc.description.abstractBurkholderia glumae, the causative agent for bacterial rice grain rot, has a LuxR-LuxI type quorum sensing (QS) system. The bacterium utilizes N-octanoyl homoserine lactone synthesized by TofI and its cognate receptor TofR to activate expression of genes for toxoflavin biosynthesis and an IclR-type transcriptional regulator gene, qsmR. Since QS is essential for pathogenicity of B. glumae, we analyzed QS-dependent proteome by two-dimensional gel electrophoresis and ESI-MS/MS. We found that a total of 79 proteins, including previously known QS-depedent proteins, were differentially expressed between the wild-type BGR1 and the tofI mutant BGS2 strains. Among this set, 59 proteins were found in the extracellular fraction, and 20 were cytoplasmic. The extracellular 34 proteins including protease, lipase, phosphatases, were secreted through the type II secretion system (T2SS). Real-time RT-PCR analysis showed that the corresponding genes of the 49 extracellular and 13 intracellular proteins are regulated by QS at the transcriptional level. The T2SS, encoded by 12 general secretion pathway (gsp) genes with three independent transcriptional units, was controlled by QS. β-Glucuronidase activity analysis of gsp::Tn3-gusA gene fusions and electrophoretic mobility shift assays revealed that the QsmR directly regulates the expression of gsp genes. The T2SS defective mutants were less virulent than the wild type in rice panicles, indicating that the T2SS-dependent extracellular proteins play important roles in B. glumae virulence.
Acyl-homoserine lactone-mediated QS regulates diverse activities in many species of Proteobacteria. QS-controlled genes commonly code for production of secreted or excreted public goods. QS affords a means of population density-dependent gene regulation. Control of public goods via QS provides a fitness benefit. Another potential role for QS is to anticipate overcrowding. As population density increases and stationary phase approaches, QS might induce functions important for existence in stationary phase. Here we provide evidence that in two related species of the genus Burkholderia QS allows individuals to anticipate and survive stationary phase stress, base toxicity. Survival requires QS-dependent activation of cellular enzymes required for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase. Our findings provide an example where QS can serve as a means to anticipate stationary phase or life at the carrying capacity of the population by activating expression of cytoplasmic enzymes, altering cellular metabolism and producing a shared resource or public good, oxalate.		-
dc.description.tableofcontentsINTRODUCTION 1
LITERATURE CITED 6
1. CHAPTER Proteomic Analysis of Quorum Sensing-Dependent Proteins in The Plant Pathogenic Bacterium Burkholderia glumae
1.1 ABSTRACT 11
1.2 INTRODUCTION 13
1.3 MATERIALS AND METHODS 18
1.3.1 Bacterial strains and growth conditions 18
1.3.2 Nucleic acid manipulations 18
1.3.3 Transposon mutagenesis, marker exchange, and Southern hybridization 19
1.3.4 Protein sample preparation for 2-DE 20
1.3.5 2-DE 20
1.3.6 2-DE analysis and MS/MS 21
1.3.7 Reverse transcription polymerase chain reaction (RT-PCR) and Real-Time RT-PCR analysis 23
1.3.8 β-glucoronidase asaay 24
1.3.9 Overexpression and purification of QsmR 24
1.3.10 Electrophoretic mobility shift assay (EMSA) 25
1.3.11 Plant inoculation 25
1.4 RESULTS 25
1.4.1 Identification of QS-dependent proteins 27
1.4.2 QS-dependent extracellular proteins and T2SS-dependent secretion of most identified extracellular proteins 28
1.4.3 QS-dependnet cellular proteins 30
1.4.4 Organization of gsp genes 31
1.4.5 QsmR controls gsp gene expression 32
1.4.6 Expression analysis of genes encoding QS-dependent proteins by real-time RT-PCR 33
1.4.7 Virulence of T2SS-deficient mutants 34
1.5 DISCUSSION 35
1.6 LITERATURE CITED 43
2. CHAPTER Quorum Sensing Coordinates the Anticipation of Stationary-Phase Stress in Burkholderia glumae.
2.1 ABSTRACT 85
2.2 INTRODUCTION 87
2.3 MATERIALS AND METHODS 90
2.3.1 Bacterial strains and growth conditions 90
2.3.2 Nucleic acid manipulations 90
2.3.3 RNA extraction and sequencing 91
2.3.4 Transposon mutagenesis and Southern hybridization 92
2.3.5 β-glucoronidase asaay 93
2.3.6 Ammonia and oxalate measurements 93
2.3.7 Electrophoretic mobility shift assay (EMSA) 94
2.4 RESULTS 96
2.4.1 QS is essential for stationary phase survival of Burkholderia 96
2.4.2 Massive population crashes are due to medium alkalization in stationary phase 96
2.4.3 QS-dependent oxalate production counteracts base toxicity 98
2.4.4 Regulation of obc genes by QsmR 100
2.4.5 B. glumae and B. thailandensis QsmR mutants behave like QS mutants 100
2.5 DISCUSSION 102
2.6 LITERATURE CITED 107
LIST OF TABLES
1. CHAPTER
1.1 Table 1. Bacterial strains and plasmids 52
1.2 Table 2. List of oligonucleotide primers used in this study 55
1.3 Table 3. Identification of extracellular proteins displaying greater than a 2.0-fold reduction in intensity in the QS mutant BGS2 relative to the wild type BGR1 58
1.4 Table 4. Identification of extracellular proteins displaying greater than a 2.0-fold increase in intensity in the QS mutant BGS2 64
1.5 Table 5. Identification of cellular proteins displaying greater than a 2.0-fold reduction in intensity in the QS mutant BGS2 66
1.6 Table 6. Identification of cellular proteins only found in the QS mutant BGS2 68
1.7 Table 7. Expression of chromosomal gsp::Tn3-gusA fusions in the tofI and qsmR mutant background 69
2. CHAPTER
2.1 Table 1. Bacterial strains and plasmids 114
2.2 Table 2. Normalized RNAseq results of B. glumae obcAB genes 115
LIST OF FIGURES
1. CHAPTER
1.1 Fig. 1. Classification of QS-dependent proteins 70
1.2 Fig. 2. Comparative 2-DE of extracellular proteins of B. glumae 72
1.3 Fig. 3. Comparative 2-DE of cellular proteins in B. glumae 74
1.4 Fig. 4. 2-DE patterns of B. glumae proteins secreted through the T2SS in culture supernatants 76
1.5 Fig. 5. Genetic organization of the gsp genes in B. glumae and agarose gel analysis and Southern hybridization analysis of RT-PCR products of the gspD-F ans gspG-N operons 78
1.6 Fig. 6. EMSAs using purified QsmR-His and a DNA fragment containing the gspD promoter region or the intergenic region between gspC and gspG 80
1.7 Fig. 7. T2SS- deficient mutant virulence assay 82
2. CHAPTER
2.1 Fig. 1. Cell viability and culture medium pH of B. glumae, and B. thailandensis grown in LB broth at 37C with shaking 116
2.2 Fig. 2. Viability and culture medium pH of B. glumae and B. thailandensis grown in LB supplemented with 100 mM HEPES [4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid] at pH 7.0 118
2.3 Fig. 3. Viability of the wild type B. glumae and B. thailandensis in LB browth at different starting culture pH values 120
2.4 Fig. 4. Ammonia and oxalate production by B. glumae and B. thailandensis 122
2.5 Fig. 5. Ammonia and oxalate levels in culture fluid of the wild type, the tofI, and the qsmR mutant of B. glumae grown in LB supplemented with 100 mM HEPES 124
2.6 Fig. 6. Massive population crashes due to alkaline toxicity in obcAB and obc1 B. glumae and B. thailandensis mutants 126
2.7 Fig. 7. Comparison of oxalate biosynthetic genes of B. glumae, B. pseudomallei, and B. thailandensis 128
2.8 Fig. 8. Regulation of obcAB genes by QsmR in B. glumae 130		-
dc.formatapplication/pdf-
dc.format.extent2563776 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectBurkholderia glumae-
dc.subjectQuorum sensing-
dc.subjectProteomics-
dc.subjectT2SS-
dc.subjectStationary phase stress-
dc.subjectoxalate-
dc.subject.ddc630-
dc.titleQuorum sensing-dependent proteomics and anticipation of stationary-phase stress in Burkholderia glumae-
dc.title.alternativeBurkholderia glumae 균의 quorum sensing 의존 단백질체 분석과 생장 정지기 스트레스에 대한 예측-
dc.typeThesis-
dc.contributor.AlternativeAuthorEunhye Goo-
dc.description.degreeDoctor-
dc.citation.pagesvii, 138-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2013-02-
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