세균성벼알마름병원균의 GluS-GluR Two-Component System의 기능 연구

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2021.8. Hwang Ingyu.Burkholderia glumae, just like any other microorganism, has a variety of adaptable biological systems that provide insight into how these organisms evolve, adapt, and function in a variety of environments. Despite the complexity of some of these systems, this work sheds light on the two-component regulatory systems (TCSs) paradigm, which serves as the basis for information flow throughout bacteria. Random mutagenesis of B. glumae BGR1 with mini-Tn5 resulted in a cell filamentation in Luria–Bertani (LB) medium in one of the mini-Tn5 derivatives. Molecular and genetic analysis revealed that gluR (BGLU 1G13360), a two-component system response regulator gene, carried the mini-Tn5 insertional mutation. A putative sensor kinase, gluS (BGLU 1G13350), was found downstream of gluR, prompting an exploratory study of the GluS-GluR TCS functional roles in B. glumae BGR1. The gluR mutant, unlike the gluS mutant formed filamentous cells in LB medium, was sensitive to 42C, and the expression of genes responsible for cell division and cell-wall (dcw) biosynthesis were elevated at transcription levels compared to the wild type, classifying GluR as an essential regulatory factor for cell division. TCSs regulate a variety of bacterial activities via an organized system in which the sensor kinase passes environmental cues to the response regulator, which decodes an appropriate cellular response. Accordingly, this study identified glutamine and glutamate as extrinsic cues that initiate cell division in B. glumae via GluR. Notably, GluR, and not GluS was also required for elicitation of the hypersensitive response in tobacco leaves, full virulence in host rice plants, and detoxification of hydrogen peroxide; all of which are important factors in the pathogenicity, survival, and fitness of B. glumae. GluR directly interacts with the type III secretion system and a manganese catalase gene katM to promote virulence and fitness of the pathogen. This study further showed that GluS-GluR is a functional TCS pair regulating β – lactam antibiotic resistance of B. glumae, but through a distinct mechanism. The inactivation of gluS or gluR conferred resistance against β-lactam antibiotics, whereas the wild type was susceptible to those antibiotics. This phenotype was supported by the significantly increased expression of genes encoding metallo-β-lactamases and penicillin-binding proteins in the TCS mutants compared to those in the wild type. Overall, this study adds to our understanding of how TCSs affect bacteria's sophisticated molecular systems, gives a new perspective on antibiotic resistance processes, and may provide a novel therapeutic approach for the successful control of bacterial pathogens.Burkholderia glumae는 다양한 미생물 유기체들이 어떻게 다양한 환경에서 진화, 적응하는지에 대한 통찰력을 제공하는 다양한 생물학적 기능 시스템들을 가지고 있다. 이러한 시스템들에 대한 일부의 복잡성에도 불구하고 본 연구는 일반적인 세균에서의 정보처리 흐름의 기초적 역할을 담당하는 two-component regulatory systems (TCS)의 패러다임을 제시하고자 한다. mini-Tn5를 사용한 B. glumae BGR1의 mini-Tn5 무작위 돌연변이 유도체 중 하나는 Luria–Bertani (LB) 배지에서 필라멘트 모양의 세포형태로 발견되었다. 이 돌연변이 유도체에 대한 분자 및 유전적 분석은 이것이 two-component regulatory systems 반응 조절 유전자인 gluR (BGLU 1G13360)에 mini-Tn5 삽입 돌연변이를 가지고 있음을 밝혔다. 이 gluR 의 전사방향 아래에서 TCS 감지-인산화효소인 gluS (BGLU 1G13350)가 발견되어 B. glumae BGR1의 GluS-GluR TCS 의 기능과 역할을 추적할 수 있게 되었다. gluR 돌연변이는 LB 배지에서 필라멘트 세포를 형성한 gluS 돌연변이와 달리 42C에 민감하며, 세포 분열 및 세포벽 (dcw) 생합성을 담당하는 유전자들의 발현을 wild type에 비해 증가되었기에 GluR을 세포 분열의 필수 조절 인자로 파악하였다. TCS는 감지-인산화효소가 환경 신호를 감지하여 반응 조절기에 전달하여 적절한 세포 반응을 유도하는 체계적인 시스템을 통해 다양한 세균 활동을 조절한다. 이 연구에선 B. glumae에서 GluR이 세포 분열을 시작하는 외부 신호로 감지하는 것을 글루타민과 글루타메이트로 확인했다. 또한, GluR은 담배 잎에서 과민성 반응의 유도와, 숙주인 벼에서의 완전한 독성발현 및 식물의 방어기작인 과산화수소의 해독을 위해 필요했다. 이 모든 것은 B. glumae의 병원성, 생존 및 환경적응의 중요한 요소들에 GluR이 관여하는 것이다. GluR은 III 형 분비 시스템 및 망간 항산화효소 유전자 katM과 직접 상호 작용하여 병원균의 독성 및 병원성을 촉진한다. 이 연구에서는 GluS-GluR이 B. glumae의 β- 락탐 항생제 내성을 조절하는 것에 기능적으로 연결되어 있으나, 서로 구별되는 메커니즘을 통해 항생제 내성이 만들어짐을 추가로 보여주었다. gluS 또는 gluR의 비활성화는 β-lactam 항생제에 대한 내성을 부여한 반면, wild type은 이러한 항생제에 민감하였다. 이러한 표현형은 wild type에 비해 TCS 돌연변이체에서 β-락탐 분해효소 및 페니실린 결합 단백질을 코딩하는 유전자들의 발현이 현저하게 증가된 것에 뒷받침된다. 전반적으로, 본 연구는 TCS가 세균의 정교한 조절 시스템에 어떻게 영향을 미치는지에 대한 이해를 더하고, 항생제 내성 반응에 대한 새로운 관점을 제공하며, 병원성 세균의 성공적인 제어를 위한 새로운 치료 방법을 제공 할 수 있다.INTRODUCTION 1 CHAPTER I. THE GLUR RESPONSE REGULATOR IS REQUIRED FOR CELL DIVISION IN THE RICE PATHOGEN BURKHOLDERIA GLUMAE 11 ABSTRACT 12 INTRODUCTION 14 MATERIALS AND METHODS 17 I. Bacterial strains and growth conditions 17 II. DNA manipulation and sequencing 17 III. Rescue mini-Tn5, Tn3-gusA, and marker-exchange mutagenesis 18 IV. Bacterial growth and viability assay 19 V. Transmission electron microscopy 20 VI. Quantitative reverse transcription-polymerase chain reaction 20 VII. Constitutive expression of ftsA gene 21 VIII. Growth and viability of B. glumae strains at 42oC 22 IX. Environmental stimuli driving GluR responses 22 X. Glutamate utilization in B. glumae 23 XI. Scanning electron microscopy 23 XII. Electrophoretic mobility shift assay (EMSA) 24 XIII. Statistical analysis 25 RESULTS 26 I. Identification of a TCS critical for normal cell division of B. glumae BGR1 26 II. Aberrant cell division due to a mutation in gluR 27 III. Direct control of genes involved in cell division by GluR 28 IV. Alleviation of aberrant cell morphology by constitutive expression of ftsA in the gluR mutant 29 V. Influence of glutamate and glutamine on GluR-mediated control of cell division 30 VI. Heat sensitivity due to altered fts gene expression in the gluR mutant 31 DISCUSSION 32 LITERATURE CITED 37 CHAPTER II. MUTATIONS IN THE TWO-COMPONENT GLUS-GLUR REGULATORY SYSTEM CONFER RESISTANCE TO Β-LACTAM ANTIBIOTICS IN BURKHOLDERIA GLUMAE 65 ABSTRACT 66 INTRODUCTION 67 MATERIALS AND METHODS 69 I. Bacterial strains and growth conditions 69 II. -lactam susceptibility test 69 III. Viability assay 69 IV. -lactamase activity assay 70 V. Detection of penicillin-binding proteins 70 VI. Quantitative reverse transcription polymerase chain reaction 71 VII. Electrophoretic mobility shift assay (EMSA) 71 VIII. Statistical analysis 72 RESULTS 73 I. Mutations in GluS-GluR TCS associated with -lactam antibiotic resistance in B. glumae 73 II. Cell viability of B. glumae strains amidst -lactam antibiotics 74 III. Increased -lactamase activity in GluS-GluR TCS mutants was responsible for the acquired resistance to carbenicillin 75 IV. BGLUS35 and BGLUR133 possessed elevated expression of PBPs 77 DISCUSSION 79 LITERATURE CITED 84 CHAPTER III. GLUR RESPONSE REGULATOR REGULATES TYPE III SECRETION SYSTEM AND BACTERIAL FITNESS IN BURKHOLDERIA GLUMAE 107 ABSTRACT 108 INTRODUCTION 110 MATERIALS AND METHODS I. Bacterial strains and growth conditions 113 II. DNA manipulation, sequencing, and mutagenesis 113 III. HR elicitation, virulence assay, and bacterial population 114 IV. Toxoflavin assay 115 V. Autoinducer assay 115 VI. Preparation of plant extracts 115 VII. RNA extraction and qRT-PCR 116 VIII. Hydrogen peroxide sensitivity assay 116 IX. Catalase activity assay 117 X. Electrophoretic mobility shift assay (EMSA) 116 XI. Protein in-vitro degradation assay 118 XII. Statistical analysis 118 RESULTS 119 I. Impact of GluS-GluR mutations on the virulence of B. glumae 119 II. GluR and Lon protease differently regulate T3SS in B. glumae 120 III. Mutations of gluR halts T3SS gene induction in in-vivo 122 IV. Lon protease does not degrade but activates gluR and inhibits hrpB 123 V. GluR mediates resistance to H2O2 killing in B. glumae 124 VI. GluR directly activates the activities of a manganese catalase, katM 125 VII. katM mutant is sensitive to exogenous H2O2 126 VIII. katM mutant showed attenuated virulence 127 DISCUSSION 128 LITERATURE CITED 134 APPENDIX 167 ABSTRACT IN KOREAN 169 ACKNOWLEGMENT 172박