耻垢分枝杆菌中KdpD/KdpE双组份系统调控钾离子泵KdpFABC的研究

摘要

钾离子维持细菌正常生理活动的必须的金属离子。Kdp-ATPase是一个对钾离子有强亲和作用的诱导型钾离子转运系统，在细菌中广泛存在，并受KdpD/KdpE双组份调节系统的调控。本研究以耻垢分枝杆菌为模式菌株，我们发现Kdp-ATPase编码基因kdpFABC在低K+浓度、高Na+或NH4+浓度下被诱导表达;而在高K+或蔗糖以及不同的pH条件下，kdpFABC的表达均不受影响。我们发现KdpE是一个转录调节因子，它能够识别并结合kdpFABC操纵子特异的22-bp的序列并正调控该操纵子的表达。并且KdpE识别位点在分枝杆菌kdpFABC操纵子中很保守。我们通过5'-RACE实验发现，在kdpFABC的转录起始位点位于MSMEG_5391基因的开放阅读框内，该转录起始位点和后面120-bp的5'-非翻译区及87-bp的kdpF基因共同组成了一个独立的转录本。我们发现，kdpE缺失会导致细菌在低K+时生长速率的改变。另外，在K+缺乏的情况下，细菌会转录同时含有kdpFABC和kdpDE操纵子的转录本kdpFABCDE。本研究首次在分枝杆菌中揭示了双组份调控系统KdpD/KdpE对kdpFABC基因的调控机制。

目录

声明

TABLE OF CONTENTS

ABSTRACT

摘要

LIST OF ABBREVATIONS

1 BACKGROUND

2 INTRODUCTION

2．2．2 Inducible K+ uptake system

2．3 Distribution of Kdp-ATPase system in bacteria

2．4 Organization of kdpFABC and kdpDE operons

2．5 KdpD／KdpE as two-component system

2．6 Kdp-ATPase expression control by KdpD／KdpE TCS

2．7 Structure and function of KdpD

2．8 Structure and function of KdpE

2．9 Proposed Stimuli for KdpD activation

2．10 Turgor pressure not induced kdp expression

2．11 Signaling model of KdpD／KdpE TCS

2．12 Role of KdpD／KdpE TCS in bacterial virulence

2．13 KdpD／KdpE TCS in different bacterial species

2．13．1 Escherichia coli

2．13．2 Staphylococcus aureus

2．13．3 Mycobacterium tuberculosis

2．13．4 Clostridium acetobutylicum

2．13．5 Salmonella typhimurium

2．14 Genus Mycobacterium

2．15 Mycobacterium smegmatis

RESEARCH OBJECTIVES AND SIGNIFICANCE

3 MATERIALS AND METHODS

3．1 Bacterial strains and growth conditions

3．1．2 E．coli

3．2 Molecular biology techniques

3．2．1 Polymerase chain reaction(PCR)

3．2．2 Agarose gel eIectrophoresis

3．2．3 Purification of DNA

3．2．4 Plasmid extraction

3．2．5 DNA digestion with restriction endonucleases

3．2．6 DNA ligation

3．2．7 Preparation of chemically competent cells of E．coil

3．2．9 Sequencing of DNA fragments

3．2．10 Expression，identification and purification of KdpE protein

3．2．11 Electro-mobility shift assay(EMSA)

3．2．12 DNase-I foot printing

3．3 Mycobacterial Special Techniques

3．3．2 M．smegmatis competent cells

3．3．3 Electroporation of plasmid DNA

3．3．4 Construction of mutants

3．3．5 Complementation of △kdpE

3．3．7 CDNA synthesis

3．3．8 Reverse transcriptase(RT-PCR) and real time quantitative PCR (RT-qPCR)

3．3．9 5'-Rapid Amplification of cDNA Ends(5'-RACE)

3．3．11 β-galactosidase assay for kdpF confirmation and functionality of kdp FABC operon under different conditions

3．3．12 Stress conditions

4 RESULTS

4．1 kdpD gene deletion

4．2 kdpE gene deletion

4．3 △kdpE complementary strain

4．4 Expression of kdpFABC operon under K+ limiting condition and its regulation by the KdpD／KdpE TCS

4．5 Co-transcription of kdpFABC and kdpDE operons under low K+ condition

4．6 Heterologous expression of KdpE

4．7 Binding of KdpE protein to PkdpF

4．8 No binding of KdpE to PkdpD

4．9 Promoter functionality β-galactosidase assay for P5391 and PkdpD

4．10 KdpE foot print in the PkdpF

4．11 Minimum KdpE binding motif in the PkdpF

4．12 KdpE binding motif in different bacterial species

4．13 KdpE binding motif in genus Mycobacterium

4．14 TSS of the kdpFABC operon

4．15 Effects of the salts on the expression of kdpFABC

4．15．1 Effects of low K+ on kdpFABC expression

4．15．2 Effects of osmotic upshift on kdpFABC expression

4．15．3 Effects of medium pH on kdpFABC expression

4．16 kdp genes expression under osmotic upshift

4．17 Requirement of KdpE for the normal growth of M．smegmatis

4．18 Trk system and other K+channels genes expression under K+ limiting condition

4．19 Proposed model of KdpFABC transcriptional regulation by KdpD／KdpE TCS

4．20 Organization of kdpFABC and kdpDE operons in mycobacterial species

5 DISCUSSION

6 CONCLUSION，NOVELITY AND FUTURE PROSPECTIVES

REFERENCES

LIST OF PUBLICATIONS

ACKNOWLEDGEMENT