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