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INTRODUCTION
... 8GENERAL INTRODUCTION ... 10
THE TRANSCRIPTION IN ESCHERICHIA COLI ... 11
THE TRANSCRIPTION MECHANISM ... 11
STRUCTURE OF THE RNA POLYMERASE ... 11
REGULATIONS OF GENE TRANSCRIPTION ... 12
MOLECULAR MECHANISM OF CSRA ACTIVITY ... 14
STRUCTURE OF CSRA ... 14
TARGET MOTIF OF CSRA ... 14
POSITIVE AND NEGATIVE REGULATION ... 15
TARGETS OF CSRA... 15
CSRA REGULATES ITS OWN EXPRESSION ... 15
CSRA REGULATES THE CENTRAL CARBON METABOLISM ... 15
CSRA REGULATES THE STRINGENT RESPONSE ... 17
CSRA REGULATES BIOFILM FORMATION... 18
MOTILITY AND CHEMOTAXIS ... 19
CSRA REGULATES QUORUM SENSING ... 19
CSRA REGULATES VIRULENCE ... 20
CSRA AND DRUG RESISTANCE ... 20
CSRA AND OTHER GLOBAL REGULATORS ... 20
IDENTIFICATION OF THE CSRA REGULON BY HIGH THROUGHPUT ANALYSIS ... 21
REGULATION OF CSRA EXPRESSION ... 23
STRUCTURE OF CSRA PROMOTER REGION ... 23
REGULATION OF CSRA ACTIVITY BY SRNAS ... 24
REGULATION OF THE TWO SRNA CSRB AND CSRC ... 26
TWO-COMPONENT SYSTEMS ... 27
GENERAL INTRODUCTION ... 27
THE BARA/UVRY TWO-COMPONENT SYSTEM REGULATES CSRB AND CSRC EXPRESSION... 28
THE TWO-COMPONENT SYSTEM CPXA-CPXR IN ESCHERICHIA COLI ... 30
ENVELOPE ORGANIZATION AND ENVELOPE STRESS RESPONSE PATHWAYS ... 30
THE CPX SYSTEM ... 31
THE CPXA SENSOR-KINASE ... 31
THE CPXR RESPONSE REGULATOR ... 32
THE CPX REGULON ... 32
EXPRESSION AND ACTIVATION OF THE CPX SYSTEM ... 33
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATION OF CPX GENES ... 33
ACTIVATION OF THE CPX STRESS RESPONSE THROUGH SIGNALS ... 35
ACTIVATION OF THE CPX PATHWAY INDEPENDENTLY OF THE CPXA SENSOR KINASE BY ACETYL-PHOSPHATE ... 36
THE ACETYL-PHOSPHATE PATHWAY AND ITS IMPLICATION IN TCS PHOSPHORYLATION ... 36
OBJECTIVES
... 40RESULTS
... 44PHENOTYPIC SCREEN ON TWO COMPONENT SYSTEMS ... 46
THE CSRA-ASSOCIATED PHENOTYPES ... 46
MUTATIONS OF THE TCS AFFECTS THE CELL MOTILITY ... 46
GLYCOGEN ACCUMULATION IS AFFECTED IN SOME TCS MUTANTS ... 47
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CONCLUSIONS OF THE PHENOTYPIC SCREEN ... 49
BETA-GALACTOSIDASE ASSAYS REPORTING PROMOTER ACTIVITY OF CSRA, CSRB AND CSRC PARTIALLY CONFIRM THE SCREEN RESULTS ... 49
THE ROLE OF THE CPX SYSTEM ON CSRA TRANSCRIPTION IN RICH MEDIUM ... 53
CSRA EXPRESSION DURING GROWTH ... 53
CPXR POSITIVELY REGULATES CSRA PROMOTER ACTIVITY ... 53
THE PHOSPHATASE ACTIVITY OF CPXA REPRESSES CSRA EXPRESSION DURING THE LOG PHASE... 53
IMPLICATION OF THE ACETYL-PHOSPHATE IN THE CSRA TRANSCRIPTION LEVEL ... 56
TRANSCRIPTION LEVEL OF THE DIFFERENT CSRA PROMOTERS ARE AFFECTED BY CPXR ... 56
PROMOTER P3 ACTIVITY IS DEPENDENT ON CPXR IN RT-QPCR ASSAYS ... 61
THE AMOUNT OF CSRA SEEMS TO NOT BE AFFECTED BY CPXR ... 62
CONCLUSION ... 63
PROBLEM OF EXPERIMENTS REPRODUCIBILITY IN LB MEDIUM ... 64
CPXR DIRECTLY UP-REGULATES CSRA TRANSCRIPTION IN CHEMICALLY-DEFINED MEDIUM IN E. COLI ... 65
CSRA TRANSCRIPTION LEVEL DURING GROWTH IN MINIMAL MEDIUM ... 65
TRANSCRIPTIONAL REGULATION OF CSRA EXPRESSION BY CPXR ... 65
CPXA DOWN-REGULATES CSRA TRANSCRIPTION DURING THE EXPONENTIAL PHASE ... 68
IMPLICATION OF THE ACETYL-PHOSPHATE ON CSRA EXPRESSION IS CPXR DEPENDENT ... 68
CPXR BINDS TO THE CSRA PROMOTER REGION IN VITRO ... 72
ITC EXPERIMENTS INDICATE THE PRESENCE OF TWO CPXR~P BINDING SITES ON THE P3 PROMOTER REGION ... 76
CONCLUSIONS ... 76
DISCUSSION AND PERSPECTIVES
... 80LIMITATIONS OF THE PHENOTYPIC SCREEN APPROACH ... 82
PROBLEM OF REPRODUCIBILITY IN LB MEDIUM... 84
THE CPXR/ATCS REGULATES CSRA PROMOTER ACTIVITY ... 84
SHED THE ENVELOPE, GROWING IN OUR PERCEPTION OF CSRA ... 87
TO GROW OR NOT TO GROW, THAT IS CSRA ... 88
INVOLVEMENT OF CSRA IN SIGNALS INTEGRATION AND ADAPTIVE RESPONSE ... 89
