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Regulation of the Pseudomonas aeruginosa amidase operon by transcription antitermination

Wachira, Munuhe Simon James; (1994) Regulation of the Pseudomonas aeruginosa amidase operon by transcription antitermination. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Previous studies have identified five genes, amiE (amidase structural gene), amiB, amiC (negative regulator gene), amiR (positive regulator gene) and amiS, in the inducible amidase operon of Pseudomonas aeruginosa. The amidase operon is regulated in part by transcription antitermination upstream of the operon at a Rho-independent terminator located between amiE and its promoter. In this study, the induction of amidase expression and the mechanism of transcription antitermination have been investigated. An inducible amidase expression system has, for the first time, been reconstituted in E. coli by making a coordinate expression vector for amiC and amiR. The amiCIR expression vector was also used to restore an amidase inducible phenotype in a Pseudomonas aeruginosa amidase-constitutive mutant and in an amidase amiC-, amiR- negative mutant. A deletion mutation within the amiB gene, previously thought to be involved in amidase induction, led to no change in amidase phenotype. It is now thought that amiB codes for the energy transducing component of an ABC type transporter system. The sequence of amiS, the potential membrane component of the putative ABC transporter has been confirmed. In an attempt to purify AmiR for in vitro studies of the transcription antitermination mechanism, an AmiR overproducing plasmid has been constructed based on a T7 expression system. AmiR has been expressed using this system to about 20% of total cellular protein. Purification of active protein was, however, not achieved due to poor binding to ion exchange columns and aggregation. Identity of the protein was however confirmed by analysis on SDS-polyacrylamide gels, Western blotting using rabbit antisera raised against an MalE-AmiR fusion and by N-terminal sequencing. The transcription antitermination mechanism has been investigated both in vivo and in vitro. Firstly, overexpression of the leader sequence in trans has been shown to reduce the direct AmiR interaction with the leader mRNA. Secondly, by cloning various lengths of the amidase leader region upstream of amiR followed by in vivo competition studies, it has been shown that AmiR mediates transcription antitermination by binding to the leader transcript within a region that runs up to the terminator upstream face. In vitro RNA/protein interaction studies have been carried out. Bandshift studies showed complex formation between 32p labelled leader RNA and a semipurified E. coli extract containing AmiR. The specific nature of the nucleoprotein complex was demonstrated by cold competition experiments. Bandshift experiments using transcripts encompassing different lengths of the leader region identified a 58b region running from +10 to +68 from the transcription start site as the binding region. The precise binding site was not determined. Extensive site directed mutagenesis within the leader region has identified sequences that are critical for the antitermination reaction without any effect on the termination reaction, and other sequences that made the terminator 'leaky' without any apparent effect on antitermination.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Regulation of the Pseudomonas aeruginosa amidase operon by transcription antitermination
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Amidase; Pseudomonas aeruginosa
URI: https://discovery.ucl.ac.uk/id/eprint/10107013
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