Norman, Richard Albert; (1998) Signal transduction in the regulation of amidase operon expression in Pseudomonas aeruginosa. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Expression of the inducible amidase operon of wild-type P. aeruginosa strain PAC1 is regulated by a two component system encoded by the amiC and amiR genes. AmiC is an amide binding (sensor) negative regulator and AmiR a transcription antitermination factor activator protein. The amiC and amiR genes from the wild-type organism and a variety of regulatory mutants have been PCR cloned and phenotypically characterised. The DNA sequences of the regulatory genes from a constitutive mutant (PAC111) and a formamide inducible mutant (PAC153) have been determined, amiC and amiR mutations identified and modeled onto the AmiC structure. Previous investigations had indicated that the mechanism of ligand dependent regulation of the antitermination activity of AmiR was mediated by steric hindrance rather than covalent modification. To investigate this the cloned amiC and amiR genes from strains PAC1, PAC111 and PACl53 have been used to overproduce the regulatory proteins in P. aeruginosa. The mutant and wild-type AmiC and AmiR proteins have been shown to form stable complexes in the presence of the amide analog co-repressor butyramide. Analytical gel filtration analysis of the wild-type AmiC/AmiR-butyramide complex have shown that it exists as a heterodimer which dissociates in the presence of the inducing ligand acetamide. This effect was enhanced in the presence of high salt indicating that the interaction between AmiC and AmiR is of an ionic nature. Furthermore, an acetamide concentration equal to the KD of AmiC-acetamide causes 50% dissociation of the AmiC/AmiR complex indicating that inducer binding to AmiC acts as a one-way switch. The three in vivo states of the AmiC/AmiR complex which regulate this inducible system have been identified: AmiC/AmiR-ligand free (non-induced), AmiC/AmiR-acetamide (induced) and AmiC/AmiR-butyramide (repressed). To investigate the structure of the regulatory molecules milligram quantities of a homogeneous AmiC/AmiR-butyramide complex have been isolated. Crystals of the complex diffract to better than 3.0 Å. The crystals have been characterised and shown to have a monoclinic space group with unit cell dimensions: a = 308Å, b = 67Å, c = 76Å, β = 103°. In vitro reconstitution of the amide dependent antitermination reaction has been achieved by RNA bandshift assays using in vitro synthesised 32P labeled run-off transcripts of the operon leader region and the purified AmiC/AmiR-butyramide complex. The addition of acetamide caused a ligand-dependent bandshift to occur due to AmiR binding to the leader RNA. AmiR binds specifically to the ami leader RNA with a calculated KD of approximately 1nM.

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