Wilson, Stuart Andrew; (1991) Regulation of Pseudomonas aeruginosa amidase expression by AmiC. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Regulation of amidase synthesis in P.aeruginosa has been studied using cloned genes from strains PACl and PAC433. Using in vitro constructed rearrangements, deletions and insertions, a new gene amiC has been identified between the amidase structural gene iamiE) and the positive regulator gene (amiE). Transcomplementation studies in E.coli and P.aeruginosa and analysis of in vitro constructed amiC- mutants has shown that AmiC is a negative regulator of amidase expression. The DNA sequence of the amiC region has been determined and two potential ntrA dependent promoters identified upstream of amiC. amiC and amiR expression vectors were constructed for complementation analysis in E.coli and P.aeruginosa. Using the amiC expression vector in P.aeruginosa, AmiC was purified to homogeneity. The purified amiC was shown to have protein kinase activity in vitro. A model has been proposed whereby the transcription antitermination activity of AmiR is modified by AmiC dependent phosphorylation. DNA sequencing studies completed the sequence of the entire 5.3kb Hin-dIII-Sa/I P.aeruginosa DNA fragment containing the amidase genes and two additional open reading frames were identified, giving the gene order for the operon, amiEYCRX. A transcription terminator was identified downstream of amiE. Transcript analysis of the amidase operon has shown constitutive expression from the amiE σ70-dependent promoter. Under inducing conditions, appproximately 40% of the amiE transcripts read through the downstream terminator sequence and into amiY, C, R, X. Studies designed to investigate the role of the ntrA dependent promoters failed to identify transcripts starting from these positions. However amidase expression from the cloned genes was shown to be regulated by the enteric ntr system. These investigations showed that σ54-holoenzyme functions to down-regulate amidase expression in E.coli and P.aeruginosa. The roles of AmiY and AmiX have not been established. However AmiY contains a consensus nucleotide binding domain and amiX shows the characteristics of an integral membrane protein.

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