Robert E. Drew

Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa

The nucleotide sequence of a 1001 bp ClaI/XhoI DNA fragment encoding the amidase regulator gene (amiR) from Pseudomonas aeruginosa has been determined. The sequence derives from strain PAC433, a constitutive high expressing amidase mutant, and contains two overlapping open reading frames. Analysis of the sequence has identified one of the reading frames as amiR. The gene encodes a 196 amino acid polypeptide which shows a strong bias towards codons with G or C in the third position. The amiR gene shows no sequence homology with other bacterial regulator proteins.

Crystallization of and preliminary X-ray data for the negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa

The negative regulator (AmiC) of the amidase operon of Pseudomonas aeruginosa has been purified from an over-expressing clone and crystalized. Crystals of diffraction quality were obtained from polyethylene glycol 4000 and ammonium sulphate. AmiC crystallizes in P4(2)2(1)2 (a = 104.4 A, c = 66.6 A) with one subunit in the asymmetric unit. Crystals diffract beyond 2.8 A.

Complementation analysis of the aliphatic amidase genes of Pseudomonas aeruginosa.

A plasmid, pCL34, capable of autonomous replication in Escherichia coli and Pseudomonas aeruginosa has been constructed which carries the promoter and structural gene (amiE) for P. aeruginosa amidase, but not the regulator gene (amiR). Plasmid pCL34 has been mobilized from E. coli to P. aeruginosa using the broad host range plasmid RP4. Complementation studies were performed in P. aeruginosa strains carrying various amidase mutations. Measurements of amidase activity in the recipients under inducing, non-inducing and repressing conditions showed trans-complementation by the chromosomally located regulator gene product. These results confirmed the positive control model for amidase gene expression. Levels of amidase expression seen during these studies were approximately threefold higher than in the parental, amidase-positive strains.

Alignment of cloned amiE gene of Pseudomonas aeruginosa with the N-terminal sequence of amidase.

A restriction enzyme map was constructed for 5.1-kb fragment of Pseudomonas aeruginosa DNA inserted into plasmid pBR322. Restriction enzyme sites were matched to the N-terminal amino acid sequence of amidase to obtain alignment of the amiE gene within the cloned fragment.

Lessons from the ami Operon

In 1958 Patricia Clarke FRS began work at UCL on the amidase system of Pseudomonas aeruginosa PAC1. At this time the majority of microbial geneticists were involved with the gut organism Escherichiacoli. It was clear however that the Pseudomonads were a catabolically much more versatile group of microorganisms with complex degradative pathways involving inducible enzymes. The reasons for choosing the amidase system were: first, that the major pathway for terminal respiration in P aeruginosa was the tricarboxylic acid cycle and thus breakdown products from acetamide hydrolysis could be rapidly metabolised; second, that aliphatic amides could serve as both carbon and nitrogen growth substrates; and finally, a number of aliphatic amides were available which would allow the type of investigation pursued by Monod and colleagues with lactose analogues studying the lac operon to be under taken. The initial studies showed that the substrate and inducer profiles of the amida se system were distinct such that acetamide, propionamide, lactamide and N-acetylacetamide were the best enzyme inducers and formamide, butyramide and phenylacetamid e the most active amide analogue corepressors of the system20. With respect to the enzyme substrate specificity, acetamide, propionamide and glycollamide were good substrates, formamide and lactamide poor substrates and trace activity was seen using butyramide and N-acetylacetamide. The enzyme was shown to have hydrolase activity, transferase activity in the presence of hydroxylamine and limited esterase activity.