Biomedical subjects
Publications and source records attributed to P H Clarke.
The regulation of amidase synthesis in P. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.
Amino acid sequence studies show that the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa PAC142 consists of a single polypeptide chain of 346 residues, giving an Mr of 38,400. The evidence from the amino acid studies is in complete agreement with that deduced from the DNA sequence of the amiE gene. Studies of the protein from Pseudomonas putida A87 show that it differs from the Ps. aeruginosa protein by about 30 amino acid substitutions. It now becomes possible to relate changes in the enzyme which result in altered specificity to structural changes in the protein.
The nucleotide sequence of the amiE gene, encoding the aliphatic amidase of Pseudomonas aeruginosa, has been determined. The sequence of 1038 nucleotides shows a strong bias in favour of codons with G or C in the third position, and only 44 different codons are utilised.
Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing subcloned DNA fragments, the amiR gene was located within a 1 kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.
R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.
Explore the source record for details and available documents.
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.
Explore the source record for details and available documents.
1. Chloroacetone (I) was shown to be an active-site-directed inhibitor of the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa strain PAC142.2. This inhibitor reacted with the enzyme in two stages: the first involving the reversible formation of an enzymically inactive species, EI, and the second the formation of a species, EX, from which enzymic activity could not be recovered. 3. Different types of kinetic experiment were conducted to test conformity of the reaction to the scheme: E + I k+1 Equilibrium k-1 EI Leads to K+2 EX A computer-based analysis of the results was carried out and values of the individual rate constants were determined. 4. No direct evidence for a binding step before the formation of EI could be obtained, as with [E]0 Less Than [I]0 the observed first-order rate constant for the formation of EI was directly proportional to the concentration of chloroacetone up to 1.2 mM (above this concentration the reaction became too rapid to follow even by the stopped-flow method developed to investigate fast inhibition). 5. The value of k+1 exhibited a bell-shaped pH-dependency with a maximum value of about 3 X 10(3) M-1. S-1 at pH6 and apparent pKa values of 7.8 and about 4.8.6. The values of k-1 and K+2 were similar and changed with the time of reaction from values of about 3 X 10(-3) S-1 (pH8.6) at short times to about one-sixth this value for longer periods of incubation. In this respect the simple reaction scheme is insufficient to describe the inhibition process. 7. The overall inhibition reaction is rapid, whether it is considered in relation to the expected chemical reactivity of chloroacetone, the rate of reaction of other enzymes with substrate analogues containing the chloromethyl group, or the rate of the amidase-catalysed hydrolysis of N-methylacetamide, a substrate that is nearly isosteric with chloroacetone. 8. Acetamide protected the amidase from inhibition by chloroacetone, and the concentration-dependence of the protection gave a value of an apparent dissociation constant similar to the Km value for this substrate. 9. Addition of acetamide to solutions of the species EI led to a slow recovery of activity. Recovery of active enzyme was also observed after dilution of a solution of EI in the absence of substrate. 10. The species EI is considered not to be a simple adsorption complex, and the possibilities are discussed that it may be a tetrahedral carbonyl adduct, a Schiff base (azomethine) or a complex in which the enzyme has undergone a structural change. The species EX is probably a derivative in which there is a covalent bond between a group in the enzyme and the C-1 atom of the inhibitor.
Biological evolution has resulted in a richness and diversity of species. Among microorganisms this is most evident in the wealth and diversity of biochemical transformations. Evidence for evolutionary relationships may be obtained from comparative studies, but with microorganisms it is also possible to follow evolution in action. Microbial populations adapt rapidly to changes in the environment and the evolution of new metabolic activities can be observed in laboratory experiments. The enzymes of many catabolic pathways are synthesized in response to the presence of inducing substrates. New catabolic activities may be acquired by mutations in regulatory genes resulting in alterations in the specificity of induction, or in enzyme synthesis in the absence of inducer. Mutations in structural genes may given rise to enzymes with altered substrate specificities. In bacteria, catabolic genes may be carried on plasmids and the exchange of plasmids among bacterial populations increases the evolutionary potential. Experiments in microbial evolution have produced strains with novel catabolic activities involving regulatory or structural gene mutations, gene duplications and plasmid exchange. Enzymes studied in this way include amidase, ribitol dehydrogenase, evolved beta-galactosidase, and enzymes of the catabolic pathways for pentoses and pentitols and haloaromatic compounds.
The amidase genes of Pseudomonas aeruginosa were inserted into a lambda replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant lambdaami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with lambdaami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of lambdaami were made by introducing Q-, S-, mutations. Cultures of E. coli infected with lambdaamiQ-S- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificty, thermal stability and immunological cross-reaction.
Anaerobic catabolism of amino acids may have provided the main source of energy for primitive microorganisms. Examples are given of amino acid catabolic reactions coupled to substrate level phosphorylations occurring in present-day anaerobes which may be biochemical fossils from a very early stage of the evolution of procaryotes.
Pseudomonas aeruginosa strain PAO1 cannot utilize L-lysine effectively as a carbon source for growth but grows on cadaverine and glutarate. Strains PAO1 and PAO1632 (Hut-Ami-) have low activities for L-lysine uptake and for L-lysine decarboxylase but both strains gave rise to mutants that grew well on L-lysine. Strain PAO2087, isolated from PAO1, had an active L-lysine uptake system and an inducible L-lysine decarboxylase. Strain PAO2070, isolated from strain PAO1632, had an active L-lysine uptake system and a constitutive L-lysine decarboxylase. We suggest that the genetic defect of strain PAO1 (and PAO1632) that prevents growth of L-lysine is in a regulatory gene controlling the expression of linked genes for L-lysine permease and L-lysine decarboxylase. Mutants unable to utilize L-lysine as a carbon source, isolated from strain PAO2070, exhibited four distinct growth phenotypes. Transductional analysis showed that the genetic defects of these mutants could be distinguished from each other and from that of strain PAO1. Group I mutants, unable to utilize glutarate, formed a single transduction linkage group and were mapped at about 20 min on the chromosome. The mutations of groups II, III and IV appeared to be in separate but linked genes. The group II mutants had no detectable L-lysine decarboxylase activity and the gene locus was mapped by interrupted mating in the 50 to 60 min region of the chromosome. The group III mutants possessed all the early enzymes of the L-lysine decarboxylase pathway and lacked only an active L-lysine uptake system.
Mutants isolated from Pseudomonas aeruginosa strain PAO1632 (Hut-Ami) were unable to utilize L-arginine or L-ornithine as the carbon source for growth. Arginine deiminase (AD), catabolic ornithine carbamoyltransferase (cOTC) and N2-acetylornithine 5-aminotransferase (ACOAT) were present in the mutants but these enzymes were not induced to higher levels by exogenous L-arginine. One group of mutants could utilize L-ornithine but not L-arginine and in these strains L-arginine induced the synthesis of ACOAT but not AD or cOTC. The mutations of the arginine utilization-negative mutants were all in genes of the same transductional linkage group and mapped in the 45 to 50 min region of the chromosome. Revertants isolated on L-arginine or L-ornithine plates were derepressed for the synthesis of ACOAT. It is suggested that L-arginine is normally catabolized by the wild-type strain via the arginine deiminase pathway and requires a threshold level of ACOAT. The regulatory factors controlling the functioning of the divergent arginine deiminase and arginine carboxylase pathways are discussed.
A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.
Mutants of Pseudomonas aeruginosa were isolated that were acetamide-negative in growth phenotype at 41 degrees C and constitutive for amidase synthesis at 28 degrees C. Two mutants were derived from the magno-constitutive amidase mutant PAC111 (C11), and a third from a mutant that had enhanced inducibility by formamide, PAC153 (F6). The three temperature-sensitive mutants produced amidases with the same thermal stabilities as the wild-type enzyme. Cultures growing exponentially at 28 degrees C, synthesizing amidase constitutively, ceased amidase synthesis almost immediately on transfer to 41 degrees C. Cultures growing at 41 degrees C were transferred to 28 degrees C and had a lag of about 0.5 of a generation before amidase synthesis became detectable. Pulse-heating for 10 min at 45 degrees C of a culture growing exponentially at 28 degrees C resulted in a lag of about 0.5 of a generation before amidase synthesis recommenced after returning to 28 degrees C. Acetamide-negative mutants that were unable to synthesize amidase at any growth temperature were isolated from an inducible strain producing the mutant B amidase PAC398 (IB10). Two mutants were examined that gave revertants producing B amidase but with novel regulatory phenotypes. It is suggested that amidase synthesis is regulated by positive control exerted by gene amiR.
In Pseudomonas aeruginosa, the synthesis of histidase, urocanase and amidase is severly repressed when succinate is added to a culture growing in pyruvate + ammonium salts medium. When growth is nitrogen-limited, catabolite repression by succinate of histidase and urocanase synthesis does not occur but succinate repression of amidase synthesis persists. Amidase synthesis is not regulated in the same way as histidase synthesis by the availability of other nitrogen compounds for growth. Growth of P. aeruginosa strain PACI in succinate + histidine media is nitrogen-limited since this strain is defective in a histidine transport system. When methyl-ammonium chloride is added to succinate + histidine media, growth inhibition occurs. Mutants isolated from succinate + histidine + methylammonium chloride plates were found to be resistant to catabolite repression by succinate even in ammonium salts media. It is suggested that the hut genes of P. aeruginosa may be regulated in the same way as in Klebsiella aerogenes, by induction by urocanate and activation by either the cyclic AMP-dependent activator protein or by glutamine synthetase.