Kinetic mechanism of the aliphatic amidase from Pseudomonas aeruginosa.

The kinetic constants for hydrolysis and transfer (with hydroxylamine as the alternate acceptor) of the aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were determined for a variety of acetyl and propionyl derivatives. The results obtained were consistent with a ping-pong or substitution mechanism. Product inhibition, which was pH dependent, implicated an acyl-enzyme compound as a compulsory intermediate and indicated that ammonia combined additionally with the free enzyme in a dead-end manner. The uncompetitive activation of acetamide hydrolysis by hydroxylamine and the observation that the partitioning of products between acetic acid and acetohydroxamate was linearly dependent on the hydroxylamine concentration substantiated these conclusions and indicated that deacylation was at least partially rate limiting. With propionamide as the acyl donor apparently anomalous results, which included inequalities in certain kinetic constants and a hyperbolic dependence of the partition ratio on the hydroxylamine concentration, could be explained by postulating a compulsory isomerisation of the acyl-enzyme intermediate prior to the transfer reaction.     

The isolation of an aliphatic amidase from Pseudomonas aeruginosa

A pilot-scale process for the isolation of an aliphatic, amidase from Pseudomonas aeruginosa has been developed. A constitutive, partially irrepressible mutant was employed to give a high initial enzyme concentration. An existing laboratory isolation procedure has been scaled up and modified particularly by substitution of polyethylene glycol for ammonium sulfate precipitation as the first stage in the conversion of the fractionation to continuous operation. Full recovery of activity was achieved with the modification. The recovery of enzyme from a subsequent chromatographic stage was 85% and the maximum overall purification was 28-fold.     

Chloroacetone as an active-site-directed inhibitor of the aliphatic amidase from Pseudomonas aeruginosa.

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.     

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.     

Inhibition of the aliphatic amidase from Pseudomonas aeruginosa by urea and related compounds.

The time-dependent inhibition of amidase from Pseudomonas aeruginosa strain AI 3 by urea, hydroxyurea and cyanate displayed saturation kinetics fitting a model for the reaction sequence in which formation of a complex in a reversible step was followed by an irreversible step. Altered amidases from mutant strains AIU 1N and OUCH 4, selected for their resistance to inhibition of growth by urea and hydroxyurea respectively, had altered kinetic constants for inhibition indicating reduced binding capacity for the inhibitors. The substrate acetamide protected AI 3 amidase against inhibition by urea,.and altered Ki values for inhibition of the mutant amidases were paralleled by alterations in Km values for acetamide indicating that urea acted at the active site. Inhibition of AI 3 amidase involved the binding of one molecule of urea per molecule of enzyme. Urea inhibited amidase slowly regained activity at pH 7.2 through release of urea.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

Summary The amidase genes of Pseudomonas aeruginosa were inserted into a replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant ami 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 ami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of ami were made by introducing Q ^-, S ^- mutations. Cultures of E. coli infected with amiQ ^- 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 specificity, thermal stability and immunological crossreaction.     

Positive regulation of amidase synthesis in Pseudomonas aeruginosa.

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.     

Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis.

Synthesis of the Pseudomonas aeruginosa aliphatic amidase was repressed severely by succinate and malate and less severely by glucose, acetate or lactate. Amidase synthesis in inducible and constitutive strains was stimulated by cyclic AMP, which also gave partial relief to catabolite repression produced by the addition of lactate to cultures growing in pyruvate medium. Mutants which were resistant to catabolite repression were isolated from succinate+lactamide medium.     

Transductional analysis of the flagellar genes in Pseudomonas aeruginosa.

Complementation in bacteriophage E79 tv-l-mediated transduction and the phenotypic properties of the flagellar genes in Pseudomonas aeruginosa PAO were investigated by using 195 flagellar mutants of this organism. A total of 15 fla. 1 mot, and 2 che cistrons were identified. At least 5 fla cistrons (fla V to flaZ) and one mot cistron resided in one region, and at least 10 fla cistrons (flaA to flaJ) and two che cistrons (cheA and cheB) resided in another. The flaC mutants exhibited cistron-specific leakiness on motility agar plates. The flaE cistron may be the structural gene for the component protein of the flagellar filament. The cheA mutations, which resulted in pleiotropic phenotypes for flagellar formation, motility, and taxis, belonged to the same complementation group as the flaF mutations; that is, we inferred that cheA and flaF are synonymous.     

Substrate interaction with recombinant amidase from Pseudomonas aeruginosa during biocatalysis

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate–amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates.     

Complementation of Methionine Auxotrophs of Pseudomonas aeruginosa from Cystic Fibrosis

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met⁻). Most (10/13) Met⁻ strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10⁻⁸ to <10⁻¹⁰). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine. Received: 2 August 1997 / Accepted: 23 September 1997     

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.     

多重耐药铜绿假单胞菌的耐药性及耐药基因检测分析

目的探讨呼吸内科病房分离的多重耐药铜绿假单胞菌相关耐药基因。方法采用纸片扩散法进行体外药敏试验,聚合酶链反应（PCR）对其中32株多重耐药铜绿假单胞菌进行相关基因TEM、SHV、CTX-M-9、DHA、VIM、PER、OXA、IMP和OprD2检测,并对耐药基因进行测序。结果 32株多重耐药铜绿假单胞菌对多黏菌素B耐药率是3.1%,对其余抗菌药物耐药率为53%～100%,β-内酰胺酶基因OXA-10、TEM-1、DHA-1、PER-1和IMP-1基因阳性率分别为46.9%、21.9%、15.6%、12.5%和31.3%,未检出SHV基因、CTX-M-9基因和VIM基因;另外OprD2基因缺失率达68.8%。结论呼吸内科病房多重耐药的铜绿假单胞菌携带多种β-内酰胺酶基因,应引起高度重视。     

Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa

The bacterial cell wall heteropolymer peptidoglycan is not a static structure as it is constantly being made and recycled throughout the bacterium's life cycle. This turnover of peptidoglycan is a highly coordinated event involving a complement of autolytic enzymes that include those with specificity for either the carbohydrate or the peptide linkages of peptidoglycan. One major class of these autolysins are the N-acetylmuramoyl-L-alanine amidases which cleave the amide linkage between the stem peptides and the lactyl moiety of muramoyl residues. They are required in the periplasm for cell separation during division and in both the periplasm and cytoplasm to trim soluble released PG fragments during turnover for recycling. The gene encoding N-acetylmuramoyl-L-alanine amidase B in Pseudomonas aeruginosa was cloned and over-expressed in Escherichia coli. The recombinant protein with a C-terminal His-tag was purified to apparent homogeneity by a combination of affinity and cation-exchange chromatographies using Ni(2+)NTA-agarose and Source S, respectively. Four separate assays involving zymography, light scattering, HPLC and MALDI-TOF mass spectrometry were used to confirm the activity of the protein as an N-acetylmuramoyl-L-alanine amidase.