Patricia H. Clarke

INDUCTION AND REPRESSION OF PSEUDOMONAS AERUGINOSA AMIDASE.

The synthesis of an inducible amidase by Pseudomonas aeruginosa 8602/a was studied in cultures growing exponentially in succinate medium. Induction by both the substrate inducer acetamide, and the non-substrate inducer N-acetylacetamide, was repressed by cyanoacetamide. Induction by 10-2mN-acetylacetamide was significantly repressed by 10-4m-cyanoacetamide, but repression of induction by 10-3m-acetamide required a tenfold excess of cyanoacetamide. Amidase synthesis in a medium in which acetamide was the sole carbon + nitrogen source was also repressed by cyanoacetamide, which under these conditions inhibited the growth of non-induced bacteria. Several tricarboxylic acid cycle intermediates, and related compounds, repressed amidase synthesis in exponentially growing organisms. Catabolite repression by propionate in succinate medium was decreased by increasing the concentration of acetamide. These findings are discussed in relation to general theories of regulation of microbial enzyme synthesis.

An Inducible Amidase Produced by a Strain of Pseudomonas aeruginosa

SUMMARY: A strain of Pseudomonas aeruginosa was obtained which was able to grow on acetamide or propionamide as sole source of carbon and nitrogen. When grown on these amides, whole bacteria and cell-free extracts rapidly hydrolysed acetamide, glycollamide, acrylamide and propionamide and slowly hydrolysed formamide and butyramide. N-Methylformamide, N-methylacetamide, N-ethylacetamide, N-acetylacetamide, N-methylpropionamide, N-ethylpropionamide, lactamide and methyl carbamate were found to be non-substrate inducers of the amidase when the organism was grown in succinate + ammonium chloride medium. N-Methylformamide, N-methylacetamide, lactamide and methyl carbamate did not inhibit propionamide hydrolysis by whole bacteria, but under the same conditions glycine amide, iodoacetamide and urea were effective inhibitors of amidase activity. N-Phenylacetamide, cyanoacetamide, glycine amide, sarcosine amide, β-hydroxy-propionamide and thioacetamide were neither substrates nor inducers of the amidase in this strain, but inhibited amidase induction by N-methylacetamide in succinate + ammonium chloride medium. Formamide also inhibited amidase induction under the same conditions.

Hydrogen Sulphide Production by Bacteria

SUMMARY: Hydrogen sulphide production by growing cultures and non-multiplying suspensions was compared and the factors influencing the sensitivity of the tests were investigated. Cysteine hydrochloride (0.01%) was added to Lemco broth to provide a medium with a standard source of sulphur. H2S was detected with lead acetate papers more readily than by lead acetate agar. Suspensions were tested with cysteine, sodium thiosulphate and sodium sulphite; the organisms investigated were mainly Bacteriaceae and seldom failed to produce H2S from cysteine; sodium thiosulphate was less readily attacked; sodium sulphite was unsuitable for this technique. Media commonly used for testing the capacity of bacteria to produce H2S are reviewed and the value of this test in bacterial classification discussed.

Biochemical classification of Proteus and Providence cultures.

Summary: Biochemical reactions of 153 Providence (29911 type) and 63 Proteus cultures were examined. Providence cultures are included in the genus Proteus, as their cultural characters are similar; this relationship is strengthened by the production of L-amino acid oxidase and glutamic acid decarboxylase by both. Proteus inconstans Ornstein nov.comb. is the name proposed for Providence cultures.

Butyramide-utilizing Mutants of Pseudomonas aeruginosa 8602 which Produce an Amidase with Altered Substrate Specificity

SUMMARY: Mutants of Pseudomonas aeruginosa 8602 were isolated which, unlike the wild type, were able to grow with butyramide as a carbon source. Six mutants derived from the constitutive strain c 11 were shown to produce an enzyme (B amidase) with altered electrophoretic mobility and altered substrate specificity. The apparent Km for butyramide of the B amidase was about a tenth of that of the A amidase and the Vmax was about ten-fold greater. A further mutation produced mutants able to grow on valeramide.

Biochemical Methods for Bacteriology

Summary: A series of biochemical methods using heavy suspensions of organisms and chemically-defined solutions are described; they include fermentations, reduction of nitrate and of methylene blue, production of indole, hydrogen sulphide and acetoin, and hydrolysis of gelatin, starch and urea. The tests use the preformed enzymes of the bacterial cells and the results are not complicated by side effects or the multiple reactions that occur in cultures growing in a nutrient medium containing the test substrate.

The occurrence of chitinase in some bacteria.

SUMMARY: A method for the detection of small quantities of chitinase in culture fluids is described and the results of a survey of representative species of the main groups of bacteria presented. Chitinase is produced by some but not all of the soil and water bacteria. Chitinase appears to be a constitutive enzyme in many species of bacteria; its occurrence may prove of diagnostic value.

Biochemical and Genetic Studies with Regulator Mutants of the Pseudomonas aeruginosa 8602 Amidase System

Surmmary Mutants of Pseudomonas aeruginosa strain 8602 were isolated which were unable to produce an aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) and could not grow on acetamide as a carbon or nitrogen source. Amidase-constitutive mutants, producing amidase in the absence of inducing amides, were isolated by selection on succinate+formamide agar. Sixteen mutants were magno-constitutive non-inducible mutants producing amidase at about the same rate or greater than the fully induced wild-type strain. Amidase synthesis in one magno-constitutive mutant was repressed by the non-substrate inducer N-acetylacetamide, but the others were not affected in any way. Six mutants were semi-constitutive, producing amidase at 10–50% of the rate of the magno-constitutive mutants and were induced by N-acetylacetamide. Most of the constitutive mutants were very sensitive to catabolite repression by succinate in pyruvate medium, but succinate produced only partial repression of one magno-constitutive mutant and three semi-constitutive mutants; one semi-constitutive mutant was not repressed except in the presence of inducer. Six mutants isolated from succinate + formamide agar had altered inducer specificity and were induced to form amidase by formamide, which is a very poor inducer for the wild-type strain. The formamide-inducible mutants were also sensitive to catabolite repression by succinate although one mutant was only partially repressed. Phage F 116 was used to transduce the amidase structural and regulator genes. In crosses between constitutive mutants of Pseudomonas aeruginosa as donors and amidase-negative mutants as recipients, the two characters were co-transduced with frequencies of 80–100%. Similarly, in crosses between formamide-inducible and amidase-negative mutants these two characters were co-transduced with frequencies of 89–96%. The amidase structural and regulator genes are considered to be closely linked.

Catabolite Repression and the Induction of Amidase Synthesis by Pseudomonas aeruginosa 8602 in Continuous Culture

SUMMARY: Pseudomonas aeruginosa 8602 was grown in continuous culture under steady-state conditions in a carbon-limited medium containing either 20 mM-acetamide or 20 mM-acetamide + 10 mM-succinate. The amidase specific activity was measured at various dilution rates and found to have a sharp peak at a dilution rate of 0.30–0.35 hr-1. Fully constitutive mutants (C 11 and L 9) gave curves for amidase activity with the highest values at very low dilution rates (0.05–0.10 hr-1) and these decreased as the dilution rate increased. A semi-constitutive mutant (C 17) gave a curve intermediate between that of the wild-type strain and the fully constitutive mutant (C 11). Mutants with decreased sensitivity to catabolite repression by succinate gave curves which declined less steeply at the higher dilution rates. Mutant L 9, a fully constitutive mutant with decreased sensitivity to catabolite repression, had higher specific activities than mutant C 11 at the equivalent dilution rates. Mutant L 11, an inducible mutant with decreased catabolite repressibility, had higher amidase specific activities at high dilution rates than the wild-type inducible strain. It is concluded that in continuous culture under steady-state conditions the specific activity of the inducible amidase of P. aeruginosa is determined by the balance between induction and catabolite repression and that catabolite repression is directly related to the growth rate of the culture.

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.