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Gel filtration of supernatant fluids, from the wild-type Clostridium perfringens, strain CN3870, and several of the mutants and reverants derived from this strain, showed that these mutants failed to product detectable amounts of still produced sialidase III activity. The reverants tested had regained the ability to produce approximately wild-type levels of the I and II forms of both activities. These results showthat there is a direct relationship between the production of the I form and hemagglutinin and sialidase activities and the production of the II form of these biologically active proteins. Models that explain the genetic basis for these results are discussed.

Freshly compared cell suspensions of clostridia (Clostridium bifermentans, C. botulinum proteolytic type A, C. difficile, C. sordellii, and C. sporogenes) and Peptostreptococcus anaerobius converted leucine to isovaleric (iV) and isocaproic (iC) acids in the absence of other amino acids. The optimal pH for conversion was between 8 and 9 at 37 degrees C. The stoichiometry of reaction was compatible with that expected for the Stickland reaction, as the ratio of iV to iC was 1:2, the amount of CO2 produced was equivalent to that of iV, and ammonium ion concentrations were equal to the total C5 and C6 acids formed. The presence of alanine and valine (proton donors in the Stickland reaction) in incubations effectively increased the concentration of iC at the expense of iV production, implying that leucine acted there primarily as a proton acceptor. Glycine and proline (proton acceptors) stimulated both iV and iC production from leucine, but increases in iV concentrations were proportionately greater than for iC so that leucine was primarily a proton donor in the presence of proton acceptors. Glucose stimulated the conversion of leucine to volatile fatty acids but favoured iC production. Production of iC from leucine was inhibited by surface active compounds (cetyltrimethylammonium bromide and desoxycholate) as well as arsenite and iodoacetate. The redox dyes methyl viologen and phenosafranine inhibited iC production more severely than iV production, as did the nitroimidazole antimicrobial agent, metronidazole.

Chloramphenicol-resistant strains of Bacteroides fragilis (minimum inhibitory concentration, 12.5 mug/ml) were isolated from a stool specimen which contained multiply resistant Escherichia coli. The enzyme responsible for resistance, chloramphenicol acetyltransferase, was produced constitutively by these strains; the specific activity was 10-fold lower than that of the E. coli enzymes. Similar activity was not detected in susceptible B. fragilis strains, nor could it be induced by growth in the presence of chloramphenicol or by mutagenesis. The enzyme had a pH optimum of 7.8 and a molecular weight of approximately 89,000. The K(m) for chloramphenicol was 5.2 muM, and the enzyme was sensitive to inhibition by 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme produced by an E. coli strain isolated from the same specimen had a similar K(m) and sensitivity to 5,5'-dithiobis-2-nitrobenzoic acid.

A study of the mechanism of interaction of acetylcholinesterase with some simple tetra-N-alkyammonium ions has been made. Kinetic schemes have been proposed which are consistent with the experimental results observed in the enzyme-tetra-N-alkylammonium system in the presence of substrate and in the presence of an organophosphorus inhibitor.

Beta-Lactamase activity was detected either biologically or using the chromogenic cephalosporin 87/312 in 20 clinical isolates of Bacteroides fragilis with penicillin G minimal inhibitory concentrations of 10 to 100 micrograms/ml. Strain AM78 (minimal inhibitory concentration, greater than 1,000 micrograms/ml) was used to optimize the conditions for production, assay, and storage of the enzyme. The enzymes are cell associated, with less than 1% of activity being found in culture fluids during growth, and can be released from the cell surface by modified osmotic shock procedure. This procedure causes concomitant release of cyclic phosphodiesterase activity. Substrate profiles and the effects of inhibitors were determined for enzymes partially purified by osmotic shock release and gel filtration. The enzymes are cephalosporinases with some penicillinase activity and are inhibited by p-chloromercuribenzoate, cloxacillin, and carbenicillin. The molecular weight, as determined by gel filtration, is 29,000 to 31,000. A method for the purification of the beta-lactamase from strain AM78 is described: the specific activity of the purified enzyme was 3,424 U/mg, about 3,000-fold that of the crude, cell-associated enzyme.

The relationship between the production of hemagglutinin and sialidase activities by Clostridium perfringens was investigated by screening for mutants producing reduced levels of hemagglutinin activity. Twelve mutants were isolated; all produced reduced levels of sialidase activity and several had other altered phenotypic markers. Revertants that regained the ability to produce active hemagglutinin were isolated. All of these revertants produced increased sialidase activity. These results show that the production of hemagglutinin activity is directly related to the production of sialidase activity. Evidence is also presented that the processes of sporulation and the production of extracellular proteins are interrelated.