Induction of penicillinase by staphylococci in vitro and in vivo

Eyckmans, Luc (Cornell University Medical College, New York, N.Y.), and Edward W. Hook. Induction of penicillinase by staphylococci in vitro and in vivo. J. Bacteriol. 91:997-1003. 1966.-Staphylococci in mice with peritoneal infection showed no significant increase in penicillinase activity 6 hr after administration of penicillin G. In contrast, induction of penicillinase was readily demonstrated in leukopenic animals under similar conditions. Induction of penicillinase by staphylococci in vitro was inhibited by including leukocytes and immune serum in the mixtures. The role of leukocytes in inhibiting induction of penicillinase by staphylococci in response to penicillin was investigated.     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.     

Cross-linking preserves conformational changes induced in penicillinase by its substrates.

Exopenicillinase of Bacillus cereus 569/H was cross-linked with toluene 2,4-diisocyanate in the presence of cephalothin, cloxacillin or no substrate. The derivatives show significant differences in susceptibility to inactivation by heat, urea, iodination or proteolysis. Such differences can be predicted from the contrasting effects of these substrates on the conformation of the enzyme.     

Transposition of the oxacillin-hydrolyzing penicillinase gene.

We have found that the oxacillin-hydrolyzing penicillinase gene (oxa) encoded by plasmid RGN238 transposes to various plasmids in a recA background. We call this transposable element Tn2603. Tn2603 encodes the genes for streptomycin, sulfonamide, and mercury resistance in addition to the oxa gene. Tn2603 has a molecular size of 19.6 kilobase pairs and appears to be flanked by small inverted repeat sequences of about 200 base pairs long.     

Antagonists of bacitracin

A device is described for the cytochemical analysis of pathogens requiring up to level 3 containment. The rapid demonstration of induced β-galactosidase in Gram-negative cells detected in a blood culture illustrates potentially wide applications of the device in clinical practice and research.     

Continuous production of bacitracin by immobilized living whole cells of Bacillus sp.

Whole cells of Bacillus sp., a bacitracin-producing bacteria, were immobilized in polyacrylamide gel. The continuous production of bacitracin by an immobilized whole-cell-containing air-bubbled reactor was examined with 0.5% peptone solution. The bacitracin productivity (28 units/ml/hr) obtained with this system was higher than that with a batch system. The effluent bacitracin concentration increased with increasing aeration rate and reached a steady-state maximum above the aeration rate of 3.0 liter/min. A high bacitracin productivity was retained for at least eight days when the gel was washed with sterilized saline at a flow rate of 250 ml/hr for 2 hr once a day. The half-life of the immobilized whole-cell system was about 10 days. Bacitracin productivity by the immobilized whole-cell reactor was higher than that by a conventional continuous fermentation process at high dilution rates.     

Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.     

Biosynthesis of bacitracin on a protein thiotemplate.

The dodecapeptide bacitracin A is the major constitutent of a family of antibacterial peptides produced by Bacillus licheniformis. The non-ribosomal biosynthesis of bacitracin has been studied in cell-free extracts. Bacitracin synthetase has been fractionated on Sephadex G 200 column into two fractions; both fractions were required for bacitracin biosynthesis. On the other hand, on a Sepharose affinity chromatography column, using L-leucine as ligand, three fractions were obtained; all three were required for bacitracin biosynthesis. During bacitracin synthesis, the enzyme components contain a number of thioester bound peptides. The nature of the peptides suggested that the synthesis proceeds towards the C-terminal end of the molecule. It is assumed that by sequential addition of thioester-bound amino acids, bacitracin A could be synthesized on the surface of the enzyme containing phosphopantetheine.