Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

Induction of penicillinase by bacitracin.

Bacitracin preparations are shown to induce penicillinase (β-lactamase I) formation in strain 569 of $\text{Bacillus cereus}$. At high bacitracin concentrations (2–4 mg/ml) the level of induced enzyme obtained reaches a maximum which is comparable to that induced by optimal concentrations (1–2 mcg/ml) of β-lactam antibiotics. Penicillinase formation induced by short exposure to bacitracin, continues at a normal rate after all free bacitracin has been removed. The inducing activity of bacitracin is highly, but not completely, resistant to β-lactamase and can be entirely eliminated by prolonged treatment with penicillinase of $\text{B. cereus}$. The site of induction by bacitracin is, however, different from that mediating induction by β-lactam antibiotics. The inducing component has been isolated by thin layer chromatography; it seems to be closely related to, but not identical with, bacitracin A,B or F.     

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.     

Characteristics of penicillinase release by washed cells of Bacillus licheniformis

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.     

High-frequency conjugal transfer of a gonococcal penicillinase plasmid.

Gonococci containing a 24 X 10(6)-dalton conjugal plasmid were able to mobilize for transfer a smaller, non-self-transmissible penicillinase (Pcr) plasmid with high frequency under appropriate conditions. In some strains, over 10% of donor colony-forming units transferred the Pcr plasmid in a mating of less than 2 h, which suggests that the conjugal system was naturally derepressed. Colony-opacity variants containing different quantities of an approximately 28,000-dalton outer membrane protein were altered in their ability to act as conjugal donors and recipients. Maximal transfer of the Pcr plasmid was observed between transparent donors and recipients, lacking appreciable amounts of the 28,000-dalton protein. Under conditions of high-frequency Pcr plasmid mobilization, no conjugal mobilization of chromosomal markers could be discerned.     

Immunoelectron microscopic localization of penicillinase in Bacillus licheniformis.

Penicillinase was localized in log-phase cells of Bacillus licheniformis 749/C by labeling with ferritin-anti-penicillinase immunoglobulin G conjugate. Mildly fixed homogenized cells, isolated subcellular fractions, and frozen thin sections were labeled. The label was distributed in discrete patches in the cell envelope. The patches extended from the inside part of the membrane to the outside part of the wall. The inside part of the membrane was labeled more extensively than the outside part. The cytoplasm also bound some ferritin-immunoglobulin G conjugate. Immunoelectrophoresis and biochemical assay of cytosol material suggest that the cytoplasmic antigenic sites are a protease-sensitive form of penicillinase.     

The amino acid sequence of Staphylococcus aureus penicillinase.

The amino acid sequence of the penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus strain PC1 was determined. The protein consists of a single polypeptide chain of 257 residues, and the sequence was determined by characterization of tryptic, chymotryptic, peptic and CNBr peptides, with some additional evidence from thermolysin and S. aureus proteinase peptides. A mistake in the preliminary report of the sequence is corrected; residues 113-116 are now thought to be -Lys-Lys-Val-Lys- rather than -Lys-Val-Lys-Lys-. Detailed evidence for the amino acid sequence has been deposited as Supplementary Publication SUP 50056 (91 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.