Intermediates in the biosynthesis of bacterial penicillinase

Abstract

A technique has been developed for the isolation in umg amounts, of exopenicillinase formed in 3-5 ml samples from cultures of the penicillinase-inducible strain 569 and the penicillinase-constitutive strain 569/H of Bacillus cereus. A search has been made for possible intermediates in the biosynthesis of exopenicillinase from amino acids by (a) measuring the degree of labeling of the exopenicillinase formed after transfer of cells, previously grown in (S35) sulfate, to fresh medium containing only unlabeled sources uf sulfur; (b) determining the specific lag in incorporation of C14- or s35-labeled amino acids from the medium into exopenicillinase formed after previous growth in unlabeled medium. The results of both types of experiment agree in showing no detectable quantities of any intermediate between "free" amino acids and exopenicillinase in the inducible strain 569; and the small quantities found in the constitutive strain 569/H probably correspond to subsequent release of the traces of penicillinase, bound to the cell surface and retained by the cells at time of transfer, into the medium as the exo-enzyme. The sensitivity of the technique permits the conclusion, based on the negative results obtained with the inducible strain 569, that with fully induced cells forming the enzyme at maximal rate, the quantity of penicillinase precursor material present could not exceed an amount corresponding to 400 molecules of penicillinase/cell. Reasons are given for believing that the true quantity may be very considerably less. These results also show (a) that the specific lag of about 15 minutes in induced penicillinase synthesis after addition of penicillin to a logarithmically growing culture of strain 569 cannot be due, in any significant degree, to the production of a specific enzyme precursor, and (b) that the intracellular "gamma-penicillinase" is unlikely normally to function as precursor of the exo-enzyme. Comparison of the results, obtained after transfer of S35-labeled cells to unlabeled medium with and without addition of excess of methionine and cystine have shown that about 10% of the sulfur of penicillinase formed by cells growing in a complete amino acid medium is derived from endogenously produced methionine or cystine, or both, probably liberated from breakdown of protein within the cells.