Analysis by Transduction of Mutations affecting Penicillinase Formation in Staphylococcus aureus

Abstract

SUMMARYSeventy-five mutants with alterations in penicillinase formation were isolated from a strain of Staphylococcus aureus inducible for penicillinase. The mutants fell into three main categories on the basis of penicillinase activity and inducibility : (i) microinducible mutants which formed decreased amounts of penicillinase but retained the property of inducibility ; (ii) penicillinase-negative variants which produced no detectable penicillinase and which showed no effect of inoculum size on penicillin resistance; (iii) strains with a wide range of penicillinase activities that produced the enzyme constitutively. Treatment of the wild-type strain with ethylmethane sulphonate increased the frequency of occurrence of microinducible and constitutive mutants but did not alter the incidence of the penicillinase-negative variants which were present in all cultures at a frequency of about Representative mutants of each class were examined for ability to revert to wild type and to give wild-type recombinants in transductional crosses. The constitutive strains and the microinducible strains behaved like point mutants in that they reverted and in that they gave wild-type recombinants. The penicillinase-negative mutants, however, behaved differently in that they were not observed to revert nor did they give wild-type recombinants in crosses, either with one another or with microinducible or constitutive mutants. A naturally occurring penicillinase-negative strain of S . aureus behaved similarly to the penicillinase-negative mutants in these respects. The possibility that the penicillinase region in s. aureus is associated with a plasmid and thus inherited extrachromosomally is considered and discussed. The properties of the penicillinase-negative variants could be explained as resulting from the loss of such a plasmid. Consistent with the plasmid hypothesis is the finding that ultraviolet irradiation of transducing phage produced an exponential decline of transducing titre for penicillinase ; against it is the failure of acridine orange to increase the frequency of the penicillinasenegative variants.

The Genetics of Penicillinase Production in Staphylococcus aureus Strain PS 80

“…The method used was the same as that described by Novick (1963). After treatment, the cultures were screened on starch agar, with or without inducer, depending on whether mutations in the structural gene or the inducibility locus were being sought.…”

“…In most penicillinase-producing strains of Staphylococcus aureus the genetic determinants for penicillinase production are on extrachromosomal particles or plasmids (Novick, 1963). In addition to the penicillinase genes these plasmids often carry genes controlling resistance to mercury (Richmond & John, I 964), arsenate, arsenite, cadmium, lead and zinc ions (Novick & Roth, 1968) and, in a few strains isolated in Japan, to erythromycin (Hashimoto, Kono & Mitsuhashi, I 964).…”

Evidence for Two Mechanisms of Plasmid Transfer in Mixed Cultures of Staphylococcus aureus

“…Bacteriophages extracted from cultures of Staphylococcus aureus are well known to mediate generalized transduction within this species (Pattee & Baldwin, 1961 ;Novick, 1963). The possibility that antibiotic resistance in S. aureus might spread between isolates in nature by transduction was strengthened by the construction of an element that contained DNA of both plasmid and phage origin (Novick, 1967).…”

The Increased Rate of Loss of Penicillinase Plasmids from Staphylococcus aureus in the Presence of Rifampicin

“…acridine orange/ml. However Novick (1963) and Richmond (1965) were unable to show any curing of staphylococcal strains using acridine dyes, and consequently the effect of these compounds must be regarded as rather variable from strain to strain. Recently Bouanchaud, Scavizzi & Chabbert (1969) have reported the elimination of pencillinase plasmids from certain staphylococcal strains with ethidium bromide, a drug known to intercalate between DNA base pairs, thus indirectly hindering the action of DNA and RNA polymerases (Waring, 1966).…”

“…All the penicillinase-less colonies that were detected had also lost the cad-r and ero-r markers, indicating that the whole penicillinase plasmid (a i-p+ cad-r ero-r), rather than the penicillinase genes alone, had been lost from the cocci. One possible origin of the high proportion of plasmid-less variants in the treated culture was that rifampicin might select the plasmid-less variants known to occur spontaneously during the growth of this strain in liquid medium (Novick, 1963). To exclude this possibility, the rates of growth of the parent, a spontaneously occurring plasmid-less variant and a plasmid-less variant that had arisen previously in the presence of rifampicin were compared.…”