A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage 011 can be transformed. Superinfection of competent cells with ,11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower. cedure for transformation, the recipient cells were grown on TSA plates and pyrimidine-requiring mutants were grown on TSA plates with added pyrimidine. Both cultures grew at 37 C overnight and then were suspended in TSB medium to an optical density at 524 nm (OD524) of 0.100, which equals 5 x 107 colony-forming units (CFU) per milliliter. The cell suspension was then diluted 10 times in TSB medium and incubated on a rotary shaker at 37 C. Maximal competence is reached after 1.5 to 2 h of growth (somewhat dependent on the strain used; see Fig. 1 and 2). The competent cells were washed once in 0.15 M NaCl and then suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris)-maleate buffer (pH 7.0) containing 0.1 M CaCl2 at a cell density of approximately 109 CFU/ml. In the transformation experiments, 0.9 ml of this cell suspension was mixed with 0.1 ml of DNA solution to give saturating concentration of DNA, i.e., 10 ug/ml (8). After incubation at 30 C for 20 min, the cells were centrifuged and suspended in 1 ml of TSB 155 on July 31, 2020 by guest
Lysogenicity with phage P11 is a requirement for competence in the presence of calcium ions in Staphylococcus aureus 8325N. The wild-type strain 8325N, lysogenic for the phages P11, P12, and P13, is also competent, but strain 8325-4, a nonlysogenic derivative of strain 8325N, as well as strains 8325-4 (P12) and 8325-4 (P13) could not develop competence. Preincubation of strain 8325-4 with culture filtrates from a competent strain can induce competence, but rabbit anti-Pll serum can neutralize the competence factor. Superinfection of competent strain 8325-4 (P11) with phage P11 at high multiplicities increases the transfection frequency. Uptake of deoxyribonucleic acid by competent cells is dependent on calcium ion concentration, pH, and temperature. Inhibition of energy metabolism or protein synthesis before and during incubation with deoxyribonucleic acid affects the binding and uptake. The ability to develop competence during bacterial growth differs between the wild-type strain (8325N) and a nuclease-deficient mutant (8325N nuc). The wild-type strain has a narrow competence maximum in the early exponential growth phase where no extracellular nuclease activity is produced. The nuc strain shows in addition competence maxima later in the exponential growth phase. 105 plaque-forming units (PFU)/,ug of DNA. MATERIALS AND METHODS Staphylococcal strains. The S. aureus strains used are listed in Table 1. The wild-type strain 8325N (P11, P12, P13) was kindly provided by M. H.
Both phage ø11 and 83A, when present as prophage or when used as helper phage, induce competence for transfection and transformation to the same level in Staphylococcus aureus , strain 8325-4. Cells lysogenized with certain temperature-sensitive ( ts ) mutants of phage ø11 show competence at the nonpermissive temperature (41 C) without production of infectious phages. Phage ø11 ts allele 31 can neither as a prophage nor as a helper phage develop competence under nonpermissive conditions. This mutant appears, therefore, to be mutated in the region of the phage genome controlling competence. The competence level for both transfection and transformation is increased by superinfecting strain 8325-4 (ø11) or 8325-4 (83A) at high multiplicities with phage ø11 with some of its mutants or with phage 83A. This superinfection enhancement appears to require protein synthesis but not deoxyribonucleic acid synthesis as judged from studies with inhibitors of macromolecular synthesis. Besides the phage particle, no extracellular or cell-bound factors so far detected can induce competence. The phage-induced product conferring competence is rapidly synthesized by strain 8325-4 ( ts ø11 31 ) after shift to permissive conditions, but requires deoxyribonucleic acid and protein synthesis to be expressed. Recombination between the sus mutants of phage ø11 of Kretschmer and Egan and ts ø11 31 indicate that competence is controlled by an early gene in the lytic cycle which may be expressed also in lysogenic cells. The phage product inducing competence appears to have a half-life of 10 to 15 min in the conditional lethal mutant at shift to nonpermissive temperature. Ultraviolet inactivation of phage ø11 infectivity occurs more rapidly than inactivation of competence induction. In fact, the number of transformants is increased at low doses of irradiation. Competence induction is, however, decreased at high does of ultraviolet irradiation.
Plasmid and probably also chromosomal characters have been genetically transformed in Staphylococcus aureus. Recipient cells show competence throughout the exponential growth phase with a maximum at early times.Recently Nomura et al. (11) reported on induction of hemolysin synthesis by transformation in Staphylococcus aureus. Earlier re-
The localization of the gene(s) mediating methicillin resistance (mecr) in Staphylococcus aureus was determined by transformation with deoxyribonucleic acid (DNA) from a natural mecr strain (DU 4916) and transformants obtained with DNA from this strain. Streptomycin resistance genes (strr) and novobiocin resistance genes (noVr) were used concurrently as representatives for chromosomal genes; penicillinase (PI2.4) and tetracycline plasmids were used as
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