Factors Affecting Competence for Transformation in Staphylococcus aureus

Abstract

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

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

“…The inhibition of E. coli K12 and S. aureus RN4220 by N5-Pan and dN5 was tested in minimal media appropriate for each bacterium. For E. coli, this medium consisted of 0.8 mM MgSO 4 , 10 mM citric acid, 60 mM K 2 HPO 4 , 20 mM NaNH 4 HPO 4 and 0.5% glucose; for S. aureus, it contained 40 mM KCl, 160 mM NaCl, 5.3 mM MgSO 4 , 30 mM (NH 4 ) 2 SO 4 , 0.11 mM CaCl 2 , 1.0 mM KH 2 PO 4 , 0.02 mM FeSO 4 , 0.04 mM MnSO 4 , 0.03 mM citric acid, 100 mM Tris, 28 mM glucose, 0.8 mM L-arginine, 1.0 mM L-proline, 1.9 mM L-glutamic acid, 1.5 mM L-valine, 1.5 mM L-threonine, 1.0 mM L-phenylalanine, 1.3 mM L-leucine, 0.78 mM L-cysteine, 0.4 lM biotin, 6.6 lM thiamine and 16 lM nicotinic acid [45]. The inhibition was determined by concentration-response analysis as described above with minor modifications.…”

Detection of a Membrane-associated Protein on Detached Membrane Ribosomes in Staphylococcus aureus

“…To obtain satisfactory incorporation of radiolabelled amino acids, cells were grown in a synthetic medium. Cells from 10 ml preculture were inoculated into 50 ml AAM-medium (Rudin et al, 1974), with the following modifications: the pH was 7.2, and glycine and serine were added to a final concentration of 500 pg ml-I each. After 4 h cultivation, 25 pCi (925 kBq) [U-14C]protein hydrolysate (Amersham) was added, and the culture was grown for another 2 h.…”

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis

“…Similarly, nuclease inactivation has also been demonstrated in Salmonella after infection with bacteriophage P22 (26). In Staphylococcus aureus (37), transformation requires lysogeny. We propose that bacteriophage )11 inactivates the ATP-dependent nuclease, or some other nuclease, in S. aureus, thereby permitting transforming DNA to enter the recipient with little degradation.…”

“…We propose that bacteriophage )11 inactivates the ATP-dependent nuclease, or some other nuclease, in S. aureus, thereby permitting transforming DNA to enter the recipient with little degradation. Essentially, lysogeny by )11 in S. aureus niay be creating the same conditions which must be artificially established to permit transformation in E. coli (7,8,30,37). Obviously, further experiments are required to elucidate the interactions between degradation and recombination enzymes during the development of competence as well as the interplay between viral and host functions.…”