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

A thymine-requiring mutant of Staphylococcus aureus, strain 8325(PIM)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated 414, which differs from phage 441 in its immunity locus. The thymineless induced lysates of strain 8325(PIn8)thy transduce the penicillinase plasmid at high frequency (10-1), whereas transduction of chromosomal markers is inefficient. A plasmid-cured derivative of strain 8325(PI,.m)thy is also lysed by thymine starvation and can be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives. on July 7, 2020 by guest

Specific transduction of inducible (eroA) and constitutive (eroB) erythromycin resistance is mediated by thymineless induced lysates from derivatives ofStaphylococcus aureus strain 8325(N)thy. Both loci can coexist in the same cell but segregate by transduction or transformation. The gene(s) is probably integrated in the recipient chromosome and excised at thymine starvation.

Objectives To evaluate the effect and mechanism of action of the flavonoid phloretin on the growth and sucrose-dependent biofilm formation of Streptococcus mutans . Methods Minimum inhibitory concentration, viability, and biofilm susceptibility assays were conducted to assess antimicrobial and antibiofilm effect of phloretin. Biofilm composition and structure were analysed with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Water-soluble (WSG) and water-insoluble glucan (WIG) were determined using anthrone method. Lactic acid measurements and acid tolerance assay were performed to assess acidogenicity and aciduricity. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the expression of virulence genes essential for surface attachment, biofilm formation, and quorum sensing. Results Phloretin inhibited S. mutans growth and viability in a dose-dependent manner. Furthermore, it reduced gtfB and gtfC gene expression, correlating with the reduction of extracellular polysaccharides (EPS)/bacteria and WIG/WSG ratio. Inhibition of comED and luxS gene expression, involved in stress tolerance, was associated with compromised acidogenicity and aciduricity of S. mutans . Conclusions Phloretin exhibits antibacterial properties against S. mutans , modulates acid production and tolerance, and reduces biofilm formation. Clinical significance Phloretin is a promising natural compound with pronounced inhibitory effect on key virulence factors of the cariogenic pathogen, S. mutans .

Seventeen mutants of a strain of Arthrobacter, producing altered amounts of extracellular protease, were isolated. One mutant had lost the ability to form wild type extracellular protease. Despite this loss, the mutant demonstrated proteolytic activity due to leakage of a thermolabile intracellular proteinase. Chemical, immunological and catalytic properties of this enzyme show that it is not related to the extracellular protease of the wild type.