Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Genetic transformation in Staphylococcus aureus: isolation and characterization of a competence-conferring factor from bacteriophage 80 alpha lysates.

The competence-conferring activity in crude lysates of the staphylococcal bacteriophage 80 alpha was concentrated and purified by (NH4)2SO4 precipitation, differential ultracentrifugation and rate-zonal centrifugation through Ficoll. This concentrated preparation exhibited lytic activity toward assay cells of Staphylococcus aureus 8325-4 that could not be attributed to the residual 80 alpha infectious particles present. Electron microscopic examination of the concentrated competence-conferring activity revealed an occasional intact but empty virion and large numbers of free phage tails. Sodium dodecyl sulfate-polyacrylamide gel analysis of this material confirmed that the competence-conferring activity contained only some, but not all, of the major virion proteins. The competence-conferring activity exhibited single-hit kinetics when assay cells and 80 alpha transfecting deoxyribonucleic acid were present in excess. The competence-conferring activity thus seems to be a unique morphogenic precursor of the 80 alpha virion that mediates transfection and transformation in the presence of 0.1 M CaCl2.     

Identification of a chromosomal determinant of enterotoxin A production in Staphylococcus aureus.

Chromosomal mapping of the determinants for enterotoxin A and enterotoxin B production in three strains of Staphylococcus aureus was attempted by using conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 as recipients. A gene governing enterotixin A production (entA+) in strain S-6 was located on the chromosome between the pur-110 and ilv-129 markers, very close to a determinant of alpha-hemolysin production, hla+. The entA+ gene of strain FRI-196E was shown not to be located at the same position; its location could not be determined. The entB+ genes of strains S-6 and C243 were not located within the known linkage groups examined. Recombinants were screened for enterotoxin production by a new procedure that combined characteristics of immune serum plate and optimal sensitivity plate procedures. The strains and methods used in this study of enterotoxin determinants should prove useful in genetic studies to locate other chromosomal determinants of S. aureus whose phenotypes are difficult to score or select for.     

Fibronectin enhances transfection of Staphylococcus aureus.

The factor in normal sera primarily responsible for the enhancement of transfection (and transformation) of Staphylococcus aureus was identified as fibronectin. Serum samples which were depleted of fibronectin by affinity chromatography showed a marked decrease in enhancing activity. Fibronectin isolated from sera of several animal species demonstrated enhancing activity.     

Temperature-Sensitive Osmotically Fragile Mutants of Staphylococcus aureus

Temperature-sensitive fragile mutants of Staphylococcus aureus which grow at the restrictive temperature only in the presence of osmotic stabilizers and appear to have conditionally defective cell wall integrity were isolated and partially characterized.