Faculty Bibliography

Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids.

A series of studies were performed on a Staphylococcus aureus strain thought to contain a pencillinase plasmid integrated into the host chromosome. Reassociation kinetics analysis of whole-cell deoxyribonucleic acid (DNA) in the presence of pure radioactive plasmid DNA revealed that plasmid-specific sequences were present at about 1 copy per chromosome equivalent as compared to 3.6 copies for the same plasmid in its autonomous state. Consistent with this observation was the finding that penicillinase activity was lower for the former strain than for the latter. It was shown further that the plasmid-specific sequences cosedimented on neutral sucrose gradients with fragments of whole-cell DNA many times larger than the plasmid. These two findings were taken as strongly confirmatory of the integrated state. Analysis of whole-cell ribonucleic acid for the presence of plasmid-specific messengers revealed that these were present in approximately the amounts expected on the basis of the DNA study

Six different Staphylococcus aureus plasmids have been examined for compatibility relationships and their intracellular DNA pools have been measured singly and in various combinations. All six were mutually compatible, but contrary to expectation, their intracellular DNA pools were not additive; instead, there appeared to be a maximum level of extrachromosomal DNA that could be supported by the cell, and the plasmids studied approached this level individually as well as in varous combinations. One exception was encountered: a plasmid encoding kanamycin/neomycin resistance was present in a small but constant number of copies regardless of the presence of other plasmids

The quantity of penicillinase plasmid deoxyribonucleic acid (DNA) in various strains of Staphylococcus aureus has been determined by DNA-DNA reassociation kinetics. Specifically, 32P- or 125I-labeled denatured probes of purified plasmid DNA were reassociated in the presence of denatured DNAs isolated from the bacterial strains in question. The number of plasmid copies per cell was calculated from the effect of the latter nucleic acid samples on the reassociation rate of the radiolabeled probe. Among the S. aureus strains examined were monoplasmid, diplasmid and replication-defective representatives, and the effect of temperature on wild-type plasmid content was also investigated.

A study has been done of reversion to thermostability of thermosensitive, replication-defective (TSR) mutant penicillinase plasmids. All three of the expected classes of reversions were encountered: back mutation, suppression, and integration. The latter class was examined in some detail and it was found that the presence of the phi 11 phophage enhance the frequency of reversion by integration some 103-fold. Prophage-dependent integration resulted in inactivation of plasmid-linked arsenate and arsenite resistance; these revertant strains gave rise to high frequency tranducing lysates where the plasmid was restored upon transduction to its original TSR state including recovery of these resistances. The integrated plasmid-prophage complexes were stable at high temperatures (43 C) but slow growing and unstable at low (32 C); loss of either plasmid or prophage restored normal growth and stability. Sometimes restoration of the plasmid to its autonomous TSR state was observed and molecular studies showed that in most cases the plasmid was essentially the same size as before integration. In some cases an excision complex was recovered that was more than twice the size of the plasmid and could have been a plasmid-phage co-integrate. Integration also took place in the absence of the l 11 prophage. These integrations retained all plasmid-linked resistances, were stable at all temperatures, and gave rise to low frequency transducing lysates in which the integrated state was retained upon transduction. On the basis of these results it is suggested that the prophage promotes integration at or near its attachment site