Faculty Bibliography

Most Staphylococcus aureus strains associated with toxic shock syndrome and producing toxic shock syndrome toxin 1 (TSST-1) require tryptophan because of a genetic defect in tryptophan biosynthesis. The association between TSST-1 production and tryptophan auxotrophy was not correlated with the phage type, the colonization site, or the disease status of the patient from whom the isolate came. Protoplast fusion and transformation mapping located the genetic determinant of TSST-1 production (tst) very close to the trp operon in such strains and very close to tyrB in a Trp+ TSST-1+ strain. Southern blot hybridization of ClaI-restricted chromosomal DNA with a tst-specific probe revealed a common homologous segment in all of the Trp+ strains with tst linked to tyrB. These results confirmed that the tst determinant in Trp- strains is located at one site, whereas in Trp+ TSST-1+ strains the determinant is located elsewhere on the S. aureus chromosome. It is suggested that the TSST-1 determinant is associated with the insertion of a transposonlike segment into several sites on the S. aureus chromosome

A monoclonal antibody, designated B-14, inhibits the nonspecific T lymphocyte mitogenicity of toxic-shock-syndrome toxin-1 (TSST-1), and the antibody binds to an internal cyanogen bromide (CNBr) fragment (Mr, 14,000) of the toxin. The epitope recognized by B-14 was further localized to include a decapeptide at the NH2-terminus of the CNBr fragment. The decapeptide inhibited the ELISA and western blot reactivity of B-14 with TSST-1, although it was approximately 10,000-fold less effective than the native toxin. The peptide also inhibited the capacity of B-14 to block TSST-1-induced mitogenicity. A conjugate, consisting of decapeptide4-ovalbumin, was used to hyperimmunize three rabbits. Serum from these rabbits reacted specifically with intact TSST-1 in ELISA and western blots and partially neutralized toxin mitogenicity; however, the serum did not prevent fever and enhancement of susceptibility to endotoxin shock typically seen in rabbits after administration of TSST-1

We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system

The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed. In addition to pT181, the family presently includes the streptomycin resistance plasmid pS194 and the chloramphenicol resistance plasmids pC221, pC223, and pUB112. Although they belong to five different incompatibility groups, the five plasmids have similar basic replicons, use the same basic copy control mechanism, and have a common structural organization. It has been demonstrated previously that pT181 and pC221 encode trans-active replication proteins (RepC and RepD, respectively) which specifically recognize the respective plasmid's origin of replication in both cases is initiated by site-specific nicking and 3' extension. The other three plasmids in this family encode similar replication proteins; 63% of the predicted amino acid residues are identical for all five and the least similar pair shows 75% identity at the amino acid level. However, despite this homology, the replication proteins and origins of replication of different members in this family did not show cross complementation in vivo. Outside of the basic replicon, which comprises about one-third of each plasmid's genome, functional organization is also conserved. The resistance determinants are all located in the same position, immediately downstream of the replication protein coding sequence, and all are transcribed in the same direction. The three chloramphenicol resistance determinants encode highly homologous chloramphenicol transacetylases which are unrelated to the tet and str gene products. Three of the five plasmids form relaxation complexes and the involved genome segments are closely related. The other two are not homologous to these three in the corresponding region, but are homologous to each other and encode a site-specific recombinase, Pre. It is suggested that the replication, resistance, and relaxation complex regions of these plasmids can be regarded as conserved segments ("cassettes") assembled in various combinations, but always with the same spatial arrangement.

Staphylococcus aureus plasmid pT181 is thought to replicate via an asymmetric rolling-circle mechanism. By studying pulse labeled replicative intermediates, here we report that pT181 replication involves: (1) a post-replicative hypersupercoiled monomer and (2) a partially replicated intermediate which lacks superhelicity but is unlike a typical rolling-circle intermediate in that only nascent strands of less than unit length are released by alkali denaturation. A model for pT181 replication is proposed to accommodate this apparent discrepancy

pS194 is a naturally occurring Staphylococcus aureus plasmid encoding streptomycin resistance. The plasmid has a copy number of about 25 per cell, and belongs to the inc5 incompatibility group. The nucleotide sequence of pS194 has been determined and consists of 4397 base pairs including four open reading frames potentially encoding proteins of greater than 100 amino acids. All four of these reading frames are on the same coding strand. The first reading frame, repE, encodes a 38 kd protein specifically required for pS194 replication. The second open reading frame, str, encodes a 34 kd polypeptide required for streptomycin resistance, probably a streptomycin adenylyltransferase. The third potential polypeptide, rlx, would be 37 kd and is probably required for relaxation complex formation and plasmid mobilization by conjugative plasmids. The fourth, orfD, overlapping the rlx reading frame, is potentially 27 kd, and may also be involved in mobilization

We have developed a rapid method to quantitate specific bacterial RNA species. The method measures the steady-state level of RNA, produces a linear response over more than a 16-fold range of RNA concentration, and can be used for Staphylococcus aureus, Escherichia coli and Bacillus subtilis. In this method, a sheared whole-cell lysate of approx. 7 x 10(8) organisms, prepared as for plasmid screening, is separated on agarose, blotted to a nitrocellulose filter, hybridized with a radiolabeled DNA probe, and autoradiographed. The RNA species are quantitated by counting the radioactive bands on the filter. We have applied the method to the measurement of mRNA induction of the genes encoding beta-lactamase, ermC rRNA methylase, and the alpha-complementing fragment of beta-galactosidase. Upon induction, a ten-fold increase in the mRNA for each gene was observed. The peak mRNA level occurred after 30 min for beta-lactamase, 20 min for beta-galactosidase, and 5 min for the ermC rRNA methylase