Faculty Bibliography

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter

The synthesis of virulence factors and other extracellular proteins by Staphylococcus aureus is globally controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide. The cognate peptides produced by some strains inhibit the expression of agr in other strains, and the amino acid sequences of peptide and receptor are markedly different between such strains, suggesting a hypervariability-generating mechanism. Cross-inhibition of gene expression represents a type of bacterial interference that could be correlated with the ability of one strain to exclude others from infection or colonization sites, or both

pT181 is a Staphylococcus aureus rolling circle plasmid that regulates its replication by controlling the synthesis of its dimeric initiator protein RepC/C and by inactivating the protein following its use in replication (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). This inactivation consists of the addition of an oligonucleotide, representing several nucleotides immediately 3' to the initiation nick site, to the active site tyrosine of one of the two subunits, generating a heterodimer, RepC/C*. Previous results suggested that the inactive form was metabolically stable and was present at a much higher level than the active form (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). In the present study we have measured total RepC antigen as a function of plasmid copy number and have analyzed the interaction of the two forms. We find that pT181-containing staphylococci contain approximately one RepC dimer per plasmid copy over a 50-fold range of copy numbers. This is consistent with previous measurements of the rate of RepC synthesis, which suggested that one RepC dimer is synthesized per replication event (J. Bargonetti, P.-Z. Wang and R. P. Novick, EMBO J. 12:3659-3667, 1993). The RepC/C* heterodimer, which is inactive for replication, is a competitive inhibitor of the replication and the topoisomerase-like and cruciform-enhancing activities of the native protein. These results suggest that the inactive form may have a specific regulatory role in vivo. Since the known plasmid-determined controls, which maintain a constant plasmid copy number, are designed to ensure the synthesis of one RepC/C dimer per plasmid replication event, it is difficult to envision any role for yet another negative regulator of replication. Conceivably, under conditions where the initiator is overproduced, such as in the absence of the normal antisense regulation of initiator production, RepC/C* could serve as a fail-safe means of preventing autocatalytic replication

Staphylococcus aureus plasmid pT181 replicates via a rolling circle mechanism. The synthesis of the pT181 initiator protein (RepC) is regulated by antisense RNAs, and RepC is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. The inactivated heterodimeric RepC/C* has been shown be unable to initiate replication in vitro (Rasooly, A., and Novick, R. P. (1993) Science 262, 1048-1050). The inactive RepC/C* has been found to be very stable and constitute about 90-95% of the total RepC antigen inside the cell. We studied the specific interaction of the RepC/C and RepC/C* complex with the pT181 double strand origin. The results indicated that RepC/C and RepC/C* footprint supercoiled DNA differently although their footprints on linear DNA are similar; we also find that RepC/C is able to enhance cruciform extrusion while RepC/C* cannot. RepC/C* binds and bends the double strand origin much more weakly than does RepC/C. These results suggest that the attached oligonucleotide induces a conformational change in the RepC/C* molecule that is responsible for its lack of activity

Previous studies have shown that glycerol monolaurate (GML), a surfactant commonly used in a wide variety of food and cosmetic products, inhibits the production of a variety of exotoxins by group A streptococci and staphylococci. Given the highly lipophilic nature of the structure of GML, it is suspected that the surfactant exerts its toxin inhibition effects via interaction with the cell membrane. The present study attempted to characterize some of the potential targets of GML action using the model system of lymphocyte activation. Results from murine splenocytes show that GML stimulates proliferation at concentrations between 10(-5) and 5 micrograms/ml/5 x 10(5) splenocytes. At concentrations greater than 5 micrograms/ml, GML inhibited lymphocyte proliferation and blocked the proliferative effects of the lymphocyte mitogens phorbol myristate acetate and concanavalin A and the potent T-cell mitogen toxic shock syndrome toxin-1. Studies using purified immune cell subsets indicated that GML at a concentration of 0.1 microgram/ml optimally induced proliferation of T cells but did not affect B cells. At higher concentrations, GML inhibited the toxic shock syndrome toxin-1 mitogenic effects on T cells, but did not inhibit the lipopolysaccharide-induced stimulation of B cells, suggesting that GML preferentially affects the T-cell population. GML-induced proliferation was blocked by the immunosuppressive drug cyclosporin A, suggesting that GML may be exerting its T-cell-proliferative effects along the calcium-dependent inositol phospholipid signal transduction pathway

Toxic shock syndrome toxin 1 (TSST-1) contains a long central alpha helix that forms the base of two grooves on opposite sides of the molecule. Previous studies indicated that residues 132, 135, and 140 along the back of the central alpha helix are important in the biological activities. We made mutations of additional central alpha-helix residues exposed along this groove on the back of TSST-1. The proteins were purified, shown not to have gross alteration in structure, and tested for both superantigenicity and ability to elicit lethal TSS, using the superantigenicity, likely to because of alteration in T-cell receptor binding. Mutants H135A, Q136A, and E132K/ Q136K lost the ability to induce lethal TSS. The mutant Q136A was most increasing because it was superantigenic, yet nonlethal