S. aureus is an important pathogen because it is common, virulent, and prone to develop resistance. As resistance to other drugs increases, clinicians are forced to rely on new agents, such as the quinolones. Quinolone antibiotics are very effective agents, but emergence of resistance to quinolones occurs readily. By understanding the mechanisms of resistance, strategies may be devised and new drugs developed to circumvent resistance. Based on work in E. coli, two general mechanisms of resistance to quinolones have been identified: 1) point mutations leading to structural alterations in the target enzyme, DNA gyrase and 2) reduced permeation of drug to its target, primarily as a result of outer membrane protein changes. Mechanisms of resistance in S. aureus are likely to be different from those in E. coli because staphylococci have no outer membrane to limit permeability. Also single step resistant mutants characteristically do not have gyrase mutations present and a novel locus, flqA, not genetically linked to gyrA or gyrB is involved. flqA also determines whether gyrase mutations, including nov (probably the same as gyrB and which determines novobiocin resistance) are expressed or not. The main thrust of the research is to identify the gene product encoded by flqA. Considerable data indicate that flqA encodes a topoisomerase IV protein. Dr. Hooper has identified two other loci, flqB and flqC, involved in expression of resistance. flqB is a point mutation upstream of norA, a gene encoding an efflux pump, over-expression of which leads to quinolone resistance by virtue of increased pumping of quinolone out of the cell. flqC is probably a resistant allele of gyrA. There are four aims: 1) Characterization of the flqA locus, its relationship to topoisomerase IV, and its interactions with flqC locus. Based on data indicating that flqA is gyrA- or gyrB-like and that mutations are present in flqA compared to flqA+, lambda clones containing these sequences will be subcloned first into E. coli (and sequenced) then into S. aureus, in the latter case via a single copy integration vector or on a plasmid, to determine effects on phenotype. Topoisomerase genes will be expressed in E. coli and purified for biochemical studies. The effects of flqA mutations on nov and flqC expression will be examined by introducing flqA mutants and flqA+ on a plasmid into S. aureus. The effect of flqC on flqA expression will be examined by transforming S. aureus with a plasmid containing beta- lactamase fused to the flqA promoter, and assaying for beta-lactamase activity. 2) Identification of flqC as an allele of gyrA and nov as an allele of gyrB. PCR amplified DNA from gyrA and gyrB will be sequenced to identify mutations associated with flqC and nov. Wild-type gyrA and gyrB cloned on a temperature sensitive plasmid will be used to transform S. aureus to integrate into the host chromosome to "cross out" resistance of flqC and nov, thereby establishing their identity with gyrA and gyrB. 3) Characterization of the regulation and normal functions of NorA. The normal norA promoter (i.e., flqB+) and the flqB mutation each will be cloned upstream of blaZ and the respective plasmids will be used to transform flqB and flqB+ S.aureus. The effect of the promoter mutation on norA transcription will be assayed by measuring beta-lactamase activity. Similar experiments will be performed to investigate the interaction of flqB with other resistance loci and the effects of environmental factors on expression. Polyclonal antiserum will be prepared against NorA for use in immunoblotting assays to characterize NorA production in clinical isolates. Studies to determine whether norA is essential will be conducted using allelic replacement or temperature sensitive plasmid constructs. 4) Defining determinants of resistance and susceptibility in clinical isolates of S. aureus. Presence of flqA and flqC in clinical isolates will be sought by complementation experiments using flqA+ and flqC+ plasmids. norA expression will be compared among clinical isolates in immunoblotting experiments.