The aim of this research is to improve the understanding of a new cell surface regulatory process (talk-back regulation) in the oral bacterium Streptococcus mutans. The physiological aspects of S. mutans that enable the organism to survive the environmental stresses of feast or famine and continuous assaults by host defense mechanisms inherent to the oral cavity are basic to understanding the persistence and pathogenicity of this organism. Elucidation of the mechanism(s) by which the cell surface (in this instance, the Gram positive cell wall) communicates its growth status to the cytoplasm seems likely to be important in this regard. In addition to further describing a basic regulatory circuit, this study will also describe molecular events leading to the secondary inhibition of RNA and protein synthesis observed in the tolerant response to Beta-lactam and other cell wall antibiotics. The experimental approach will utilize established bacteriological and biochemical methods to analyze molecular events subsequent of exposure to growing bacterial cultures and cultures of forming, growing, and reverting protoplasts of S. mutans FA-1 to benzylpenicillin and other inhibitors of cell wall synthesis. Kinetic and dose response analyses of inhibition of peptidoglycan, rhamnose-containing accessory wall polymers, RNA, protein and DNA synthesis following antibiotic treatment will be coupled with determinations of cytoplasmic pools of the regulatory molecule guanosine tetraphosphate (ppGpp) in growing cell and protoplast cultures. The involvement of an intact cell wall in the talk-back circuit will be determined both by timing the release from growth inhibition of cultures exposed to cell wall inhibitors relative to hydrolysis of the cell wall during protoplast formation and by establishing the kinetics of accumulation versus secretion of cell wall polymers concomitant with reacquisition of drug sensitivity in cultures of reverting protoplasts. The long-range objectives of this research are to define the molecular aspects of talk-back regulation and the involvement of cell surface regulation in the tolerant and non-lytic death response to cell wall antiobiotics.
