The research proposed is in the general area of gene regulation studied in a model system in which erythromycin specifically and efficiently induces resistance to itself and other macrolides, to the lincosamides, and to the streptogramin B (MLS) antibiotics. Synthesis of the inducible 23S ribosomal RNA methylase responsible for MLS resistance is controlled at the translational level by an attenuator whose conformation regulates expression by determining availability of the ribosome loading site for translation initiation of the resistance methylase. One of the main unanswered questions in this work is: What mechanism accounts for the apparent wide variability of induction efficiency of different subsets of MLS antibiotics in different bacteria despite the apparently equal efficiency of these antibiotics as inhibitors? The long term objective of this proposal is to study and test models of regulation of gene expression with emphasis on genes that specify antibiotic resistance;
The specific aims of this proposal are to study regulation of inducible MLS resistance model systems by: (2) determining induction specificity in vivo following insertion of chemically synthesized model leader sequences into expression vectors constructed for this work, (2) determining amino acid sequence or codon usage alterations in mutants that involve the leader peptide and show possible associated altered specificity of induction, and (3) studying induction specificity and attenuator function in new bacterial strains. These studies of induction provide a model for understanding rapid development of constitutive MLS resistance following a short course of treatment with MLS antibiotics in a clinical setting. Inducible MLS resistance is also found in Streptomyces that produce MLS antibiotics and in some instances resistance is not expressed before the cells enter antibiotic production. It is our working hypothesis that in nature, leader peptides serve to optimize the coupling between antibiotic production and development of resistance, or more generally, between inductive stimulus and increased expression of specific genes. Methods that will be used in these studies include recombinant DNA technology and DNA sequencing applied to cloning model DNA attenuator sequences synthesized by chemical means and analyzing new control sequences from natural resources.
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