SITE-SPECIFIC DNA INVERSION IN MORAXELLA LACUNATA

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DNA rearrangements, involving deletion, inversion, transposition, or insertion, have been shown to control gene expression and development in numerous systems ranging from integration/excision of bacteriophage lambda to rearrangement of the gene segments of the mammalian immunoglobulin genes. the study of relatively simple DNA rearrangements in prokaryotes not only reveals the mechanisms controlling, for example, phase variation of surface antigens in microbial pathogens such as Borrelia hermsii, Neisseria gonorrhea, and Salmonella typhimurium but also provides models for understanding the regulation of gene expression in higher organisms. The objective of the work described in this proposal is to define the molecular details of the site-specific DNA inversion system of Moraxella lacunata. In M. lacunata, a causative agent in human conjunctivitis and keratitis, inversion of a chromosomal segment regulates the expression of two type 4 pilin genes. Preliminary characterization of the M. lacunata inversion system suggests that a novel mechanism for site-specific DNA inversion is utilized. The specific goals of this project are to determine the protein and DNA components of this inversion system and characterize how these elements interact to form a productive synaptic complex. A three step approach will be taken to accomplish these goals: 1) Define the essential genes and DNA sites for the recombination reaction in vivo and in vitro by insertion and deletion/substitution mutagenesis analyses of the subcloned inversion region, over-expression of identified recombination factors, and development of a defined in vitro system for the inversion reaction; 2) Characterize the interactions among the essential components of the inversion system using nuclease/chemical protection/interference assays; 3) Elucidate the molecular details of the recombination reaction using biochemical/physical assays of recombination intermediates, and mutational analyses of the recombinase and recombination sites.