GENETIC RECOMBINATION IN MYCOPLASMAS

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Mycoplasmas, the smallest free-living microorganisms, arc widely distributed in nature and commonly produce disease in plants, insects, and animals, including man. Although the economic impact of these diseases is great, little information is available concerning mechanisms of pathogenesis and effective methods of control are unavailable. The surface properties of mycoplasmas vary at a high frequency of about 103 per cell per generation. High-frequency phenotypic variations affect the structure of surface antigens, colony morphology, the susceptibility of the organism to mycoplasma viruses, and the adsorption of mycoplasmas to red blood cells. High-frequency changes in surface properties have also been shown to be important to disease pathogenesis. Some high-frequency phenotypic variations in Mycoplasma pulmonis have been correlated with site-specific DNA rearrangements that are the focus of this proposal. Two DNA rearrangements that occur concurrently are of particular interest One is the inversion of a novel DNA element encoding a restriction and modification (R-M) system. The other involves DNA sequences encoding epitopes present in variable surface antigens. The long-range goals are to understand the molecular basis of mycoplasmal chromosomal and phenotypic variation, the role these phenomena play in disease pathogenesis, and the role they have played in mycoplasmal evolution. A tentative hypothesis is that R-M systems regulate chromosomal DNA rearrangements in mycoplasmas via double-strand break repair pathways. Such a recombination mechanism is fundamentally different from known pathways of recombination in prokaryotes, and these studies may have considerable impact on current thinking regarding genetic variation in pathogenic bacteria The goals of this current proposal are to characterize the R-M systems of M. pulmonis, to characterize the genes encoding the variable surface antigens, and to determine how DNA rearrangements regulate expression of these systems. The first aim is to clone and characterize at the nucleotide level a genetic locus that is highly homologous to the invertible element. This locus may encode a second R-M system. The second aim is to determine how DNA inversion affects R-M by examining the transcriptional regulation of the pertinent loci. The third aim is to inactivate the R-M systems in M. pulmonis in order to determine whether R-M enzymes play a causal role in high- frequency DNA rearrangements and phenotypic switching in mycoplasmas. The fourth aim is to determine how DNA rearrangements affect expression of variable surface antigens. This will be accomplished by characterizing both the genes encoding variable surface antigens and the DNA rearrangements that occur in their vicinity. The fifth and final aim is to expand our repertoire of subclones containing DNA inversions and other rearrangements in order to more fully characterize the potential for genetic variation in this system.