The overall objectives of this proposal are to determine (by DNA sequence analysis) the primary structure of selected membrane surface proteins of the arthritogenic pathogen Mycoplasma hyorhinis, and to define a reiterated genomic element that may contribute to genomic diversity within this species and reflect transmission of genetic material among mycoplasmas. M. hyorhinis is one of over 70 species in the genus Mycoplasma, a group of diverse, rapidly evolving small wall-less procaryotes (750 kb genomes), phylogenetically related to gram positive eubacteria. It causes chronic degenerative arthritis in swine. M. hyorhinis interacts with the surface of host cells through structures exposed on its single limiting membrane. These are also targets of immune responses contributing to disease processes, and include a major group of recently defined membrane proteins covalently modified by lipids. Important phenotypic differences occur among strains of M. hyorhinis, including arthritogenic potential, degree of cytadsorption and variation of surface membrane antigens. Unique features of membrane protein structure and processing are not understood, nor is the genetic basis of phenotypic variation in M. hyorhinis or other mycoplasma known.
The first aim of the proposed research is to obtain primary DNA sequence and expression of recently identified cloned genes encoding integral membrane proteins of M. hyorhinis with potentially novel sites of lipid acylation, or encoding membrane subunits of ATPase. Critical features of mycoplasma membrane protein processing, anchorage and surface antigenic structure will be determined, and comparison of highly conserved functional membrane proteins (ATPase) will be made with analogous eubacterial counterparts.
The second aim i s to define the structure and possible transmission of a recently identified repetitive genomic element, that is highly reiterated in variable patterns among strains of M. hyorhinis, and is distributed in selecting strains of other mycoplasma species. This is important as a potential generator of genomic diversity within M. hyorhinis, and may be useful in understanding transmission of genetic material among mycoplasmas. Standard techniques of restriction mapping and Sanger dideoxy sequencing will be to characterize existing DNA fragments cloned from recombinant genomic libraries of M. hyorhinis. Expression and characterization of mycoplasma proteins from cloned fragments will utilize phage- or plasmid-directed synthesis in E. coli minicells or high expression host-vector systems, deletion analysis, antigenic analysis and tryptic mapping of recombinant protein products. Complementation analysis will be used to assess mycoplasma gene function in E. coli.
