Mycoplasmas are unique bacteria characterized by their lack of a cell wall and minimal genomes. They are the causative agent of many important human and animal diseases such as atypical pneumonia and contagious bovine pleuropneumonia. A general requirement of successful infection is the attachment of the bacteria to the tropic tissue by adhesins that are often poorly understood. We have designed a novel method of gene-specific recombineering in Mycoplasmas in an attempt to better understand their pathogenesis at the molecular level. Due to their lack of many biosynthetic pathways, growth of Mycoplasmas is tenuous at best making this research an arduous task. To overcome this obstacle, we will first test and optimize the experimental conditions in the model organism Escherichia coli. A unique tetracycline resistance cassette will be constructed by PCR and introduced site-specifically into RecA+ and RecA- E. coli using exogenous RecA-catalyzed recombination. Successful recombinants will be selected for by tetracycline resistance and screened for ß-galactosidase activity. Appropriate mutants will be antibiotic resistant and Lac– demonstrating the knockout of the ß-galactosidase gene lacZ. The outcome of these experiments will provide a flexible new technique for the study of Mycoplasmas.