Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Fundamental aspects of mycoplasma cell and molecular biology remain poorly understood, despite the impact of mycoplasmas on public health and agriculture. More effective means of prevention, control and resolution of infectious disease requires elucidation of the fundamental biological rocesses of the etiological agents involved. With the M. pneumoniae genome sequence complete, this will require continued pursuit and application of approaches to manipulate the mycoplasma genome in novel ways. M. pneumoniae colonization of the respiratory epithelium (cytadherence) is a complex process that includes proteins directly involved in receptor binding, as well as those having an accessory role involving assembly of the attachment organelle. The focus of this proposal is the structure, function and regulation of several mycoplasma proteins having very different roles in the adherence process. The adhesin P1 is concentrated at the attachment organelle, and the applicant will investigate by deletion analysis and green fluorescent protein fusions, the domains of P1 essential for trafficking to the attachment organelle and association with the mycoplasma cytoskeleton. Protein P30 is required for cytadherence, but loss of P30 is also associated with a developmental defect. This phenomenon will be explored in more detail, focusing in particular on nucleoid condensation and partitioning in P30 mutants, as well as identification of domains important in P30 function and subcellular localization. Finally, HMW1 is required for development of the attachment organelle and for P1 trafficking to this structure. The C-terminus of HMW1 is essential for function and is also targeted for accelerated proteolysis in hmw2 mtants. Structure-function studies will be pursued with HMW1, including identification of the target site(s) for proteolysis and analysis of the role of proteolysis in controlling HMW1 function in cell development.