Type 1 of Escherichia coli and other gram negative enteric bacteria are filamentous, proteinaceous appendages composed of a repeating subunit, pilin. Type 1 pili mediate a mannose sensitive adhension of bacteria to a variety of eucaryotic cells and are involved, as a colonization factor, in extraintestinal infections caused by E. coli and Klebsiella pneumoniae. The long-term goal of this project is to establish the molecular nature of the genetic control, assembly an receptor-binding activity of Type 1 pili in E. coli and in doing so, contribute to the understanding of the control and assembly of supramolecular structures and the molecular nature of receptor-ligand interactions in general. Also, it is hoped this work will lead to a better understanding of the role of bacterial adhesion in the pathogenesis of infectious disease. The specific research herein proposed utilizes a variety of genetic and biochemical techniques to discern the molecular nature of; (i) The control of pilus assembly. Pilus assembly is regulated by a trans-acting polypeptide encoded by a gene (hyp) adjacent to the structural gene (pilA). Fusions of lacZ with pilA have suggested that the hyp gene product regulates piliation by repressing transcription of pilA. The proposed research utilizes the conditional lethal character of hyp mutations and pilA-lacZ fusions to identify sites in the hyp gene product and in the pilA promotor that effect pilA transcription. (ii)Pilus assembly. The products of at least two genes, pilB and pilC are involved in pilus assembly. The proposed research is designed to determine if these gene products are involved in direct interaction with pilin during polymerization by identifying mutations in pilB and pilC and then isolating Pil+ pseudorevertants having lesions in pilA; (iii) Receptor binding. Pilin interacts with a mannose containing receptor or eucaryotic cells. Experiments are designed to detect the domains of pilin involved in receptor binding by taking advantage of the nucleotide sequence of pilA and employing site-directed mutagenesis. (iv) The metastable nature of pili expression. Pili expression is regulated in a metastable manner in E. coli, resulting in variation between piliated and non-piliated states. This variation will be examined by cloning the genes for type 1 piliation from E. coli K12 and E. coli Bam onto a low copy number plasmid and determining which of the genes involved in piliation is controlled in a metastable manner.
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