We will take a molecular genetic approach to study the genes involved in the lysogenic development of temperate phage Mx8 of the complex prokaryote, Myxococcus xanthus. Using a combination of classical phage genetics and recombinant D&A analysis, we will identify and characterize the Mx8 genes involved in site-specific recombination and superinfection immunity. Like phages lambda and P22, Mx8 can integrate its genome as prophage at a preferred attachment site (attB) on the M. xanthus genome. Unlike lambda and P22, Mx8 encodes multiple proteins required for integration. The mechanism underlying Mx8 superinfection immunity is also novel and complex, and involves a hierarchy of at least three genes, one of which may encode a DNA methylase with novel specificity. We will sequence an 8.2 kbp cloned region of Mx8 which encodes the functions required for both integration and immunity. To correlate genes with functions, we will sequence mutations that affect the decision between lytic and lysogenic development. We will also make site-directed mutations that inactivate potential open reading frames and sites involved in integration and immunity, and determine the phenotypes of these mutations on plasmids and phages. Temperature.sensitive mutations in the primary repressor gene of Mx8 will be isolated, and a fragment with a ts repress or gene and strong promoter will be used to construct an inducible, high-level expression system for M. xanthus. Two results will emerge from the successful completion of this work. First, we will begin to understand the biology of the myxophages, about which little is known. The study of a variety of phages has revealed novel mechanisms that regulate gene expression; we have shown that Mx8 offers new surprises. Second, a basic understanding of Mx8 biology will allow us to develop a more powerful genetics to facilitate mapping, cloning, and sequencing studies in M. xanthus. M. xanthus is a major source of natural antibiotics, as well as a model system for the study of cell-cell interactions critical for both gliding motility and multiple, complex developmental cycles.
