The sequence of the G+C-rich temperate bacteriophage Mx8 genome shows that it is unlike any phage examined in detail to date. Our continuing studies of Mx8 genes and their functions will likely reveal new regulatory paradigms that are also used by its M. xanthus host during the process of multicellular development. We will try to uncover the secret of a unique phage-host adaptation: how is the Mx8 prophage maintained stably throughout M. xanthus multicellular development, despite the dramatic, successive, global changes in gene regulation that occur during this cycle? We will complete the sequence of the remaining third of the 50 kb Mx8 genome, to identify the Mx8 genes involved in lysogenic development, lytic development, and the stability of the Mx8 prophage throughout the multicellular developmental cycle of its host. The structure and function of a divergent pair of Mx8 promoters will be dissected, to learn what sequence elements are critical for strong promoter function in M. xanthus. A single-copy plasmid will be used to trap vegetative M. xanthus promoters, to expose their consensus sequence elements. Phage promoters will be cloned, to learn when they are activated during the lytic cycle of phage growth, or from an Mx8 prophage during the multicellular development of an M. xanthus lysogen. Mutations will be made in the genes controlling Mx8 superinfection immunity, to understand its novel dosage-dependent mechanism, in the repressor gene, to permit the inducible expression of genes from a strong Mx8 promoter and in the phage terminase, to construct high-frequency generalized transducing mutants. Temperature-sensitive mutations in Mx8 will be isolated to characterize its essential functions, and we will attempt to use conditionally-polar insertions in new ways to identify both nonessential phage functions, and to correlate phage genes with their protein products.
