FUNCTIONAL ANALYSIS OF THE MYXOCOCCUS XANTHUS GENOME

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Using an innovative set of genetic approaches, we will analyze the functions of the genes required for the multicellular development of Myxococcus xanthus on its 9.5 megabase pair genome. Segments (35-40 kb) of the M. xanthus genome subcloned into a cosmid vector will be crossed onto Salmonella phage P22 as our cloning vector, and maintained as single-copy P22+insert prophages in S. typhimurium. Because induction of P22 lysogens yields a high burst of particles (5,000 to 10,000/cell) which are easily purified, induced lysates are rich sources of double-stranded M. xanthus DNA inserts for restriction and sequence analyses. More important, these P22+insert particles are powerful genetic elements that allow us to order a genomic library of M. xanthus inserts by the functional approach of complementation of Salmonella auxotrophies. Using auxonography to construct an P22+insert overlap-clone map of the M. xanthus genome will identify 200 new functions on the M. xanthus genome encoding the enzymes of central carbon metabolism, some of which play central roles in the M. xanthus multicellular development cycle. Starting with an assembled P22+insert overlap-clone map, consisting of about 300 ordered segments of the M. xanthus genome, we will use the well-established method of transitory cis-complementation to make insertions of a mini-Mu transposon in essentially every M xanthus gene. Pools of insertions in each segment will be crossed onto the M. xanthus genome, to identify the subset of 300 genomic segments with genes encoding functions critical for development. Segments with developmental genes will be sequenced, by an ordered, one-pass strategy. This strategy, using P22+insert DNAs as templates and primers specific to the ends of the Mu will yield not only the sequence of the M. xanthus genome, but also the sequences of the sites of insertions in M. xanthus genes. These defined insertion mutations will be crossed onto the M. xanthus chromosome to make targeted gene disruptions, then reporter fusion substitutions that can be used to identify regulatory genes governing development. The successful application of our combined functional approaches to analyze the M. xanthus genome will demonstrate that functional analysis can and should motivate sequence analysis, and will reduce the cost of sequencing by more than 10-fold when compared to current more labor-intensive methods. Our efforts will show that the sequences of the genomes of most pathogenic and other interesting microbes can be completed at a much more reasonable cost than current efforts entail.