An in vivo molecular vehicle system for B. subtilis will be developed which will allow the replication and transposition of selected fragments of DNA. Isolated specific fragments of the B. subtilis chromosome will be analyzed genetically to select for sporulation genes. The organization of sporulation genes within the spore loci will be studied. The gene amplification system will consist of a host bacterium, B. subtilis; and a molecular vehicle, a temperate Bacillus phage genome cleaved once by restriction enzymes. Temperate phage cloning vehicles which are able to replicate themselves and/or integrate into the host genome, will be selected genetically. The vehicle will be ligased directly to isolated B. subtilis DNA fragments via natural cohesive ends generated by the restriction enzymes being used. A putative temperate phage molecular cloning vehicle with a single Sal GI site and a single Bgl II site is currently under study. Specific fragments of the B. subtilis chromosome have already been isolated using agarose electrophoresis and restriction enzymes. A large number of these fragments will be characterized by transformation to determine which contain sporulation genes. A search for regulatory genes will include in vitro mutagenesis of fragments containing sporulation genes. Development of this system will provide a powerful tool for B. subtilis, making it even more valuable in areas where it has made landmark contributions. Cloning of sporulation genes will provide a unique approach to the study of bacterial morphogenesis.
NIH ExPorter
NIH ExPorter