Bacterial sporulation is a developmental process with well-defined stages. At least 40 genes are involved. As yet, very little is known about sporulation-specific products and the regulation of their synthesis. To disentangle sporulation-specific processes from other processes which occur concurrently with sporulation, it is useful to clone spo+ genes. In this way sporulation-specific products can be individually studied, apart from the complex program of sporulation. Advances in our understanding of the control of sporulation have already resulted from studies on the transcription of cloned spo+ genes. We propose to isolate, by means of recombinant DNA technology, genes which show developmental regulation in Bacillus subtilis. The major emphasis in this project will be on cloning genetic determinants involved in the process of sporulation. However, other enzyme systems, i.e., the Krebs cycle and a secretory system which controls the synthesis and export of several extracellular enzymes, are also induced at the end of the exponential phase of growth. We seek to clone representative genes from these pathways, as well. B. subtilis has an extensively described genetic map with well characterized mutations in the above systems and powerful cloning systems. Consequently, we plan to isolate our desired clones by inserting """"""""shotgun"""""""" restriction fragments of B. subtilis DNA into B. subtilis plasmid vectors with selection by biological complementation of appropriate mutations. Clones obtained by these techniques, as well as several clones involved in the sporulation process which our laboratory has previously isolated, will be characterized as their DNA sequence and transcriptional and translational products. Our ultimate aim is to analyze the interaction between these sporulation genes.
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