Molecular Cloning and Expression of the Pyrimidine B Gene From Bacillus Subtilis

145 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.The aspartate transcarbamylase of Bacillus subtilis is an example of a "vegetative" gene product which ceases to be synthesized once the cells begin to sporulate. To study the regulation of expression of this enzyme, the gene which encodes it (pyrB) was cloned from B. subtilis. Chromosomal DNA from wild type B. subtilis was digested with BamHI and ligated into the BamHI site of pHV14, a chimeric plasmid bearing chloramphenicol resistance and capable of replicating in either E. coli or B. subtilis. The recombinant plasmid pBS205 was selected by its ability to complement an E. coli pyrB deletion mutant (TB2). pBS205 contained a 5.8 kb fragment of B. subtilis DNA. The aspartate transcarbamylase made by TB2 carrying pBS205 was immunochemically indistinguishable from B. subtilis aspartate transcarbamylase. The cloned fragment was capable of transforming various B. subtilis pyr mutants to uracil prototrophy. As a result of the transformation experiments and complementation experiments in E. coli pyrC and pyrD hosts, it was determined that the fragment also carried the intact pyrC gene but not the entire pyrD gene.The cloned fragment does not carry a promoter for either the pyrB or pyrC. The genes are probably expressed from a plasmid-borne promoter. It remains to be shown whether the promoter for the pyrD gene lies on this fragment. Aspartate transcarbamylase synthesized by TB2 carrying pBS205 was not sensitive to repression by exogenous uridine. There are two in vitro transcripts which hybridize to pBS205. One transcript, 4.5 kb in length, hybridizes to the pyrCB portion of the fragment. The other transcript, 2.5 kb in length, hybridizes to the pyrD portion of the fragment. These transcripts appear during logarithmic growth in the absence of uridine and disappear in the stationary phase of growth. The transcripts are undetectable in cells cultured in the presence of uridine. They do not appear to contain poly-adenylated sequences. These results show that the pyrimidine cluster of B. subtilis is not expressed as a single polycistronic message and also that the regulation of expression is at the level of transcription.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD