Cloning inEscherichia coli of aBacillus subtilis arginine repressor gene through its ability to confer structural stability on a fragment carrying genes of arginine biosynthesis

Summary

The structural stability of a previously isolated recombinant plasmid pUL720 was examined. pUL720 contains an insert in pBR322 of 23.8 kbp comprising 4 EcoR1 fragments of sizes 12 kbp and 6 kbp, both of which are homologous to theB. subtilis genome, and 5 kbp and 0.8 kbp (of unknown origin). The 12 kbp fragment, which encodes the arginine biosynthesis genesargA-F-cpa, cannot be cloned in isolation in a high copy vector inE. coli but can be inserted into a low copy vector pGV1106 to generate pUL800. Deletion analysis of pUL720 indicated that the 5 kbp and 0.8 kbp fragments were not necessary to maintain plasmid stability. The 6 kbp fragment, when cloned into the EcoR1 site in pACYC184 to generate pUL2030, permitted the cloningin trans in pBR322 of the 12 kbp fragment or subclones containing the instability region. The minimum inhibitory concentration of kanamycin determined in theB. subtilis argC-neo transcriptional fusion pUL730 and expression of theargF gene product, ornithine carbamoyltransferase (OCTase), in pUL800 were reduced by approximately 3 and 2 fold respectively under conditions of arginine excess and in the presence of pUL2030.B. subtilis partial diploids were constructed by transforming parental and arginine hydroxamate resistant (Ahr) mutants with pUL2100, a plasmid generated by inserting the 6 kbp fragment into the integration vector pJH101. The 6 kbp fragment complemented and restored parental type levels of OCTase inahrC mutants. These data suggest that the 6 kbp fragment, fortuitously isolated in pUL720, stabilises the 12 kbp fragment cloned into a high copy vector inE. coli by encoding aB. subtilis arginine repressor which regulates a promoter upstream toargC.