Site-directed mutagenesis of the Escherichia coli chromosome near oriC: identification and characterization of asnC, a regulatory element in E. coli asparagine metabolism

摘要

We developed a new method for the specific mutagenization of the E. coli chromosone. This method takes advantage of the fact that a pBR322 plasmid containing chromosomal sequences is mobilizable during an Hfr-mediated conjugational transfer, due to an homologous recombination between the E. coli Hfr chromosome and the pBR322 derivative. Transconjugants are screened with a simple selection procedure for integration of mutant sequences in the chromosome and loss of pBR322 sequences. Using this method we specifically inactivated several genes near the E. coli replication origin oriC. We found that a gene coding for a protein of 17kD has a regulatory role in transcription of the gene coding for asparagine synthetase A. This regulatory mechanism was investigated in detail by determining in vivo regulation of asnA promoter activity by the 17kD protein under different growth conditions. Results obtained also suggest a general regulatory role of the 17kD protein in E. coli asparagine metabolism. Therefore the 17kD gene is proposed to be renamed asnC.