Open AccessSite-directed mutagenesis of theEscherichia colichromosome nearoriC: identification and characterization ofasnC, a regulatory element inE. coliasparagine metabolism
Author(s) -
Niels de Wind,
Marten de Jong,
Michiel Meijer,
Antoine R. Stuitje
Publication year - 1985
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/13.24.8797
Subject(s) - biology , gene , plasmid , genetics , asparagine , escherichia coli , mutagenesis , mutant , pbr322 , chromosome , regulatory sequence , regulation of gene expression , amino acid
We developed a new method for the specific mutagenization of the E. coli chromosome. 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 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.