[Cloning of the gpp gene of Escherichia coli and the use of recBC, sbcB cells for inserting its mutant allele into the chromosomal structure]

Abstract

The gpp gene involved in the pppGpp conversion into ppGpp in Escherichia coli cells was cloned and localized within the multicopy pBR322 plasmid. Amplification of the gpp gene leads to the decline of the intracellular level of pppGpp, which implies enhanced activity of the corresponding enzyme, guanosine pentaphosphatase. To inactivate the cloned gene, a fragment of the pUC4K plasmid containing the kan gene was inserted within the gpp gene. The functional chromosomal allele of the gpp gene was replaced by its inactivated gpp::kan allele, taking advantage of homologous recombination during the transformation of recBC, sbcB cells with the intact hybrid plasmid. This procedure is accompanied by plasmid elimination and may be used for the replacement of other loci of bacterial chromosome with appropriate cloned alleles.