Inhibition of cell growth and stable DNA replication by overexpression of the bla gene of plasmid pBR322 in Escherichia coli.
A composite plasmid comprising the mini-F and pBR322 replicons was found to inhibit cell growth of a host with conditional mutations in dnaA and rnh under restrictive conditions, where the normal initiation of replication from oriC was inactivated, but the alternative replication initiation from oriK was active. It was further shown that the composite plasmid inhibited stable DNA replication (SDR) which occurs constitutively in cells mutant for rnh. Neither pBR322 nor mini-F alone produced these inhibitory effects. Deletion analyses revealed that the mini-F segment responsible for the inhibition of both processes was the promoter region of the sopA gene which had been cloned into a site upstream of the bla gene on pBR322 in such an orientation as to cause overexpression of bla. Inserting the promoter of the Escherichia coli lac gene into the same site had the same effect. Introduction of a deletion and a frameshift mutation into bla abolished the inhibition. Thus, the inhibition of growth and SDR appear to be due to overproduction of the bla gene product, beta-lactamase.
Katayama
T
T
Institute for Virus Research, Kyoto University, Japan.
Nagata
T
T
D002702:Chloramphenicol Resistance / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005144:F Factor; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D010957:Plasmids; D011401:Promoter Regions, Genetic; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics
D004269:DNA, Bacterial; D001618:beta-Lactamases