Demonstration by a Novel Genetic Technique That Leader Peptidase Is an Essential Enzyme of Escherichia coli

Abstract

It was previously shown that two separate regions of DNA are required for expression of the cloned leader peptidase gene on plasmid pTD101 (T. Date and W. Wickner, Proc. Natl. Acad. Sci. U.S.A. 78: 6106-6110, 1981). Both loci have been mapped in detail, and their roles have been established. A 1.3-kilobase region, termed the L region, encodes the 37,000-dalton leader peptidase protein. Another region, termed the P region, is about 1.5 kilobases away from the L region and is less than 350 base pairs long. The P region acts in cis to the L region, suggesting that it plays a role as a promoter. A technique for inactivation of the leader peptidase gene on the Escherichia coli chromosome has been developed to examine whether the leader peptidase which we had cloned is essential for cell growth. A specific plasmid (P − L − ) which deletes both the P region and a substantial portion of the L region was constructed and transformed into a polA mutant strain. The plasmid cannot replicate in this strain; thus, the plasmid-borne ampicillin resistance is lost unless the plasmid DNA recombines into the chromosome. Integration of the P − L − plasmid did not yield any viable ampicillin-resistant cells, whereas the three control plasmids, P + L + , P + L − , and P − L + , did. When the P − L − plasmid was transformed into the polA (Ts) strain, the strain could only grow in the presence of ampicillin at a permissive temperature, suggesting that integration of the plasmid into the host chromosome leads to inactivation of the chromosomal leader peptidase gene. Southern hybridization analysis demonstrated that the integration of plasmids into the chromosome occurred at the homologous site. This study demonstrates that expression of the leader peptidase gene is critical for cell growth.