Amplification of the put genes and identification of the put gene products in Escherichia coli K12

Abstract

The utilization of L-proline as C or N source for the growth of E. coli K12 requires the activities of an L-proline porter (PP-I) and a bifunctional L-proline dehydrogenase-.DELTA.1-pyrroline carboxylate dehydrogenase. PP-I is inactivated by mutations at putP and the bifunctional dehydrogenase is encoded in the adjacent locus, putA, at 22 min on the chromosome map. Two additional loci, proP (at 92 min) and proT (at 82 min), were also implicated in L-proline transport. Four ColE1/E. coli K12 hybrid plasmids from the plasmid bank prepared by Clarke and Carbon. Each of these plasmids was shown previously to complement a L-proline transport defect in E. coli. Genetic complementation analysis and biochemical assays of L-proline transport and L-proline dehydrogenase activity show that 3 of these hybrid plasmids bear the putPA region of the E. coli chromosome (plasmids pLC4-45, pLC10-29 and pLC43-41). The 4th plasmid, pLC35-38, specifically enhances the L-proline transport activity of its host bacteria but not their L-proline dehydrogenase activity. It probably encodes putP. These plasmids were used in an E. coli minicell system to identify the putA and putP gene products.