Locations of the genes from pepD through proA on the physical map of the Escherichia coli chromosome.

Peptidase D is one of at least six peptide-hydrolyzing enzymes of Escherichia coli (9). By sequence analysis of the 5' flanking and amino-terminal regions of the corresponding gene, pepD, we were able to show that it is contiguous to the adjacent gpt locus (4). Combination of this sequence with the already-known sequences of the gpt (10), phoE (11), and proBA (2) genes and of two additional open reading frames (49kDa and 15kDa, encoding 49and 15-kDa proteins, respectively) (10, 11) yielded a continuous 6,752-bp stretch of DNA sequence extending from the 3' end of proA to the 5' end of the reversely transcribed pepD gene (Fig. 1A). Applying a computer analysis, Watanabe and Kunisawa (12) found that the restriction maps predicted from this sequence matched the physical map of Kohara et al. (6) in the region of the previously reported genetic map position (1) around 5.7 min. In order to experimentally verify the location of the pepD gene, we used several digoxigenin-labeled DNA fragments, isolated from plasmid pJK11 (5), to probe DNA from three lambda clones of the Kohara "miniset" library (6, 7) (Fig. 1C). As we had recently sequenced the entire pepD gene (3),