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In the course of our studies on the glutamine synthetase regulatory cascade, we have characterized the physical location of the glnD gene, encoding the uridylyl-transferase which is thought to be the primary sensor of cellular nitrogen status (13). This gene is linked to dapD, and we therefore analyzed two lambda phages from the Kohara library (9, 9a), 9H2 and 21C8, mapping in the dapD region (3.8 min, 190 kb) (12). To identify the physical location ofginD, we made use of a glnD::TnlO insertion in strain RB9040 (3). Southern blotting of PstI-, EcoRI-, and EcoRI-SalI-digested genomic DNAs of RB9040 and its wild-type parent RB9010, using 21C8 phage DNA as a probe, showed that the physical map of the hybridizing region of the chromosome corresponds to the physical map of 21C8, indicating the absence of rearrangement in phage 21C8. This experiment also located the TnlO in strain RB9040 within a 2.8-kb PstI fragment, a 17-kb EcoRI fragment, and an 8-kb EcoRI-SalI fragment. The 8-kb EcoRI-SalI fragment was cloned from phage 21C8 into the Bluescript vector pBSK+, to obtain pWVH46. Figure 1 shows the hybridization pattern of EcoRIand Sall-digested DNAs obtained with pWVH46 as a probe. This experiment located the TnlO at 4 kb from the EcoRI site (Fig. 2), because the alternative location, at 1 kb from the EcoRI site and in the opposite orientation, falls outside the 2.8-kb PstI fragment. The physical map of pWVH46 was found to correspond with the physical map of an independently derived glnD-containing plasmid pXG204 (4a). pWVH46 was also found to complement the Glnbradytrophy of RB9040 (bradytrophy is defined as a leaky auxotrophy). Thus, the glnD gene was localized to the 2.8-kb segment separating dapD and map (4, 10; Fig. 2). Considering the molecular mass of uridylyl-transferase (95 kDa; 5), the glnD gene should be approximately 2.7 kb long. Therefore, the TnlO insertion in RB9040, which maps 1.2 kb to the right of dapD, indeed appears to disrupt the glnD coding region (Fig. 2). The orientation of ginD was determined on the basis of previous work (4a, 14) and was found to be from map towards dapD, counterclockwise on the Escherichia coli chromosome. A single inconsistency remained; the size of the EcoRI fragment hybridizing with pWVH46 was 17 kb (Fig. 1) instead of the 7.9 kb predicted from the Kohara map (9a). We therefore reevaluated this portion of the map by verifying the overlap between phages 21C8 and 9H2. By physical mapping and by hybridization experiments between fragments from both phages, we obtained the revised map shown in Fig. 2. The resulting corrected genomic map is fully consistent with the estimated size of the glnD-containing

Localization of the glnD gene on a revised map of the 200-kilobase region of the Escherichia coli chromosome