Vol. 173, No. 5

JOURNAL OF BACTERIOLOGY, Mar. 1991, p. 1572-1573

0021-9193/91/051572-02$02.00/0 Copyright © 1991, American Society for Microbiology

Localization of the Escherichia coli amtA Gene to 95.8 Minutes A. JAYAKUMAR,1 KENNETH E. RUDD,2 JOHN M. FABINY,1 AND EUGENE M. BARNES, JR.'* Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030,1 and Division of Bacterial Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 208922

between the sites at 4520.4 and 4538.4 kb as depicted on the genomic restriction map (3; Fig. 1), we would not have seen any hybridization. Thus, our data reveal that clone ElH3[657] must actually contain the chromosomal DNA between the EcoRI sites at 4517.8 and 4538.4 kb. Using the published restriction map for the cpdB gene encoding the enzyme cyclic nucleotide phosphodiesterase (4), we have positioned the cpdB gene at a PvuII site at 4516.1 kb (Fig. 1). This alignment predicts that the cpdB

The amtA gene of Escherichia coli, previously designated amt 1 (2), is required for active transport of ammonium and methylammonium ions. The plasmid pJHlamtA+ was isolated by using genetic complementation of the amtAl: :TnlO mutation (2). Using a recently described restriction map alignment program, MapSearch (5), and Southern blot hybridizations to DNA from bacteriophage lambda clones from the Kohara library of genomic clones (3), we were able to quickly and precisely position the amtA gene on the E. coli SA

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FIG. 1. Restriction map of the E. coli chromosome from 4500.0 to 4540.0 kb. The inserts from various Kohara clones and the plasmids pJH1 and pJHlA and their locations on the genomic map are depicted. The positions of GenBank sequences (ECORPSRFI [X04022], ECOCPDB [M13464]) and the amtA gene were determined by using MapSearch (5). The figure was drawn with the PrintMap program (6). B, BamHI; D, HindlIl; E, EcoRI; F, EcoRV; G, BglI; Q, KpnI; S, PstI; V, PvuII.

gene is transcribed in the counterclockwise orientation. This corrects the previously published cpdB MapSearch alignment (5), which relied on DNA sequence information alone. Fragmentary EcoRV information in this region of the ge-

chromosome. MapSearch was used to scan the E. coli genomic restriction map (3, 5) for regions matching the published restriction map of plasmid pJH1. The highest ranking alignment is the only significant one (P = 0.056) and includes an EcoRI site at 4517.8 kb and a PstI site at 4518.5 kb. By using the pJHlA subclone (2), the corresponding sites in pJH1 have been shown to be very close to or within the amtA gene. In order to confirm the genomic map location of the amtA gene, we performed Southern blot hybridization experiments (data not shown). The 32P-labeled HindIII-SalI chromosomal DNA fragment cloned in pJHlA was used to probe HindIII and EcoRI digests of DNA from five clones. The control clones 1OH6[425] and 14G6[464] showed no hybridization, but the clones from the 4518-kb region, 7E9[655], 5B5[656], and ElH3[657], were all positive. The EcoRI fragments correspond to those predicted from the genomic DNA (Fig. 1) and the lambda vector restriction map (Table 1). If clone ElH3[657] contained only the EcoRI fragment

TABLE 1. Observed and predicted chromosomal DNA fragments cloned in pJHlA kb

Clone Enzyme

Observed Predicted

4517.8, 4518.5 (EMBL4 mcs) 4510.2, 4517.8 Hindlll 4507.9, left end 656 EcoRI 4517.8, 4520.4 4517.8, 4512.0 (EMBL4 mcs) HindIII 4519.6, left end 657 EcoRI 4517.8, 4520.4 HindIII 4519.6, 4517.8 (2001 mcs) Restriction site addresses refer to genomic map positions determined by Kohara et al. (3) and are depicted in Fig. 1. mcs refers to the multiple cloning 655

EcoRI

0.7 7.2 25.0 2.6 6.4 28.0 2.6 1.8

0.7 7.6 29.9 2.6 5.8 26.9 2.6 1.8

a

*

Sites' (kb)

sites in the EMBL4 or lambda 2001 vector (3).

Corresponding author. 1572

VOL. 173, 1991

E. COLI MAP

nomic restriction map caused the previous error. The cpdB gene is placed at 95.7 min on the genetic map (1); therefore, we place the amtA gene at 95.8 min. The nucleotide sequence of pJHlA confirms that the cpdB gene is immediately

adjacent to the amtA

gene

(submitted for publication).

REFERENCES 1. Bachmann, B. J. 1990. Linkage map of Escherichia coli K-12, edition 8. Microbiol. Rev. 54:130-197. 2. Jayakumar, A., S. J. Hwang, J. M. Fabiny, A. C. Chinault, and E. M. Barnes, Jr. 1989. Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and comple-

mentation

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by the cloned gene. J. Bacteriol. 171:996-1001. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid anaylsis and sorting of a large genomic library. Cell 50:495-508. 4. Liu, J., D. M. Burns, and I. R. Beacham. 1986. Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3'cyclic phosphodiesterase. J. Bacteriol. 165:1002-1010. 5. Rudd, K. E., W. Miller, J. Ostell, and D. A. Benson. 1990. Alignment of Escherichia coli K12 DNA sequences to a genomic 3.

restriction map. Nucleic Acids Res. 18:313-321.

6.

Rudd, K. E., and C. Warner. Unpublished data.