Jones, D. H. A., and C. M. Thomas. Unpublished data. 7. Kohara, Y., K. Akiyama, ... Sasse-Dwight, S., and J. D. Gralla. 1990. Role of eukaryotic-type functional ...
Vol. 175, No. 5
JOURNAL OF BACrERIOLOGY, Mar. 1993, p. 1548-1549
0021-9193/93/051548-02$02.00/0 Copyright © 1993, American Society for Microbiology
E. COLI MAP Location of the rpoN Gene on the Physical Map of Escherichia coli MIKE MERRICK,"* DAVID H. A. JONES,2 AND CHRISTOPHER M. THOMAS2 AFRC Nitrogen Fixation Laboratory, University of Sussex, Brighton BN1 9RQ,' and School of Biological Sciences, University of Birmingham, Birmingham B15 217,2 United Kingdom
The Escherichia coli rpoN (glnfl gene encodes the novel RNA polymerase sigma factor &e' (4, 5, 9). During the isolation of the homologous gene from Klebsiella pneumoniae, we purified R-prime DNA carrying K pneumoniae rpoN from an E. coli K-12 strain and cloned the gene by complementation (10). Two classes of plasmid were obtained, one (pMM17) with a 1.8-kb ClaI insert carrying K pneumoniae rpoN and one (pMM18) with a 4.6-kb ClaI insert carrying E. coli rpoN. Both plasmids have since been used in a number of laboratories to characterize rpoN homologs in other bacterial species (6, 12). In E. coli and other enteric species rpoN has been genetically mapped at around 70 min (2, 3, 11), and in order to map the gene precisely on the E. coli physical map, we have determined the restriction map of pMM18 and compared it with the Kohara restriction map (7) and the nucleotide sequence of E. coli rpoN (6a, 13). These comparisons (Fig. 1) unambiguously locate rpoN at 3411 to 3413 kb on the E. coli physical map, in that every restriction site predicted by Kohara et al. (7) to occur within pMM18 is present and no
3415 kb
3408 kb II
I
11 III'
rpoN BamHI Hindlil EcoRI EcoRV
BgIl
: ~~
USA 82:8453-8457. 6. Inouye, S., M. Yamada, A. Nakazawa, and T. Nakazawa. 1989. Cloning and sequence analysis of the ntrA (rpoN) gene of
Kpnl
Pseudomonasputida. Gene 85:145-152. 6a.Jones, D. H. A., and C. M. Thomas. Unpublished data. 7. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell
PstI Pvull Clal
50:495-508. 8. Merrick, M. J., and J. R. Coppard. 1989. Mutations in genes
pMM18
downstream of the rpoN gene (encoding or") of Klebsiella
FIG. 1. Restriction map of the cloned fragment from pMM18, showing the rpoN locus and adjacent open reading frames (arrows indicate gene orientations) aligned with the Kohara map (7) for the appropriate region. *
REFERENCES 1. Castano, I., and F. Bastarrachea. 1984. glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation. Mol. Gen. Genet. 195:228-233. 2. Gaillardin, C. M., and B. Magasanik. 1978. Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in KIebsiella aerogenes. J. Bacteriol. 133:1329-1338. 3. Garcia, E., S. Bancroft, S. G. Rhee, and S. Kustu. 1977. The product of a newly identified gene, gInF, is required for synthesis of glutamine synthetase in Salmonella. Proc. Natl. Acad. Sci. USA 74:1662-1666. 4. Hirschman, J., P. K. Wong, K. Sei, J. Keener, and S. Kustu. 1985. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc. Natl. Acad. Sci. USA 82:7525-7529. 5. Hunt, T. P., and B. Magasanik. 1985. Transcription of glnA by
purified Escherichia coli components: core RNA polymerase and the products of glnF,glnG, and glnL. Proc. Natl. Acad. Sci.
1
Clal
unpredicted sites were found. The only minor differences are the presence of two adjacent EcoRV sites between 3412 and 3413 kb and the reversal of the order of the EcoRV and PstI sites between 3410 and 3411 kb, both of which were determined by sequencing. These data also confirm that the gene is transcribed clockwise as proposed by Castano and Bastarrachea (1). Sequence analysis of the region downstream of rpoN (6a unpublished) has identified three additional open reading frames, all of which are transcribed in the same direction as rpoN (Fig. 1). The predicted translation products of these three genes are homologous to open reading frames 95, 162, and 193, which lie immediately downstream of rpoN in K pneumoniae (8).
pneumoniae affect expression from o54-dependent promoters. Mol. Microbiol. 3:1765-1775. 9. Merrick, M. J., and J. R. Gibbins. 1985. The nucleotide se-
quence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 13:76077620.
Corresponding author. 1548
VOL. 175, 1993 10. Merrick, M. J., and W. D. P. Stewart. 1985. Studies on the regulation and function of the Klebsiella pneumoniae ntrA gene. Gene 35:297-303. 11. Pahel, G. A., A. D. Zelenetz, and B. M. Tyler. 1978. gltB gene and regulation of nitrogen metabolism in Escherichia coli. J. Bacteriol. 133:139-148. 12. Romermann, D., J. Warrelmann, R. A. Bender, and B.
E. COLI MAP
1549
Friedrich. 1989. An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation. J. Bacteriol. 171:1093-1099. 13. Sasse-Dwight, S., and J. D. Gralla. 1990. Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor oJ5'. Cell 62:945-954.
