Journal of Bacteriology, September 2002, p . 4930-4932, Vol . 184, No . 17

The metD D-Methionine Transporter Locus of Escherichia coli Is an ABC Transporter Gene Cluster

József Gál,^* Attila Szvetnik, Róbert Schnell,^, and Miklós Kálmán

Institute for Biotechnology, Bay Zoltán Foundation for Applied Research, and Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary

Received 9 April 2002/ Accepted 5 June 2002

We have identified the abc-yaeE-yaeC gene cluster (now renamed metNIQ genes) as a likely candidate for the metD locus in the fhuA-proA region . The abc gene was previously found in a search for ABC transporter ATP-binding domains (1) . The PROSITE program (6) indicated that the Abc protein harbors an ATP- and GTP-binding site motif A (P-loop) (24) and an ABC transporter family signature (4) . Gene yaeE encodes a putative membrane protein with a high sequence similarity to several bacterial amino acid transporters and contains a binding protein-dependent transport system inner membrane component signature (6, 25) . The abc and yaeE open reading frames (ORFs) overlap by 8 nucleotides . The yaeC ORF is located 39 nucleotides downstream of the yaeE stop codon and was found to possess a probable signal sequence as well as a prokaryotic membrane lipoprotein lipid attachment site (6, 8), suggesting that it could be a periplasmic amino acid-binding protein .

A consensus MET box (5'-AGACGTCT-3'), the binding site of the MetJ repressor (2, 20, 22), was identified upstream of abc (28) . There is a 62.5% consensus MET box next to the 100% box (Fig . 1) . Recently, a conformational model-based prediction also identified the MetJ-binding site upstream of abc (15) . This suggested that the abc-yaeE-yaeC cluster might be part of the MET regulon .

 
Using a neural network promoter prediction algorithm (http://searchlauncher.bcm.tmc.edu/seq-search/gene-search.html) (21), a very likely 70 promoter was predicted upstream of abc (positions 2690 to 2726, reverse strand of the sequence registered under GenBank accession no . AE000129 [3]) . Its spacer region between the -35 and -10 boxes is almost completely made up of the tandem MET boxes (Fig . 1) . Even the -10 box of the putative promoter is part of a third, 50% MET box .

Uptake of D-methionine. To determine whether the abc-yaeE-yaeC putative ABC transporter gene cluster was involved in the ability of D-methionine to satisfy a methionine requirement, we deleted the cluster . The genomic region of the wild-type E . coli K-12 strain MG1655 corresponding to positions 90 to 2643 of the sequence with GenBank accession no . AE000129 was replaced with the kanamycin resistance cassette from pUC4K (Pharmacia) by using ET recombination (18), resulting in strain MK1958 . The deletion was transduced into the methionine auxotroph strain MTD23 (metE metH) (27) by using P1vir (17) with selection for kanamycin resistance, resulting in strain MK1962 .

E . coli strains unable to synthesize L-methionine are known to grow in the presence of D-methionine (5, 11, 14) . It was found that unlike the parental strain, strain MK1962 was unable to grow on M9 minimal plates (23) containing 0.2% glucose and 10 µg of D-methionine/ml (Sigma) .

Plasmids expressing the individual abc, yaeE, and yaeC ORFs were constructed on the basis of the pBAD18 and pBAD33 arabinose-inducible expression vectors (7) . A plasmid expressing the abc-yaeE gene cluster was also generated . The expression from the pBAD-based plasmids was induced by the addition of 0.05% arabinose to the medium . Complementation studies on M9 glucose minimal plates showed that the expression of the three individual genes one at a time or two at a time in any combination did not enable MK1962 to grow on D-methionine . The ability to grow in the presence of 10 µg of D-methionine/ml was restored only by the expression of all three genes, showing that all are necessary for the function of the transport system .

Uptake of -methyl methionine. The growth of strain MG1655 on M9 glucose minimal medium is severely inhibited by -methyl methionine, a toxic methionine analog . The analog is thought to be transported by the system encoded by the metD locus (11) . Unlike strain MG1655, strain MK1958, harboring a deletion of the abc-yaeE-yaeC cluster, was resistant to 250 µg of -methyl methionine/ml (Sigma) . Complementation studies on M9 glucose minimal plates showed that the sensitivity to the analog was restored only by the expression of all three genes .

Uptake of L-methionine. There was no apparent difference in the growths of strains MK1962 and MTD23 in liquid M9 glucose minimal medium supplemented with L-methionine at concentrations ranging from 3.3 to 100 µg/ml . It has been reported that there are at least two uptake systems for L-methionine in E . coli, a high-affinity system and a low-affinity system (9, 12) . Therefore, it could not be ruled out that the Abc-YaeE-YaeC system transports L-methionine .

It has been hypothesized that the MmuP S-methylmethionine permease could also transport L-methionine (27) . The abc-yaeE-yaeC deletion was transduced into MTD234 (metE metH mmuP) (27), resulting in strain MK2053 . The growth of MK2053 was indistinguishable from that of MTD23, MTD234, and MK1962 in liquid M9 glucose minimal medium supplemented with L-methionine as described above . However, because of the potential existence of another system(s) transporting L-methionine, we cannot exclude the possibility that the Abc-YaeE-YaeC system is one of the L-methionine transporters . The search for systems transporting L-methionine is under way and should be facilitated by the deletion of the abc-yaeE-yaeC cluster .

Regulation of transcription of the metD locus. To test whether the sequence shown in Fig . 1 is a promoter under the control of the MetJ repressor, it was cloned into the EcoRI and BamHI sites of the pRS415 ß-galactosidase-based promoter-probe vector (26), resulting in pROMET1 . The expression of ß-galactosidase from pROMET1 was assayed in the E . coli strain JM109 (29) harboring pBAD33 (7) or pMJ33, a pBAD33-derived, pROMET1-compatible plasmid expressing the metJ gene under the control of its native promoter . The strains were grown in liquid M9 minimal medium containing 0.2% glucose and 10 µg of thiamine-HCl per ml, with or without 100 µg of L-methionine per ml . The ß-galactosidase specific activities of the cultures were determined by using the o-nitrophenyl-ß-D-galactopyranoside substrate (Sigma) (16) . The data shown in Table 1 are the averages of three measurements . Assays with E . coli strain TN1, a metJ mutant derivative of JM109 (19), failed because of the very slow growth of the strains .

 
The results show that the segment behaves as a promoter . Its expression decreased about threefold upon the addition of L-methionine to the medium and about twofold when the metJ gene was present in multicopy . When both L-methionine was added and metJ was present in multicopy, the expression decreased about 12-fold . This suggests that the promoter is repressed by the MetJ repressor . The expression of the abc-yaeE-yaeC cluster is probably similarly regulated .

This work was supported by grants from the Bay Zoltán Foundation for Applied Research .

Present address: Department of Microbiology, Stockholm University, Stockholm, Sweden S-10691 .

1. Allikmets, R., B . Gerrard, D . Court, and M . Dean. 1993 . Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance . Gene 136:231-236.
2. Belfaiza, J., C . Parsot, A . Martel, C . B . de la Tour, D . Margarita, G . N . Cohen, and I . Saint-Girons. 1986 . Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region . Proc . Natl . Acad . Sci . USA 83:867-871.
3. Blattner, F . R., G . Plunkett III, C . A . Bloch, N . T . Perna, V . Burland, M . Riley, J . Collado-Vides, J . D . Glasner, C . K . Rode, G . F . Mayhew, J . Gregor, N . W . Davis, H . A . Kirkpatrick, M . A . Goeden, D . J . Rose, B . Mau, and Y . Shao. 1997 . The complete genome sequence of Escherichia coli K-12 . Science 277:1453-1474.
4. Blight, M . A., and I . B . Holland. 1990 . Structure and function of haemolysin B,P-glycoprotein and other members of a novel family of membrane translocators . Mol . Microbiol . 4:873-880.
5. Cooper, S. 1966 . Utilization of D-methionine by Escherichia coli . J . Bacteriol . 92:328-332.
6. Falquet, L., M . Pagni, P . Bucher, N . Hulo, C . J . Sigrist, K . Hofmann, and A . Bairoch. 2002 . The PROSITE database, its status in 2002 . Nucleic Acids Res . 30:235-238.
7. Guzman, L . M., D . Belin, M . J . Carson, and J . Beckwith. 1995 . Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter . J . Bacteriol . 177:4121-4130.
8. Hayashi, S., and H . C . Wu. 1990 . Lipoproteins in bacteria . J . Bioenerg . Biomembr . 22:451-471.
9. Kadner, R . J. 1974 . Transport systems for L-methionine in Escherichia coli . J . Bacteriol . 117:232-241.
10. Kadner, R . J. 1975 . Regulation of methionine transport activity in Escherichia coli . J . Bacteriol . 122:110-119.
11. Kadner, R . J. 1977 . Transport and utilization of D-methionine and other methionine sources in Escherichia coli . J . Bacteriol . 129:207-216.
12. Kadner, R . J., and W . J . Watson. 1974 . Methionine transport in Escherichia coli: physiological and genetic evidence for two uptake systems . J . Bacteriol . 119:401-409.
13. Kadner, R . J., and H . H . Winkler. 1975 . Energy coupling for methionine transport in Escherichia coli . J . Bacteriol . 123:985-991.
14. Kuhn, J., and R . L . Somerville. 1971 . Mutant strains of Escherichia coli that use D-amino acids . Proc . Natl . Acad . Sci . USA 68:2484-2487.
15. Liu, R., T . W . Blackwell, and D . J . States. 2001 . Conformational model for binding site recognition by the E . coli MetJ transcription factor . Bioinformatics 17:622-633.
16. Miller, J . H. 1972 . Experiments in molecular genetics, p . 352-355 . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
17. Miller, J . H. 1992 . A short course in bacterial genetics, p . 263-274 . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
18. Muyrers, J . P., Y . Zhang, G . Testa, and A . F . Stewart. 1999 . Rapid modification of bacterial artificial chromosomes by ET-recombination . Nucleic Acids Res . 27:1555-1557.
19. Nakamori, S., S . Kobayashi, T . Nishimura, and H . Takagi. 1999 . Mechanism of L-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of L-methionine biosynthetic enzymes . Appl . Microbiol . Biotechnol . 52:179-185.
20. Phillips, S . E., I . Manfield, I . Parsons, B . E . Davidson, J . B . Rafferty, W . S . Somers, D . Margarita, G . N . Cohen, I . Saint-Girons, and P . G . Stockley. 1989 . Cooperative tandem binding of met repressor of Escherichia coli . Nature 341:711-715.
21. Reese, M . G. 2001 . Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome . Comput . Chem . 26:51-56.
22. Saint-Girons, I., N . Duchange, G . N . Cohen, and M . M . Zakin. 1984 . Structure and autoregulation of the metJ regulatory gene in Escherichia coli . J . Biol . Chem . 259:14282-14285.
23. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed., p . A.3 . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
24. Saraste, M., P . R . Sibbald, and A . Wittinghofer. 1990 . The P-loop—a common motif in ATP- and GTP-binding proteins . Trends Biochem . Sci . 15:430-434.
25. Saurin, W., W . Koster, and E . Dassa. 1994 . Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins . Mol . Microbiol . 12:993-1004.
26. Simons, R . W., F . Houman, and N . Kleckner. 1987 . Improved single and multicopy lac-based cloning vectors for protein and operon fusions . Gene 53:85-96.
27. Thanbichler, M., B . Neuhierl, and A . Böck. 1999 . S-Methylmethionine metabolism in Escherichia coli . J . Bacteriol . 181:662-665.
28. Thieffry, D., H . Salgado, A . M . Huerta, and J . Collado-Vides. 1998 . Prediction of transcriptional regulatory sites in the complete genome sequence of Escherichia coli K-12 . Bioinformatics 14:391-400.
29. Yanisch-Perron, C., J . Vieira, and J . Messing. 1985 . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors . Gene 33:103-119.

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