| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biology, Ursinus College, PO Box 1000, Collegeville, PA 19426, USA
2 Biology Department, Hanover College, PO Box 890, Hanover, IN 47243, USA
Correspondence
Jeffrey A. Hughes
hughes{at}hanover.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Present address: Little Britain Veterinary Services, 281 Sleepy Hollow Road, Nottingham, PA 19362, USA.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; reviewed by Greene, 1996). The combined activities of MetA, MetB and MetC replace the hydroxyl group of L-homoserine with a thiol moiety to form L-homocysteine. Simultaneously, GlyA and MetF transfer and reduce one carbon from serine to tetrahydrofolate (THF), producing N5-methyl-THF. Either of two methyltransferases, the products of the metE and metH genes, transfers the methyl group from THF to homocysteine to form methionine. Methionine then either participates in protein synthesis or is condensed by the metK-encoded SAM synthetase with ATP to produce the ubiquitous nucleotide S-adenosyl-L-methionine (SAM).
|
). Mutants of the more common type have decreased in vivo pools of SAM as a result of impaired SAM synthetase activity, and the defects map to metK (Greene et al., 1973; Hafner et al., 1977). Mutations of the second class map to metJ, and genetic and biochemical evidence identified the corresponding small protein as a DNA-binding aporepressor of met gene transcription (Su & Greene, 1971). These results suggested that a complex of MetJ and SAM binds a common nucleotide sequence in the promoters of target genes to prevent their transcription (Greene et al., 1970, 1973; Hobson & Smith, 1973). Identification of a consensus repressor binding site (‘met box’; Belfaiza et al., 1986) and crystallographic studies (Rafferty et al., 1989) all support this model. Genes most induced by defects in either metJ or metK include metA, B, C, E, F, K, L (as the downstream gene in the metBL operon) and metR (a regulatory protein that, by itself or when bound with homocysteine, regulates expression of a variety of genes in the met regulon to coordinate the activities of the two branches of the methionine biosynthetic pathway; again, see Greene, 1996). Cells thus regulate methionine biosynthesis through a feedback mechanism that monitors levels of SAM to activate met gene expression before the concentration of methionine decreases to levels that could impair protein synthesis. In addition to the MetJ–SAM repressor complex, other regulatory mechanisms, including the effects of the activator MetR protein (Urbanowski et al., 1987) and regulation of metA expression by heat shock (Biran et al., 1995) and of MetA stability by proteolysis (Biran et al., 2000), make regulation of elements of the met regulon highly complex.
Though well supported by genetic and biochemical data, the role of SAM has not been directly tested in vivo. This is due to three problems inherent in studies involving SAM in bacteria: SAM typically cannot cross bacterial cell membranes (including those of Escherichia coli), SAM synthetase inhibitors (e.g. methionine analogues such as ethionine) induce side effects unrelated to SAM deprivation (Alix, 1982; Pine, 1978), and known viable SAM synthetase mutants are leaky even under restrictive conditions (Hafner et al., 1977; Mulligan et al., 1982). We circumvented these difficulties by transforming cells with plasmid expression vectors containing the cloned coliphage T3 SAM hydrolase (SAMase) gene, resulting in in vivo SAMase activity not otherwise found in E. coli. This enzyme cleaves SAM into homoserine and 5'-methylthioadenosine, removes SAM from the cell, and inhibits a variety of SAM-related activities (Hughes et al., 1987; Posnick & Samson, 1999; Val & Cronan, 1998). Introducing SAMase activity should specifically remove the co-repressor without affecting the MetJ aporepressor and demonstrate the effect of reduced SAM concentrations on the expression of elements of the met regulon. Results from our assays of met gene expression in cells containing in vivo SAMase activity firmly support the role of SAM as the co-repressor. They also indicate that other factors – including and possibly in addition to MetR – play roles in influencing SAM-related met gene regulation.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; the YT complete and M9 defined media used are those of Miller (1972). Assays of enzyme activities other than the expression of met : : lacZ fusions were performed on BW545. As described by Mulligan et al. (1982), the GW strains were identified by screening for defects in methionine metabolism after Mud : : Ap,lac-mediated insertional mutagenesis of BW545, and the metJ : : Tn5 and metK : : Tn5 markers were inserted into the originally identified and stabilized met reporter strains by P1vir transduction. BWmJ and BWmK are metJ : : Tn5 and metK : : Tn5 derivatives of BW545 produced by P1vir transduction (Mares et al., 1992) of the respective markers from GW2529 and GW2533. They were identified by screening for resistance to 50 µg kanamycin ml–1 on YT agar plates after P1vir transduction and subsequent demonstration of elevated MetC activity using the assay described below. E. coli BW545, BWmJ (metJ : : Tn5) and BWmK (metK : : Tn5) lysogenic for bacteriophage 
gt2 constructs containing Salmonella typhimurium met gene promoter : : lacZ fusions were assayed for metB (Blac; Urbanowski & Stauffer, 1986), metE (Elac; Plamann et al., 1988), metF (Flac; Stauffer & Stauffer, 1988), metH (Hlac; Urbanowski & Stauffer, 1989b), metJ (Jlac; Urbanowski & Stauffer, 1986) and metR (Rlac; Urbanowski & Stauffer, 1987) expression. In all cases, cells used to inoculate liquid cultures came from colonies raised overnight on agar plates inoculated with either freshly transformed cells or transformants stored in 15 % (v/v) glycerol at –70 °C and streaked for purification. Prior to assay, cultures were grown overnight with shaking at 32 °C in test tubes containing 3 ml liquid medium containing 100 µg ampicillin ml–1 and/or 15 µg tetracycline ml–1.
|
. The SAMase gene cloned into M13mp8 that produced M13hb1 (Hughes et al., 1987) was removed by EcoRI and HindIII digestion and ligated into pUC18 (Yanisch-Perron et al., 1985) to produce pHBF1UC. Because we questioned the ability of ampicillin to enforce long-term maintenance of this plasmid, pHBF1UC was cut with PstI and ligated to PstI-cut pBR322 (Bolivar et al., 1977) to form pHBBR2 and gain the tetracycline resistance that ensured plasmid maintenance. Also, the promoterless EcoRI–BamHI SAMase gene removed from M13hb1 was inserted into pBR322 to produce pHBF1BR, a stably propagated plasmid under ampicillin selection that directs weak SAMase expression, apparently from a cryptic promoter.
Enzyme assays.
Cystathionine synthetase (MetB) activity was determined in toluenized cells by the O-succinylhomoserine-dependent oxidation of reduced nicotinamide adenine dinucleotide, measured as A340 (Holloway et al., 1970; Kaplan & Flavin, 1966). Cystathionase (MetC) activity was assayed by the cystathionine-dependent reduction of Ellman's reagent, measured as A410 (Flavin, 1962; Holloway et al., 1970). 
-Galactosidase expression of met : : lacZ fusion genes was monitored by the rate of ONPG hydrolysis (Miller, 1972). SAMase was assayed by passing reaction mixes inoculated with 14COOH-SAM (Amersham Biosciences) over Affi-gel 601 (Bio-Rad) cis-diol affinity columns and determining the net percentage of total counts that failed to bind the column and were assumed to be in 14COOH-L-homoserine (Hughes et al., 1987). SAM synthetase activity was measured as the percentage of [35S]methionine (Amersham Biosciences) converted to labelled cis-diol (Affi-gel 601)-binding compounds (e.g. SAM and its metabolites S-adenosylhomocysteine, 5'-methylthioadenosine and 5'-methylthioribose) after incubation with toluenized cells under conditions for the SAM synthetase assay (Hafner et al., 1977; Hughes et al., 1987). Protein concentrations were determined by the Lowry method. In all cases, assays were conducted on at least five cultures from independently transformed cells, grown to stationary phase, performed according to minor modifications of published procedures, and quantified spectrophotometrically (with a Spectronic 21 or Beckman DU-64) or by liquid scintillation (Beckman LS5000TD) using Beckman ReadySafe scintillation cocktail. Statistical comparisons employed Student's t test, with P<0·05 indicating significant differences between paired samples.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Effect of in vivo SAMase expression in met wild-type cells in a complete medium
Three systems were used initially to survey the consequence on met gene expression of the in vivo expression of SAMase in E. coli K-12 BW545. The results of these assays are presented in Table 2.
|
Secondly, we expressed SAMase in the BW545-derived GW strains that carry the lacZ gene fused behind the host's promoters for metA, metE and metF. The use of these met–lac fusion strains simplified the assays and expanded our survey to include another three genes. Introduction of SAMase activity into GW2517 (metA : : lacZ), GW2521 (metF : : lacZ) and GW2522 (metE : : lacZ) also greatly enhanced expression of these genes.
We then lysogenized BW545 with recombinant gt2 phage carrying metB, E, F, H, J or R gene promoters from S. typhimurium fused to lacZ, constructs that have been used extensively to assay met gene expression in E. coli (see Methods for appropriate references). metB, E, F and R showed dramatic induction of activity while metJ and metH showed much smaller but still significant increases. Both the E. coli and S. typhimurium promoters for metE and metF responded similarly to the introduction of in vivo SAMase activity, although the S. typhimurium promoters directed a somewhat higher basal level of gene expression. While results from the cystathionine synthetase (MetB) assays showed less enhanced enzyme expression than those revealed by assays of MetB activity gauged through the S. typhimurium promoter met–lac fusions, in both cases the assays indicated substantial SAMase-mediated enzyme induction.
Because of the similarity of results seen using E. coli and S. typhimurium promoters, the simplicity of the reporter assays, and the fact that BW545 lysogens are met wild-type strains while GW strains are methionine auxotrophs, we used these lysogens for all subsequent experiments. We deemed the use of met wild-type cells to be especially important. For example, defects in homocysteine biosynthesis in MetA– strains or in its use in MetE– or MetF– strains would likely alter MetR-mediated gene activation (Byerly et al., 1990; Cowen et al., 1993; Mares et al., 1992; Urbanowski et al., 1987) and introduce complex and unpredictable effects beyond those provoked by SAMase expression.
Effect of in vivo SAMase expression in met wild-type cells in a methionine-limited medium
Because cells transformed with pHBBR2 grow poorly in defined media (data not shown), we routinely cultured cells in antibiotic-supplemented YT complete medium. On the other hand, it was necessary to grow cells in a methionine-deficient medium to determine the impact of methionine on met gene expression. To ensure reliable growth, cells were first cultured overnight in YT medium, washed and diluted 10-fold into YT medium, M9 minimal medium or M9 medium supplemented with L-methionine, and then cultured once again overnight. This regimen allowed growth in defined media and showed a reliable pattern of methionine-influenced gene expression (Table 3). With the exception of metH and metJ, the tested genes were significantly induced in cells grown without methionine in the absence of SAMase, while in vivo SAMase activity provoked a significant induction of expression for every gene tested under all conditions. The metA, B, E, F and R genes were most dramatically affected by either the absence of methionine from the culture medium or the presence of the SAMase expression vector. metC expression showed less dramatic results, although it is possible the different and possibly less sensitive method used to assay metC activity accounted in part for its apparently lower degree of sensitivity to either condition.
|
illustrates the results derived from met : : lacZ fusion strains expressing low or high levels of SAMase upon their transformation with pHBF1BR or pHBBR2, respectively. In all cases, genes that showed dramatic induction in cells containing high levels of in vivo SAMase activity (metB, metE, metF, and metR from Table 2
) were induced to a lesser degree in cells expressing the nearly 10-fold lower levels of SAMase activity resulting from the introduction of pHBF1BR. Expression of metH and metJ, genes only weakly induced by pHBBR2-directed SAMase activity, was not significantly induced in cells expressing the lower levels of SAMase directed by transformation with pHBF1BR.
|
) with pHBBR2 to determine whether in vivo SAMase expression in met gene derepressed cells would have a significant effect on met gene expression. Table 5 shows the results of these assays. Assays on metJ : : Tn5 cells revealed an approximately 1·5–4-fold increase in the activity of five of the six tested met genes in the presence of in vivo SAMase activity; metJ itself is the exception. Assays of met gene expression in the metK : : Tn5 background showed over 10-fold induction for metB, metE, metF and metR; the increases in metH expression resembled the lower levels of induction seen in all of the genes assayed in the MetJ– cells, and metJ expression in this case was significantly enhanced.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Pine, 1978); the relative sensitivity of met gene promoters to induction under SAM-limiting conditions (Table 4; Greene et al., 1973; Holloway et al., 1970); and in our metJ : : Tn5 (no aporepressor) and metK : : Tn5 (limited SAM synthesis) cells (Table 5; Greene et al., 1973; Su & Greene, 1971).
The current model explains the trends in expression of genes shown to be sensitive to SAM levels and/or the absence of the MetJ aporepressor, but understanding the low but significant SAMase-mediated induction of metJ requires more thought. metJ is autoregulated, as shown by the repressive effect of excess levels of the aporepressor on metJ expression with or without added SAM (Shoeman et al., 1985; Urbanowski & Stauffer, 1986). SAM enhances this autoregulatory activity, so the removal of SAM by SAMase may simply decrease the effectiveness of this regulation, requiring higher concentrations of MetJ to regulate its own gene. This concept is further supported by both the higher level of metJ expression in MetJ-deficient cells and the lack of further metJ induction in cells lacking the aporepressor (Table 5).
It is more difficult to explain the enhanced induction of all met genes in response to SAMase activity in metJ : : Tn5 and metK : : Tn5 backgrounds through the activity of the MetJ–SAM repressor alone. MetJ– cells lack the MetJ aporepressor and, consequently, should fully and constitutively express genes regulated solely by the MetJ–SAM complex. However, cells bearing these Tn5-inactivated metJ and metK alleles – as well as other metJ and metK alleles that allow constitutive expression of met genes (data not shown) – consistently show enhanced expression of all tested met genes in the presence of in vivo SAMase activity with the single exception of metJ in MetJ-deficient cells noted above. Some or all of the enhanced met expression in cells with this particular metK : : Tn5 insertion may be due to residual SAM synthesis that occurs in these cells (e.g. DNA from these cells is fully Dam methylated and SAM is detectable at <5 % of wild-type levels; personal observations), and it would be interesting to test the expression of these genes under SAM-limiting conditions known to be lethal (Newman et al., 1998). At the same time the hyperinduction of met genes by SAMase suggests a need to dissect the impact of other factors known to regulate met gene expression under these conditions and to search for other sources of regulation.
The impact that SAMase-enhanced levels of MetR and/or homocysteine might have on met gene expression warrants the most careful consideration. MetR by itself activates metH expression (Urbanowski & Stauffer, 1989a; Urbanowski et al., 1987), and a MetR–homocysteine complex in the absence of the MetJ–SAM repressor is needed for efficient expression of metE (Plamann et al., 1988; Urbanowski et al., 1987). In addition, MetR– cells are impaired to some degree in expression of metA and metF, each of which is downregulated when homocysteine levels increase, a result presumably of increased levels of the MetR–homocysteine complex and a concomitant decrease of the free MetR activator protein in the cell (Cowen et al., 1993; Mares et al., 1992). It is also possible that MetR, free or bound to homocysteine, is needed in some way for efficient expression of other met genes. Consequently, assuming that levels of MetR are normally sufficient only to induce submaximal expression of its target genes, the induction of metR directed by SAMase activity could boost expression of MetR-activated genes.
The above logic is contradicted by the observation that metR expression is also induced in metJ : : Tn5 and metK : : Tn5 cells lacking SAMase. These cells should therefore also exhibit enhanced met gene expression comparable to cells with high levels of SAMase if increased levels of MetR synthesis are responsible for the extra level of expression of these genes. SAMase also presumably enhances synthesis of homocysteine through induction of metA, B and C, and this could also play a role in enhancing met gene expression. Depending on the fate of any extra homocysteine produced as a consequence of SAMase expression, this should lead to a higher level of the MetR–homocysteine activator complex (and less free MetR). Increased levels both of MetR and homocysteine would presumably also occur in both MetJ- and MetK-deficient cells, both of which are induced for metA, B and C with or without in vivo SAMase activity. These and other conflicting and unresolved possibilities surrounding SAMase-mediated induced met gene induction can only be resolved by further investigation of the role of MetR activity and/or homocysteine levels, or by searching for other unidentified regulatory elements that influence met gene activity such as revealed by the role of SAM in the S-box system of Bacillus subtilis (Murphy McDaniel et al., 2003).
Explanations for this effect might also be found outside of the met regulon. One possible explanation for the increased expression of met genes in metJ- and metK-deficient cells would include any positive effect on gene transcription resulting from SAMase-mediated hypomethylation of chromosomal DNA. Methylation of promoter sequences, particularly in studies with -GATC- sequence methylation mediated by the Dam methylase, results in reduced transcription for some genes in E. coli (e.g. Stauffer & Stauffer, 1988; reviewed by Palmer & Marinus, 1995; Plumbridge, 1987). Reduction of SAM pools to levels below those needed for Dam- or Dcm-mediated DNA methylation has been achieved with the in vivo expression of recombinant SAMase (Hughes et al., 1987; Macintyre et al., 2001; Posnick & Samson, 1999). Should met gene promoters or those of genes whose activity impacts met gene expression be hypomethylated, or should any other consequence of hypomethylation affect DNA packing or structure, an increase in the transcription of their corresponding genes could result. Answers to these possibilities await studies with Dam- or Dcm-deficient strains.
|
ACKNOWLEDGEMENTS |
|---|
gt2 met gene reporter phage constructs were gratefully received from George Stauffer, University of Iowa. |
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Belfaiza, J., Parsot, C., Martel, A., de la Tour, C. B., Margarita, D., Cohen, G. N. & Saint-Girons, I. (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 U S A 83, 867–871.
Biran, D., Brot, N., Weissbach, H. & Ron, E. Z. (1995). Heat shock-dependent transcriptional activation of the metA gene of Escherichia coli. J Bacteriol 177, 1374–1379.
Biran, D., Brot, N., Weissbach, H. & Ron, E. Z. (2000). Control of methionine biosynthesis in Escherichia coli by proteolysis. Mol Microbiol 37, 1436–1443.[CrossRef][Medline]
Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95–112.[Medline]
Byerly, K. A., Urbanowski, M. L. & Stauffer, G. V. (1990). Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response. J Bacteriol 172, 2839–2843.
Cowen, J. M., Urbanowski, M. L., Talmi, M. & Stauffer, G. V. (1993). Regulation of the Salmonella typhimurium metF gene by the MetR protein. J Bacteriol 175, 5862–5866.
Flavin, M. (1962). Microbial transsulfuration: the mechanism of an enzymatic disulfide elimination reaction. J Biol Chem 237, 768–777.
Greene, R. C. (1996). Biosynthesis of methionine. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd edn, pp. 542–560. Edited by F. Neidhardt and others. Washington, DC: American Society for Microbiology.
Greene, R. C., Su, C.-H. & Holloway, C. T. (1970). S-adenosylmethionine synthetase deficient mutants of Escherichia coli K-12 with impaired control of methionine biosynthesis. Biochem Biophys Res Comm 38, 1120–1126.[CrossRef][Medline]
Greene, R. C., Hunter, J. S. V. & Coch, E. H. (1973). Properties of metK mutants of Escherichia coli K-12. J Bacteriol 115, 57–67.
Hafner, E. W., Tabor, C. W. & Tabor, H. (1977). Isolation of a metK mutant with a temperature-sensitive S-adenosylmethionine synthetase. J Bacteriol 132, 832–840.
Hobson, A. C. & Smith, D. A. (1973). S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium. Mol Gen Genet 126, 7–18.[CrossRef][Medline]
Holloway, C. T., Greene, R. T. & Su, C. H. (1970). Regulation of S-adenosylmethionine synthetase in Escherichia coli. J Bacteriol 104, 734–747.
Hughes, J. A., Brown, L. R. & Ferro, A. J. (1987). Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polymamine biosynthesis in Escherichia coli. J Bacteriol 169, 3625–3632.
Kaplan, M. M. & Flavin, M. (1966). Cystathionine 
-synthetase of Salmonella. Catalytic properties of a new enzyme in bacterial methionine biosynthesis. J Biol Chem 241, 4463–4471.
Macintyre, G., Atwood, C. V. & Cupples, C. G. (2001). Lowering S-adenosylmethionine levels in Escherichia coli modulates C-to-T transition mutations. J Bacteriol 183, 921–927.
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mares, R., Urbanowski, M. L. & Stauffer, G. V. (1992). Regulation of the Salmonella typhimurium metA gene by the MetR protein and homocysteine. J Bacteriol 174, 390–397.
Miller, J. H. (1972). Experiments in Molecular Genetics, pp. 352–355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mulligan, J. T., Margolin, W., Krueger, J. H. & Walker, G. C. (1982). Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions. J Bacteriol 151, 609–619.
Murphy McDaniel, B. A., Grundy, F. J., Artsimovitch, I. & Henkin, T. M. (2003). Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc Natl Acad Sci U S A 100, 3083–3088.
Newman, E. B., Budman, L. I., Chan, E. C., Greene, R. C., Lin, R. T., Woldringh, C. L. & D'Ari, R. (1998). Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli. J Bacteriol 180, 3614–3619.
Palmer, B. R. & Marinus, M. G. (1995). The dam and dcm strains of Escherichia coli: a review. Gene 143, 1–12.
Pine, M. J. (1978). Comparative physiological effects of incorporated amino acid analogs in Escherichia coli. Antimicrob Agents Chemother 13, 676–685.
Plamann, L. S., Urbanowski, M. L. & Stauffer, G. V. (1988). Salmonella typhimurium metE operator-constitutive mutations. Gene 73, 201–208.[CrossRef][Medline]
Plumbridge, J. (1987). The role of dam methylation in controlling gene expression. Biochemie 69, 439–443.[Medline]
Posnick, L. M. & Samson, L. D. (1999). Influence of S-adenosylmethionine pool size on spontaneous mutation, Dam methylation, and cell growth of Escherichia coli. J Bacteriol 181, 6756–6762.
Rafferty, J. B., Somers, W. S., Saint-Girons, I. & Phillips, S. E. V. (1989). Three-dimensional crystal structures of Escherichia coli met repressor with and without co-repressor. Nature 341, 705–710.[CrossRef][Medline]
Shoeman, R., Redfield, B., Coleman, T., Greene, R., Smith, A., Saint-Girons, I., Brot, N. & Weissbach, H. (1985). Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL, and metJ genes. Biochem Biophys Res Commun 133, 731–739.[CrossRef][Medline]
Stauffer, G. V. & Stauffer, L. T. (1988). Salmonella typhimurium LT2 metF operator mutations. Mol Gen Genet 212, 32–36.
Su, C. H. & Greene, R. C. (1971). Regulation of methionine biosynthesis in Escherichia coli: mapping of the metJ locus and properties of a metJ+-metJ– merodiploid. Proc Natl Acad Sci U S A 68, 367–371.
Urbanowski, M. L. & Stauffer, G. V. (1986). Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene. J Bacteriol 165, 740–745.
Urbanowski, M. L. & Stauffer, G. V. (1987). Regulation of the metR gene of Salmonella typhimurium. J Bacteriol 169, 5841–5844.
Urbanowski, M. L. & Stauffer, G. V. (1989a). Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol 171, 3277–3281.
Urbanowski, M. L. & Stauffer, G. V. (1989b). The control region of the metH gene of Salmonella typhimurium LT2: an atypical met promoter. Gene 73, 193–200.
Urbanowski, M. L., Stauffer, L. T., Plamann, S. & Stauffer, G. V. (1987). A new methionine locus, metR, that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J Bacteriol 169, 1391–1397.
Usuda, Y. & Kurahashi, O. (2005). Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli. Appl Environ Microbiol 71, 3228–3234.
Val, D. L. & Cronan, J. E. (1998). In vivo evidence that S-adenosylmethionine and fatty acid synthesis intermediates are the substrates for the LuxI family of autoinducer synthases. J Bacteriol 180, 2644–2651.
Yanisch-Perron, C., Vieira, C. J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.[CrossRef][Medline]
Received 4 September 2005;
revised 25 January 2006;
accepted 27 January 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
