Vol. 172, No. 6
JOURNAL OF BACTERIOLOGY, June 1990, p. 2839-2843
0021-9193/90/062839-05$02.00/0 Copyright © 1990, American Society for Microbiology
Escherichia coli metR Mutants That Produce a MetR Activator Protein with an Altered Homocysteine Response KYNA A. BYERLY, MARK L. URBANOWSKI, AND GEORGE V. STAUFFER* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received 8 November 1989/Accepted 2 March 1990 Using an Escherichia coli lac deletion strain lysogenized with a A phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression. In Escherichia coli and Salmonella typhimurium the methylation of homocysteine to form methionine is catalyzed by either the MetE (vitamin B12-independent) or the MetH (vitamin B12-dependent) transmethylase (for a review, see reference 11). This reaction links the serine-glycine and methionine biosynthetic pathways (Fig. 1). The cell controls expression of the genes encoding the methionine biosynthetic enzymes by both negative and positive regulatory mechanisms. The MetJ repressor, with S-adenosylmethionine acting as a corepressor, negatively regulates all of the methionine biosynthetic genes, with the exception of metH (11). The MetR gene product, a DNA-binding protein (2, 18), is necessary for the activation of both metE and metH expression (2, 20). Homocysteine modulates this activation by stimulating metE expression and inhibiting metH expression (19). The MetJ repressor system, however, can override activation by the MetR protein (20). The metR gene itself is negatively autoregulated, with homocysteine acting as the corepressor (16). Using a XmetH-lacZ fusion phage in an E. coli strain that accumulates high endogenous levels of homocysteine, we isolated mutants in which homocysteine no longer inhibits metH-lacZ expression. Using metH-lacZ, metE-lacZ, and metR-lacZ gene fusions to measure expression of the metH, metE, and metR genes, respectively, we found that the mutations affect the regulation of all three genes. Our data suggest that the mutations lie in the metR gene, altering the ability of the MetR activator protein to use homocysteine as a modulator of gene expression.
MATERIALS AND METHODS Bacterial strains, bacteriophages, and plasmids. All bacterial strains used were derived from E. coli K-12 and are described in Table 1. Construction of the lacZ fusion phages XElac (10), XHlac (17), XRlac (16), and XRElac (19) has been described previously. Plasmid pGSmetR (16) carries the S. typhimurium metR gene on the single-copy vector pDF41
GM and LM were always supplemented with phenylalanine (50 ,ug/ml) and vitamin B1 (1 jig/ml). Additional supplements were added where indicated at the following concentrations: L-methionine and D-methionine, 50 ,ug/ml; vitamin B12, 1 ,ug/ml; D,L-homocysteine (prepared as described previously [19]), 100 jig/ml; kanamycin, 20 ,ug/ml; phenylethyl-p-Dthiogalactoside (TPEG), 0.8 mg/ml; 5-bromo-4-chloro-3-indolyl-p3-D-galactoside, 40 ,ug/ml. All A lysogens produce a temperature-sensitive lambda repressor due to the cI857 mutation and therefore were grown at 30°C. All other strains were grown at 370C. Construction of A lysogens. Appropriate strains were lysogenized with XElac, XHlac, ARlac, and XRElac fusion phages as described previously (15). After purification, the lysogens were tested for a single copy of the A phage by infection with phage X cI90c17 (12). Mutant isolation. Strain GS723 lysogenized with XHlac (723XHlac) was grown overnight in 1 ml of LB. The cells were washed twice with lx minimal salts and then suspended in 0.5 ml of lx minimal salts; 0.2 ml was plated on LM plates supplemented with L-methionine and TPEG. TPEG, a competitive inhibitor of 3-galactosidase (8), was added to reduce background growth due to the leaky Lac' phenotype of 723XHlac on the selection plates. Colonies that arose after 48 h were streaked for purity on L-agar plates and then retested for the Lac' phenotype on LM plates supplemented with L-methionine and TPEG. 3-galactosidase enzyme assays. P-Galactosidase activity of mid-log-phase cultures was measured as described by Miller (8) with the chloroform-sodium dodecyl sulfate lysis procedure. Each sample was done in triplicate, and each assay was done at least twice. P1 transduction. P1 transductions were performed with the P1 cml clr-JOO phage as described previously (8). RESULTS Mutant selection and analysis. A regenerative pathway that produces homocysteine from S-adenosylmethionine exists in E. coli (3) (Fig. 1). Due to metB and metC mutations, strain GS723 can produce homocysteine only through this regenerative pathway from exogenously added methionine. Because this strain also carries a metF mutation and cannot produce 5-methyltetrahydrofolate, homocysteine cannot be utilized and accumulates in the cell. Previous experiments
(6).
Media and growth conditions. Luria agar (L agar), Luria broth (LB), and glucose minimal medium (GM) have been described previously (13). Lactose minimal medium (LM) was identical to GM, except that lactose replaced glucose. *
Corresponding author. 2839
2840
BYERLY ET AL.
J. BACTERIOL.
hooserine
I
,-galactosidase levels due to the presence of two or more copies of the AmetH-lacZ phage. These multiple lysogens were not characterized further. The four Lac' colonies carrying a single copy of the XHlac phage were each tested to determine whether the Lac' phenotype was phage associated. Phage isolated from each Lac' colony by temperature induction were used to relysogenize the parental strain GS723. After'single-colony purification, the lysogens were spotted on LM plates supplemented with L-methionine and
uMO
O-succinylhomoserine
I
_U
cystathionine
I homocysteine mtF
5-methylTHF S- 5,10-methylene THIF
S-ribosylhoaocysteine
serine
_jlvA
glycine metE or
methionine
I
.w.adenine
SAH
SAlLM
SAM-dependent cellular
methylases
FIG. 1. Methionine biosynthetic pathway in E. coli and S. typhimurium (3, 11).
have shown that the MetR-mediated activation of the metH gene is inhibited by high levels of homocysteine (19). Accordingly, the metH-lacZ fusion in lysogen 723XHlac produces insufficient levels of P-galactosidase to grow on LM plates supplemented with methionine and TPEG. Using Iysogen 723XHlac, we selected for mutants with elevated metH-lacZ expression by their ability to grow on LM plates supplemented with L-methionine and TPEG. Colonies that grew on the selection plates were tested with the A c190c17 phage for the presence of a single copy of the XHlac phage (12); 14 of 18 Lac+ colonies had elevated
Strain
GS244
GS719 GS723 GS792 GS793 GS794 GS795 GS796 GS808 GS809 GS810 GS811
GS812
TABLE 1. E. coli strains used in this investigation Genotype' AmetR::Mu metBI metJ97 metBI metC162::TnlO metJ97 AmetF::Mu ilv metBI metC162::TniO metJ97 AmetF::Mu metRI metBI metC162::TrlO metJ97 AmetF::Mu metR2 metBI metC162::TnlO metJ97 AmetF::Mu metR3 metBI metC162::TnlO metJ97 AmetF::Mu metR4 metBi metC162::TnlO metJ97 metBI metC162::TnlO metJ97 metRI metBI metC162::TnlO metJ97 metR2 metBI metC162::TnlO metJ97 metR3 metBI metC162::TnlO metJ97 metR4
a In addition, all strains carry the pheA905, -thi, araD129, rspL, and
AlacU169 mutations. All strains were isolated during this study or were obtained from the laboratory collection.
TPEG. None of the lysogens was' able to grow on these plates, suggesting that the'mutations were not phage associated. The four original Lac' lysogens, presumably carrying chromosomal mutations, were cured of XHlac (21) and designated GS793, GS794, GS795, and GS796. These strains were relysogenized with wild-type XHlac phage, and the resulting lysogens (793XHlac, 794XHlac, 795XHlac, and 796XHlac, respectively), along with the parental lysogen (723AHlac), were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine. The cultures were then assayed for ,-galactosidase activity. In the absence of exogenously added homocysteine, ,-galactosidase levels were four- to sixfold higher in the mutant strain. The addition of homocysteine to the growth media did not significantly lower these levels in either the mutant or parental strain (Table 2). To determine whether the mutations specifically alter metH-lacZ gene expression, the parental and mutant strains were each lysogenized with X phage carrying a metE-lacZ gene fusion (XElac), so that the effects of the mutations on metE expression could be determined. The resulting lysogens (723XElac, 793XElac, 794XElac, 795XElac, 'and 796XElac) were grown in GM supplemented with L-methionine or L-methionine plus homocysteine. The cultures were then assayed for P3-galactosidase activity. metE-lacZ expression was 5- to 22-fold lower in the mutant lysogens (793XElac, 794XElac, 795XElac, and 796XElac) than in the parental lysogen (723XElac) (Table 2), indicating that the mutations were not specific for the metH-lacZ gene fusion. In addition, there was considerable variation in the levels of metE-lacZ expression among the four mutant lysogens, indicating that strains with different mutations had been isolated. The metE and metR genes share a common control region with overlapping, divergently transcribed promoters (9). In addition, the metR gene is'negatively autoregulated and uses homocysteine as a corepressor (16). Since metE-lacZ expression is altered in the mutants, we tested the effects of the mutations on expression of a metR-lacZ gene fusion (XRlac). The parental and mutant strains were lysogenized with the XRlac phage, and the resulting lysogens were grown in GM supplemented with L-methiomnne or L-methionine plus homocysteine. The cultures were then assayed for ,B-galactosidase activity. metR-lacZ expression was elevated 9- to 20-fold in the mutant XRlac lysogens (Table 2), indicating that the mutations interfere with the homocysteine-mediated negative autoregulation of metR. Complementation of the mutants by a wild-type metR gene. Since the mutations caused low metE-lacZ expression and high metH-lacZ and metR-lacZ expression in the presence of accumulated homocysteine, we hypothesized that the mutations might be in the metR gene, causing the synthesis of a MetR protein unable to respond to homocysteine. To test this hypothesis, we lysogenized the parental strain and the four mutant strains with XRElac phage. XRElac (19) contains an intact wild-type metR gene plus -80 base pairs down-
VOL. 172, 1990
ALTERED HOMOCYSTEINE RESPONSE IN metR MUTANTS
TABLE 2. Effects of the metR mutations on metH-lacZ, metE-lacZ, and metR-lacZ expression and complementation of the mutations by an intact copy of the metR gene
TABLE 3. P1 transduction results showing linkage of the mutations to the ilv locus Donor
1-Galactosidase Lysogena
metH-lacZ 723XHlac 793XHlac 794XHlac 795XHlac 796XHlac
723XHlac(pGSmetR) 793XHlac(pGSmetR) 794XHlac(pGSmetR) 795XHlac(pGSmetR) 796XHlac(pGSmetR) metE-lacZ 723XElac 793XElac 794XElac 795XElac 7%XElac
723XRElac 793XRElac 794XRElac 795XRElac 796XRElac metR-lacZ 723XRlac 793XRlac 794XRlac 795XRlac 796XRlac
metR
genotype
metR+ metRI metR2 metR3 metR4
metR+lmetR+ metRl/metR+ metR2/metR+ metR31metR+ metR41metR+
132 215 215 272 220
metR+lmetR+ metR+lmetRI metR+lmetR2 metR+lmetR3 metR+lmetR4
metR+ metRI metR2 metR3 metR4
6,995 724 568 316 1,295
With HC 78 408 385 586 303
ND ND ND ND ND
6,785 1,014 977
302 2,006
10,504 9,313 8,323 7,370 7,313
9,238 9,500 9,340
161 2,714 1,442 2,400 2,092
108 2,000 1,200 2,230 1,330
8,252 7,807
a All lysogens were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine (HC), as indicated. b Units are Miller units (8). ND, Not determined.
stream of the translation stop codon and the same metE-lacZ
fusion as in AElac. These lysogens (723XRElac, 793XRElac, 794XRElac, 795XRElac, and 796ARElac) were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine and then assayed for ,Bgalactosidase levels. The intact copy of the S. typhimurium metR gene carried on XRElac complemented the mutations, restoring metE-lacZ expression to approximately the levels seen in the parental strain (Table 2). A XHlac phage carrying a wild-type metR gene was not available for complementation experiments. Therefore, a single-copy plasmid (pGSmetR) carrying the S. typhimurium metR gene was used for metH complementation tests. Plasmid pGSmetR contains the TnS kan gene (1), the S. typhimurium metR gene, -80 base pairs downstream of the metR translation stop codon, the metE control region, and the first 283 codons of the metE structural gene. The mutant and parental XHlac lysogens were transformed (7) with Kmr plasmid pGSmetR, and the resulting transformants were grown in GM supplemented with kanamycin and L-methionine or with kanamycin, L-methiofline, and homocysteine. The cultures were then assayed for ,B-galactosidase activity. This plasmid partially complemented the mutations, lower-
gene
No. of colonies on:
Recipienta GM
GM + methionine
93 87
95 92
87
90
84b
87
activityb
Without HC 110 444 444 601 401
metR+ metRI metR2 metR3 metR4
2841
GS793 (metRI) GS794 (metR2) GS795 (metR3) GS79S (metR4)
GS792 (ilv) GS792 (ilv) GS792 (ilv) GS792 (ilv)
Ilv+
transductants were selected on GM supplemented with L-methionine; colonies were then scored on GM and GM supplemented with Lmethionine. b The three colonies scored as having a methionine requirement grew slowly after 12 h and were well established by 18 hr. a
ing metH-lacZ expression two- to threefold (Table 2). The failure to observe complete complementation with pGSmetR is probably due to underproduction of a wild-type MetR protein. In a related experiment, only partial activation of the metH gene was seen in a metR deletion strain transformed with pGSmetR, presumably because of insufficient production of the MetR protein (unpublished results). Pl transduction experiments. In previous P1 transduction experiments, the metR gene was found to cotransduce with the ilv locus at min 85 on the E. coli chromosome at a frequency of 39% (20). In the transduction experiment reported, the P1 lysate was prepared from the ilv strain (GS38) with the metR strain as the recipient. Although the exact nature of the ilv mutation in strain GS38 is unknown, it is presumed to be a deletion because of its nonreverting phenotype. Thus the reported 39o value represents an abnormally high cotransduction frequency between ilv and metR. In reciprocal transduction experiments done later, when the P1 lysate was prepared from known metR point mutants, a metR-ilv cotransduction frequency of 2 to 3% was observed (unpublished results). We tested for linkage of the new mutations to ilv by using strain GS792 (metJ+ ilv), a derivative of strain GS38, as a recipient. Because strain GS792 has a functional MetJ repressor, synthesis of the metE gene product (homocysteine transmethylase) is much lower in this strain than in the original GS723 strain (metJ metB metC metF). We predicted that introduction of the new metR mutations, shown to cause a 5- to 22-fold drop in metE-lacZ expression (Table 2), would significantly lower the levels of the metE-encoded transmethylase in strain GS792, resulting in methionine auxotrophy. GS792 was transduced with P1 phage prepared from the metR+ strain, GS723, and each of the four mutant strains (GS793, GS794, GS795, and GS796), and Ilv+ transductants were selected on GM plates supplemented with L-methionine. Ilv+ colonies were scored on GM plates and on GM plates supplemented with L-methionine. When GS723 was sued as a donor, no Ilv+ transductants required methionine for growth. Approximately 2 to 5% of the Ilv+ transductants from each transduction with a metR mutant required methionine for growth (Table 3). It is worth noting that the GS796 colonies scored as having a methionine requirement grew slowly after 12 h and were well established by 18 h. This corresponds to the level of metE-lacZ expression in lysogen 796AElac, which is the highest of the four mutant strains (Table 2). To demonstrate that the Met- phenotype is due to a mutation in the metR gene, one Met- transformant isolated with GS793 (metRI) as a donor was transformed with the single-copy plasmid pGSmetR and retested on GM plates and GM plates supplemented with L-methionine. The intact
2842
J. BACTERIOL.
BYERLY ET AL.
copy of the metR gene in pGSmetR complemented the Mettransductant, resulting in a Met+ phenotype.
Since the
new
mutations
were
TABLE 4. Effects of homocysteine on metH-lacZ and metE-lacZ expression in parental and mutant strains
cotransducible with ilv at a
frequency comparable to known metR mutants and were complemented by a wild-type metR gene (see above), we conclude they lie in the metR gene. The mutations in strains GS793, GS794, GS795, and GS796 were designated metRi, metR2, metR3, and metR4, respectively. Effect of homocysteine on metE and metH expression in the mutant strains. To confirm that the altered MetR proteins being synthesized were unable to respond to homocysteine, we needed to look at metE-lacZ and metH-lacZ expression in both a low- and a high-homocysteine environment. The high internal homocysteine levels in the GS723 background due to the metF mutation prevented analysis of expression of the lac fusions in a low-homocysteine environment. Therefore, we transduced a wild-type metF gene into the parental and mutant strains, so that homocysteine could be utilized and would no longer accumulate in the cell. The effects of homocysteine on metH-lacZ and metE-lacZ expression could then be determined by manipulating homocysteine levels in the cell through medium supplementation. P1 phage from GS719 (metB metJ) was used to transduce strains GS723, GS793, GS794, GS795, and GS796. metF+ transductants were selected on GM supplemented with homocysteine. Since metF and metB are closely linked on the chromosome, the transductants were then spotted on GM and GM supplemented with homocysteine to confirm that homocysteine was required for growth. The functional metF gene product in these strains allows exogenously added homocysteine to be converted to methionine by the metE gene product. These new strains were designated GS808 (parental), GS809 (metRI), GS810 (metR2), GS811 (metR3), and GS812 (metR4). These five strains were lysogenized separately with XHlac and XElac phage. The lysogens were grown in GM supplemented with D-methionine or with D-methionine plus homocysteine. D-Methionine was used instead of L-methionine because it limits the cell for methionine, resulting in lower levels of internal homocysteine produced via the regenerative pathway. The cultures were then assayed for 3-galactosidase activity. In the parental lysogen, 808XHlac, homocysteine cause a fourfold drop in metH-lacZ expression, whereas in the mutant XHlac lysogens there was no significant drop in metH-lacZ expression (Table 4). In the parental lysogen 808XElac, homocysteine caused a sixfold increase in metE-lacZ expression, whereas none of the mutants responded significantly. However, in lysogens of strain GS812 (metR4), homocysteine had a greater effect on both metHlacZ and metE-lacZ expression than in the other three mutants. This experiment supports our hypothesis that the metR mutations cause the production of a MetR protein with an altered response to homocysteine. DISCUSSION Activation of both the metE and metH genes is dependent on the metR gene product (20) and is regulated by intracellular homocysteine (19). For both genes, the MetR protein has been shown in vitro to bind specifically to a region of the promoter containing an 8-base-pair interrupted palindrome (2, 18). Formation of the MetR-DNA complex does not require homocysteine under these conditions. Although the mechanism of MetR activation and homocysteine involvement is not known, we have hypothesized that homocysteine modulates the expression of the target genes by causing a
metR
Lysogena Lysogen genotype gntp
13-Galactosidase activityb
Without HC
With HC
808XHlac 809XHlac 81OXHlac 811AHlac 812XHlac
metR+ metRI metR2 metR3 metR4
442 482 532 508 488
101 409 518 435 391
808XElac 809XElac 810XElac 811AElac 812AElac
metR+ metRI metR2 metR3 metR4
1,456 592 1,124 440 900
8,544 655 1,363 306 1,495
a All lysogens carry metJ metB mutations and therefore do not accumulate homocysteine. Cells were grown in GM supplemented with either D-methionine or D-methionine plus homocysteine (HC), as indicated. b Units are Miller units (8).
conformational change in MetR protein already bound at its target site (18). This altered conformation is proposed to inhibit the ability of MetR to activate metH expression while enhancing its ability to activate metE expression, possibly through direct contact with RNA polymerase. In this report we describe the isolation and characterization of a set of mutants in which the MetR protein fails to properly regulate metH-lacZ, metE-lacZ, and metR-lacZ gene fusions. Expression of the metE-lacZ fusion, which is normally activated in the presence of homocysteine, was significantly lowered in the mutant strains (Table 2), although the levels were still 100- to 400-fold above those seen with a metR deletion mutant (20). The mutant phenotype cannot be explained as simply due to a partially active MetR protein, since metH-lacZ expression was four- to sixfold higher in the mutants than in the parent lysogen (Table 2). This latter result suggests that the altered MetR proteins can still bind efficiently to operator DNA, at least in the metH promoter region. However, we cannot rule out the possibility that the mutations have altered the affinity of the MetR protein for its target DNA. If so, the mutations must cause an increased affinity for the metH promoter region and a decreased affinity for the metE promoter region. Alternatively, the phenotypes of the mutants may result from an inability of the altered MetR proteins to respond properly to the coactivator. In XHlac and AElac lysogens of strains GS809, GS810, GS811, and GS812, which have low internal levels of homocysteine, the addition of homocysteine to the growth medium had a diminished effect on metH-lacZ and metE-lacZ expression, as compared with that in lysogens of the parental strain, GS808 (Table 4). Furthermore, in lysogens 793XElac, 794XElac, and 796X Elac, in which intracellular homocysteine accumulates to high levels, exogenously added homocysteine caused a small but reproducible increase in activation of metE-lacZ expression (Table 2). In contrast, in the parent lysogen 723XElac, exogenously added homocysteine did not cause a further increase in metE-lacZ expression. If homocysteine modulates gene expression by binding directly to the MetR protein, mutations metRI, metR2, and metR4 may involve a change in the homocysteine binding site on the metR protein, resulting in a lowered affinity for homocysteine. A third possible explanation is that that mutant MetR proteins are locked into a conformation that activates metHlacZ expression while preventing activation of the metElacZ fusion.
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ALTERED HOMOCYSTEINE RESPONSE IN metR MUTANTS
Examples of activator mutants similar to the four metR mutants isolated here have been reported in the literature. Certain bacteriophage lambda cI repressor mutations greatly reduce the ability of the repressor to activate promoter PRM, without altering the affinity of the repressor for its specific operator DNA (5). The mutant phenotype presumably results from the loss of a favorable protein-protein contact between RNA polymerase and DNA-bound repressor. However, unlike the metR system, no coactivator is involved in the k system. In another case, mutations in the crp gene (cyclic AMP receptor protein) of E. coli have been shown to produce an altered Crp protein that no longer requires the presence of the effector molecule, cyclic AMP, to exert positive control over catabolite-sensitive operons (4). The altered apoprotein appears to be locked into a conformation resembling the wild-type cyclic AMP-CRP complex. The CRP system may not be entirely analogous to the MetR system, however, since cyclic AMP confers a positive effect on CRP function, whereas homocysteine not only positively regulates metE but also negatively regulates metH. We plan to clone the mutant metR genes to facilitate purification of the altered proteins. Using the purified proteins, we will attempt to (i) set up a homocysteine binding assay to assess the relative affinities of the mutant and wild-type MetR proteins for the coactivator and (ii) use an in vitro DNA binding assay to determine the DNA operator site affinities. These studies should help us to understand more specifically how homocysteine interacts with the MetR protein to regulate metE, metH, and metR gene expression. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant GM38912 from the National Institute of General Medical Sciences. 1.
2.
3.
4.
5.
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JOURNAL OF BACTERIOLOGY, June 1990, p. 2839-2843
0021-9193/90/062839-05$02.00/0 Copyright © 1990, American Society for Microbiology
Escherichia coli metR Mutants That Produce a MetR Activator Protein with an Altered Homocysteine Response KYNA A. BYERLY, MARK L. URBANOWSKI, AND GEORGE V. STAUFFER* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received 8 November 1989/Accepted 2 March 1990 Using an Escherichia coli lac deletion strain lysogenized with a A phage carrying a metH-lacZ gene fusion, we isolated trans-acting mutations that result in simultaneous 4- to 6-fold-elevated metH-lacZ expression, 5- to 22-fold-lowered metE-lacZ expression, and 9- to 20-fold-elevated metR-lacZ expression. The altered regulation of these genes occurs in the presence of high intracellular levels of homocysteine, a methionine pathway intermediate which normally inhibits metH and metR expression and stimulates metE expression. P1 transductions and complementation tests indicate that the mutations are in the metR gene. Our data suggest that the mutations result in an altered MetR activator protein that has lost the ability to use homocysteine as a modulator of gene expression. In Escherichia coli and Salmonella typhimurium the methylation of homocysteine to form methionine is catalyzed by either the MetE (vitamin B12-independent) or the MetH (vitamin B12-dependent) transmethylase (for a review, see reference 11). This reaction links the serine-glycine and methionine biosynthetic pathways (Fig. 1). The cell controls expression of the genes encoding the methionine biosynthetic enzymes by both negative and positive regulatory mechanisms. The MetJ repressor, with S-adenosylmethionine acting as a corepressor, negatively regulates all of the methionine biosynthetic genes, with the exception of metH (11). The MetR gene product, a DNA-binding protein (2, 18), is necessary for the activation of both metE and metH expression (2, 20). Homocysteine modulates this activation by stimulating metE expression and inhibiting metH expression (19). The MetJ repressor system, however, can override activation by the MetR protein (20). The metR gene itself is negatively autoregulated, with homocysteine acting as the corepressor (16). Using a XmetH-lacZ fusion phage in an E. coli strain that accumulates high endogenous levels of homocysteine, we isolated mutants in which homocysteine no longer inhibits metH-lacZ expression. Using metH-lacZ, metE-lacZ, and metR-lacZ gene fusions to measure expression of the metH, metE, and metR genes, respectively, we found that the mutations affect the regulation of all three genes. Our data suggest that the mutations lie in the metR gene, altering the ability of the MetR activator protein to use homocysteine as a modulator of gene expression.
MATERIALS AND METHODS Bacterial strains, bacteriophages, and plasmids. All bacterial strains used were derived from E. coli K-12 and are described in Table 1. Construction of the lacZ fusion phages XElac (10), XHlac (17), XRlac (16), and XRElac (19) has been described previously. Plasmid pGSmetR (16) carries the S. typhimurium metR gene on the single-copy vector pDF41
GM and LM were always supplemented with phenylalanine (50 ,ug/ml) and vitamin B1 (1 jig/ml). Additional supplements were added where indicated at the following concentrations: L-methionine and D-methionine, 50 ,ug/ml; vitamin B12, 1 ,ug/ml; D,L-homocysteine (prepared as described previously [19]), 100 jig/ml; kanamycin, 20 ,ug/ml; phenylethyl-p-Dthiogalactoside (TPEG), 0.8 mg/ml; 5-bromo-4-chloro-3-indolyl-p3-D-galactoside, 40 ,ug/ml. All A lysogens produce a temperature-sensitive lambda repressor due to the cI857 mutation and therefore were grown at 30°C. All other strains were grown at 370C. Construction of A lysogens. Appropriate strains were lysogenized with XElac, XHlac, ARlac, and XRElac fusion phages as described previously (15). After purification, the lysogens were tested for a single copy of the A phage by infection with phage X cI90c17 (12). Mutant isolation. Strain GS723 lysogenized with XHlac (723XHlac) was grown overnight in 1 ml of LB. The cells were washed twice with lx minimal salts and then suspended in 0.5 ml of lx minimal salts; 0.2 ml was plated on LM plates supplemented with L-methionine and TPEG. TPEG, a competitive inhibitor of 3-galactosidase (8), was added to reduce background growth due to the leaky Lac' phenotype of 723XHlac on the selection plates. Colonies that arose after 48 h were streaked for purity on L-agar plates and then retested for the Lac' phenotype on LM plates supplemented with L-methionine and TPEG. 3-galactosidase enzyme assays. P-Galactosidase activity of mid-log-phase cultures was measured as described by Miller (8) with the chloroform-sodium dodecyl sulfate lysis procedure. Each sample was done in triplicate, and each assay was done at least twice. P1 transduction. P1 transductions were performed with the P1 cml clr-JOO phage as described previously (8). RESULTS Mutant selection and analysis. A regenerative pathway that produces homocysteine from S-adenosylmethionine exists in E. coli (3) (Fig. 1). Due to metB and metC mutations, strain GS723 can produce homocysteine only through this regenerative pathway from exogenously added methionine. Because this strain also carries a metF mutation and cannot produce 5-methyltetrahydrofolate, homocysteine cannot be utilized and accumulates in the cell. Previous experiments
(6).
Media and growth conditions. Luria agar (L agar), Luria broth (LB), and glucose minimal medium (GM) have been described previously (13). Lactose minimal medium (LM) was identical to GM, except that lactose replaced glucose. *
Corresponding author. 2839
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BYERLY ET AL.
J. BACTERIOL.
hooserine
I
,-galactosidase levels due to the presence of two or more copies of the AmetH-lacZ phage. These multiple lysogens were not characterized further. The four Lac' colonies carrying a single copy of the XHlac phage were each tested to determine whether the Lac' phenotype was phage associated. Phage isolated from each Lac' colony by temperature induction were used to relysogenize the parental strain GS723. After'single-colony purification, the lysogens were spotted on LM plates supplemented with L-methionine and
uMO
O-succinylhomoserine
I
_U
cystathionine
I homocysteine mtF
5-methylTHF S- 5,10-methylene THIF
S-ribosylhoaocysteine
serine
_jlvA
glycine metE or
methionine
I
.w.adenine
SAH
SAlLM
SAM-dependent cellular
methylases
FIG. 1. Methionine biosynthetic pathway in E. coli and S. typhimurium (3, 11).
have shown that the MetR-mediated activation of the metH gene is inhibited by high levels of homocysteine (19). Accordingly, the metH-lacZ fusion in lysogen 723XHlac produces insufficient levels of P-galactosidase to grow on LM plates supplemented with methionine and TPEG. Using Iysogen 723XHlac, we selected for mutants with elevated metH-lacZ expression by their ability to grow on LM plates supplemented with L-methionine and TPEG. Colonies that grew on the selection plates were tested with the A c190c17 phage for the presence of a single copy of the XHlac phage (12); 14 of 18 Lac+ colonies had elevated
Strain
GS244
GS719 GS723 GS792 GS793 GS794 GS795 GS796 GS808 GS809 GS810 GS811
GS812
TABLE 1. E. coli strains used in this investigation Genotype' AmetR::Mu metBI metJ97 metBI metC162::TnlO metJ97 AmetF::Mu ilv metBI metC162::TniO metJ97 AmetF::Mu metRI metBI metC162::TrlO metJ97 AmetF::Mu metR2 metBI metC162::TnlO metJ97 AmetF::Mu metR3 metBI metC162::TnlO metJ97 AmetF::Mu metR4 metBi metC162::TnlO metJ97 metBI metC162::TnlO metJ97 metRI metBI metC162::TnlO metJ97 metR2 metBI metC162::TnlO metJ97 metR3 metBI metC162::TnlO metJ97 metR4
a In addition, all strains carry the pheA905, -thi, araD129, rspL, and
AlacU169 mutations. All strains were isolated during this study or were obtained from the laboratory collection.
TPEG. None of the lysogens was' able to grow on these plates, suggesting that the'mutations were not phage associated. The four original Lac' lysogens, presumably carrying chromosomal mutations, were cured of XHlac (21) and designated GS793, GS794, GS795, and GS796. These strains were relysogenized with wild-type XHlac phage, and the resulting lysogens (793XHlac, 794XHlac, 795XHlac, and 796XHlac, respectively), along with the parental lysogen (723AHlac), were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine. The cultures were then assayed for ,-galactosidase activity. In the absence of exogenously added homocysteine, ,-galactosidase levels were four- to sixfold higher in the mutant strain. The addition of homocysteine to the growth media did not significantly lower these levels in either the mutant or parental strain (Table 2). To determine whether the mutations specifically alter metH-lacZ gene expression, the parental and mutant strains were each lysogenized with X phage carrying a metE-lacZ gene fusion (XElac), so that the effects of the mutations on metE expression could be determined. The resulting lysogens (723XElac, 793XElac, 794XElac, 795XElac, 'and 796XElac) were grown in GM supplemented with L-methionine or L-methionine plus homocysteine. The cultures were then assayed for P3-galactosidase activity. metE-lacZ expression was 5- to 22-fold lower in the mutant lysogens (793XElac, 794XElac, 795XElac, and 796XElac) than in the parental lysogen (723XElac) (Table 2), indicating that the mutations were not specific for the metH-lacZ gene fusion. In addition, there was considerable variation in the levels of metE-lacZ expression among the four mutant lysogens, indicating that strains with different mutations had been isolated. The metE and metR genes share a common control region with overlapping, divergently transcribed promoters (9). In addition, the metR gene is'negatively autoregulated and uses homocysteine as a corepressor (16). Since metE-lacZ expression is altered in the mutants, we tested the effects of the mutations on expression of a metR-lacZ gene fusion (XRlac). The parental and mutant strains were lysogenized with the XRlac phage, and the resulting lysogens were grown in GM supplemented with L-methiomnne or L-methionine plus homocysteine. The cultures were then assayed for ,B-galactosidase activity. metR-lacZ expression was elevated 9- to 20-fold in the mutant XRlac lysogens (Table 2), indicating that the mutations interfere with the homocysteine-mediated negative autoregulation of metR. Complementation of the mutants by a wild-type metR gene. Since the mutations caused low metE-lacZ expression and high metH-lacZ and metR-lacZ expression in the presence of accumulated homocysteine, we hypothesized that the mutations might be in the metR gene, causing the synthesis of a MetR protein unable to respond to homocysteine. To test this hypothesis, we lysogenized the parental strain and the four mutant strains with XRElac phage. XRElac (19) contains an intact wild-type metR gene plus -80 base pairs down-
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ALTERED HOMOCYSTEINE RESPONSE IN metR MUTANTS
TABLE 2. Effects of the metR mutations on metH-lacZ, metE-lacZ, and metR-lacZ expression and complementation of the mutations by an intact copy of the metR gene
TABLE 3. P1 transduction results showing linkage of the mutations to the ilv locus Donor
1-Galactosidase Lysogena
metH-lacZ 723XHlac 793XHlac 794XHlac 795XHlac 796XHlac
723XHlac(pGSmetR) 793XHlac(pGSmetR) 794XHlac(pGSmetR) 795XHlac(pGSmetR) 796XHlac(pGSmetR) metE-lacZ 723XElac 793XElac 794XElac 795XElac 7%XElac
723XRElac 793XRElac 794XRElac 795XRElac 796XRElac metR-lacZ 723XRlac 793XRlac 794XRlac 795XRlac 796XRlac
metR
genotype
metR+ metRI metR2 metR3 metR4
metR+lmetR+ metRl/metR+ metR2/metR+ metR31metR+ metR41metR+
132 215 215 272 220
metR+lmetR+ metR+lmetRI metR+lmetR2 metR+lmetR3 metR+lmetR4
metR+ metRI metR2 metR3 metR4
6,995 724 568 316 1,295
With HC 78 408 385 586 303
ND ND ND ND ND
6,785 1,014 977
302 2,006
10,504 9,313 8,323 7,370 7,313
9,238 9,500 9,340
161 2,714 1,442 2,400 2,092
108 2,000 1,200 2,230 1,330
8,252 7,807
a All lysogens were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine (HC), as indicated. b Units are Miller units (8). ND, Not determined.
stream of the translation stop codon and the same metE-lacZ
fusion as in AElac. These lysogens (723XRElac, 793XRElac, 794XRElac, 795XRElac, and 796ARElac) were grown in GM supplemented with either L-methionine or L-methionine plus homocysteine and then assayed for ,Bgalactosidase levels. The intact copy of the S. typhimurium metR gene carried on XRElac complemented the mutations, restoring metE-lacZ expression to approximately the levels seen in the parental strain (Table 2). A XHlac phage carrying a wild-type metR gene was not available for complementation experiments. Therefore, a single-copy plasmid (pGSmetR) carrying the S. typhimurium metR gene was used for metH complementation tests. Plasmid pGSmetR contains the TnS kan gene (1), the S. typhimurium metR gene, -80 base pairs downstream of the metR translation stop codon, the metE control region, and the first 283 codons of the metE structural gene. The mutant and parental XHlac lysogens were transformed (7) with Kmr plasmid pGSmetR, and the resulting transformants were grown in GM supplemented with kanamycin and L-methionine or with kanamycin, L-methiofline, and homocysteine. The cultures were then assayed for ,B-galactosidase activity. This plasmid partially complemented the mutations, lower-
gene
No. of colonies on:
Recipienta GM
GM + methionine
93 87
95 92
87
90
84b
87
activityb
Without HC 110 444 444 601 401
metR+ metRI metR2 metR3 metR4
2841
GS793 (metRI) GS794 (metR2) GS795 (metR3) GS79S (metR4)
GS792 (ilv) GS792 (ilv) GS792 (ilv) GS792 (ilv)
Ilv+
transductants were selected on GM supplemented with L-methionine; colonies were then scored on GM and GM supplemented with Lmethionine. b The three colonies scored as having a methionine requirement grew slowly after 12 h and were well established by 18 hr. a
ing metH-lacZ expression two- to threefold (Table 2). The failure to observe complete complementation with pGSmetR is probably due to underproduction of a wild-type MetR protein. In a related experiment, only partial activation of the metH gene was seen in a metR deletion strain transformed with pGSmetR, presumably because of insufficient production of the MetR protein (unpublished results). Pl transduction experiments. In previous P1 transduction experiments, the metR gene was found to cotransduce with the ilv locus at min 85 on the E. coli chromosome at a frequency of 39% (20). In the transduction experiment reported, the P1 lysate was prepared from the ilv strain (GS38) with the metR strain as the recipient. Although the exact nature of the ilv mutation in strain GS38 is unknown, it is presumed to be a deletion because of its nonreverting phenotype. Thus the reported 39o value represents an abnormally high cotransduction frequency between ilv and metR. In reciprocal transduction experiments done later, when the P1 lysate was prepared from known metR point mutants, a metR-ilv cotransduction frequency of 2 to 3% was observed (unpublished results). We tested for linkage of the new mutations to ilv by using strain GS792 (metJ+ ilv), a derivative of strain GS38, as a recipient. Because strain GS792 has a functional MetJ repressor, synthesis of the metE gene product (homocysteine transmethylase) is much lower in this strain than in the original GS723 strain (metJ metB metC metF). We predicted that introduction of the new metR mutations, shown to cause a 5- to 22-fold drop in metE-lacZ expression (Table 2), would significantly lower the levels of the metE-encoded transmethylase in strain GS792, resulting in methionine auxotrophy. GS792 was transduced with P1 phage prepared from the metR+ strain, GS723, and each of the four mutant strains (GS793, GS794, GS795, and GS796), and Ilv+ transductants were selected on GM plates supplemented with L-methionine. Ilv+ colonies were scored on GM plates and on GM plates supplemented with L-methionine. When GS723 was sued as a donor, no Ilv+ transductants required methionine for growth. Approximately 2 to 5% of the Ilv+ transductants from each transduction with a metR mutant required methionine for growth (Table 3). It is worth noting that the GS796 colonies scored as having a methionine requirement grew slowly after 12 h and were well established by 18 h. This corresponds to the level of metE-lacZ expression in lysogen 796AElac, which is the highest of the four mutant strains (Table 2). To demonstrate that the Met- phenotype is due to a mutation in the metR gene, one Met- transformant isolated with GS793 (metRI) as a donor was transformed with the single-copy plasmid pGSmetR and retested on GM plates and GM plates supplemented with L-methionine. The intact
2842
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BYERLY ET AL.
copy of the metR gene in pGSmetR complemented the Mettransductant, resulting in a Met+ phenotype.
Since the
new
mutations
were
TABLE 4. Effects of homocysteine on metH-lacZ and metE-lacZ expression in parental and mutant strains
cotransducible with ilv at a
frequency comparable to known metR mutants and were complemented by a wild-type metR gene (see above), we conclude they lie in the metR gene. The mutations in strains GS793, GS794, GS795, and GS796 were designated metRi, metR2, metR3, and metR4, respectively. Effect of homocysteine on metE and metH expression in the mutant strains. To confirm that the altered MetR proteins being synthesized were unable to respond to homocysteine, we needed to look at metE-lacZ and metH-lacZ expression in both a low- and a high-homocysteine environment. The high internal homocysteine levels in the GS723 background due to the metF mutation prevented analysis of expression of the lac fusions in a low-homocysteine environment. Therefore, we transduced a wild-type metF gene into the parental and mutant strains, so that homocysteine could be utilized and would no longer accumulate in the cell. The effects of homocysteine on metH-lacZ and metE-lacZ expression could then be determined by manipulating homocysteine levels in the cell through medium supplementation. P1 phage from GS719 (metB metJ) was used to transduce strains GS723, GS793, GS794, GS795, and GS796. metF+ transductants were selected on GM supplemented with homocysteine. Since metF and metB are closely linked on the chromosome, the transductants were then spotted on GM and GM supplemented with homocysteine to confirm that homocysteine was required for growth. The functional metF gene product in these strains allows exogenously added homocysteine to be converted to methionine by the metE gene product. These new strains were designated GS808 (parental), GS809 (metRI), GS810 (metR2), GS811 (metR3), and GS812 (metR4). These five strains were lysogenized separately with XHlac and XElac phage. The lysogens were grown in GM supplemented with D-methionine or with D-methionine plus homocysteine. D-Methionine was used instead of L-methionine because it limits the cell for methionine, resulting in lower levels of internal homocysteine produced via the regenerative pathway. The cultures were then assayed for 3-galactosidase activity. In the parental lysogen, 808XHlac, homocysteine cause a fourfold drop in metH-lacZ expression, whereas in the mutant XHlac lysogens there was no significant drop in metH-lacZ expression (Table 4). In the parental lysogen 808XElac, homocysteine caused a sixfold increase in metE-lacZ expression, whereas none of the mutants responded significantly. However, in lysogens of strain GS812 (metR4), homocysteine had a greater effect on both metHlacZ and metE-lacZ expression than in the other three mutants. This experiment supports our hypothesis that the metR mutations cause the production of a MetR protein with an altered response to homocysteine. DISCUSSION Activation of both the metE and metH genes is dependent on the metR gene product (20) and is regulated by intracellular homocysteine (19). For both genes, the MetR protein has been shown in vitro to bind specifically to a region of the promoter containing an 8-base-pair interrupted palindrome (2, 18). Formation of the MetR-DNA complex does not require homocysteine under these conditions. Although the mechanism of MetR activation and homocysteine involvement is not known, we have hypothesized that homocysteine modulates the expression of the target genes by causing a
metR
Lysogena Lysogen genotype gntp
13-Galactosidase activityb
Without HC
With HC
808XHlac 809XHlac 81OXHlac 811AHlac 812XHlac
metR+ metRI metR2 metR3 metR4
442 482 532 508 488
101 409 518 435 391
808XElac 809XElac 810XElac 811AElac 812AElac
metR+ metRI metR2 metR3 metR4
1,456 592 1,124 440 900
8,544 655 1,363 306 1,495
a All lysogens carry metJ metB mutations and therefore do not accumulate homocysteine. Cells were grown in GM supplemented with either D-methionine or D-methionine plus homocysteine (HC), as indicated. b Units are Miller units (8).
conformational change in MetR protein already bound at its target site (18). This altered conformation is proposed to inhibit the ability of MetR to activate metH expression while enhancing its ability to activate metE expression, possibly through direct contact with RNA polymerase. In this report we describe the isolation and characterization of a set of mutants in which the MetR protein fails to properly regulate metH-lacZ, metE-lacZ, and metR-lacZ gene fusions. Expression of the metE-lacZ fusion, which is normally activated in the presence of homocysteine, was significantly lowered in the mutant strains (Table 2), although the levels were still 100- to 400-fold above those seen with a metR deletion mutant (20). The mutant phenotype cannot be explained as simply due to a partially active MetR protein, since metH-lacZ expression was four- to sixfold higher in the mutants than in the parent lysogen (Table 2). This latter result suggests that the altered MetR proteins can still bind efficiently to operator DNA, at least in the metH promoter region. However, we cannot rule out the possibility that the mutations have altered the affinity of the MetR protein for its target DNA. If so, the mutations must cause an increased affinity for the metH promoter region and a decreased affinity for the metE promoter region. Alternatively, the phenotypes of the mutants may result from an inability of the altered MetR proteins to respond properly to the coactivator. In XHlac and AElac lysogens of strains GS809, GS810, GS811, and GS812, which have low internal levels of homocysteine, the addition of homocysteine to the growth medium had a diminished effect on metH-lacZ and metE-lacZ expression, as compared with that in lysogens of the parental strain, GS808 (Table 4). Furthermore, in lysogens 793XElac, 794XElac, and 796X Elac, in which intracellular homocysteine accumulates to high levels, exogenously added homocysteine caused a small but reproducible increase in activation of metE-lacZ expression (Table 2). In contrast, in the parent lysogen 723XElac, exogenously added homocysteine did not cause a further increase in metE-lacZ expression. If homocysteine modulates gene expression by binding directly to the MetR protein, mutations metRI, metR2, and metR4 may involve a change in the homocysteine binding site on the metR protein, resulting in a lowered affinity for homocysteine. A third possible explanation is that that mutant MetR proteins are locked into a conformation that activates metHlacZ expression while preventing activation of the metElacZ fusion.
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ALTERED HOMOCYSTEINE RESPONSE IN metR MUTANTS
Examples of activator mutants similar to the four metR mutants isolated here have been reported in the literature. Certain bacteriophage lambda cI repressor mutations greatly reduce the ability of the repressor to activate promoter PRM, without altering the affinity of the repressor for its specific operator DNA (5). The mutant phenotype presumably results from the loss of a favorable protein-protein contact between RNA polymerase and DNA-bound repressor. However, unlike the metR system, no coactivator is involved in the k system. In another case, mutations in the crp gene (cyclic AMP receptor protein) of E. coli have been shown to produce an altered Crp protein that no longer requires the presence of the effector molecule, cyclic AMP, to exert positive control over catabolite-sensitive operons (4). The altered apoprotein appears to be locked into a conformation resembling the wild-type cyclic AMP-CRP complex. The CRP system may not be entirely analogous to the MetR system, however, since cyclic AMP confers a positive effect on CRP function, whereas homocysteine not only positively regulates metE but also negatively regulates metH. We plan to clone the mutant metR genes to facilitate purification of the altered proteins. Using the purified proteins, we will attempt to (i) set up a homocysteine binding assay to assess the relative affinities of the mutant and wild-type MetR proteins for the coactivator and (ii) use an in vitro DNA binding assay to determine the DNA operator site affinities. These studies should help us to understand more specifically how homocysteine interacts with the MetR protein to regulate metE, metH, and metR gene expression. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant GM38912 from the National Institute of General Medical Sciences. 1.
2.
3.
4.
5.
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