A New Methionine Locus , metR , That Encodes a trans-Acting Protein Required for Activation of metE and metH in Escherichia coli and Salmonella typhimurium We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase , the metF gene product .
Transduction analysis indicates that ( i ) the mutant carries normal metE , metH , and metF genes ; ( ii ) the phenotype is due to a single mutation , eliminating the possibility that the strain is a metE metH double mutant ; and ( iii ) the new mutation is linked to the metE gene by P1 transduction .
Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation , even when the plasmidborne metE gene is inactivated .
Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression , a moderate decrease in metH gene expression , and a disruption of the metH-mediated vitamin-B12 repression of the metE and metF genes .
Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin-B12-independent ( metE ) and vitamin-B12-dependent ( metH ) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine .
We propose that this mutation defines a positive regulatory gene , designated metR , whose product acts in trans to activate the metE and metH genes .
The methylation of homocysteine to form methionine can be carried out by either of two transmethylases in Salmo-nella typhimurium and Escherichia coli ( for a review , see reference 15 ) .
The first is a vitamin-B12-independent enzyme , the product of the metE gene ; the second is a vitamin-B12-dependent enzyme , the product of the metH gene .
The methyl donor for both enzymes is 5-methyltetrahydrofolate , produced by the metF gene product at a point of convergence of two major pathways , the methionine biosynthetic pathway and the C1 pathway ( Fig. 1 ) .
The cell regulates the flow of C1 units through this convergence point on several levels to balance the requirements for protein synthesis , methylation reactions , and nucleic acid synthesis .
The genes in the nonfolate branch of the methionine pathway ( metA , metB , metC , and metK ) and those in the folate branch of the pathway ( metF , metE , and , to a small extent , metH ) are all negatively controlled by the metJ repressor system .
In addition , the metH gene product is involved in repression of the metE and metF genes when the cells are grown in medium containing vitamin-B12 .
We report here the finding of a third regulatory mechanism at the methionine-C , convergence point , namely , the positive activation of the and metE metH genes .
MATERIALS AND METHODS Bacterial strains , plasmids , and bacteriophages .
All bacterial strains used are derivatives of E. coli K-12 and are described in Table 1 .
Plasmids pGS47 and pGS69 , and their metE : : Tn5 derivatives have been described previously ( 16 ) .
Plasmid pMC1403 ( 4 ) was from M. Casadaban .
Bacteriophage Xgt2 ( 13 ) was from R. Davis .
Plasmid pBR322 has been described previously ( 9 ) .
Plasmid pGS191 , the lacZ fusion plasmids , and lacZ fusion phage were isolated during this investigation .
Luria broth ( LB ) , Luria agar ( L-agar ) and glucose minimal ( GM ) medium have been described previously ( 19 ) .
Supplements were added at the following concentrations : amino-acids , 50 jig/ml , except for D-methionine ( 150 , ug/ml ) ; vitamins B1 and B12 , 1 , ug/ml ; ampicillin , 100 , ug/ml ; tetracycline , 10 , ug/ml ; kanamycin , 25 , ug/ml .
Transductions were performed with the P1 cml clr-100 phage as described ( 10 ) .
Mutant isolation and classification .
Strain GS162 was mutagenized with phage Mu cts ( 3 ) and penicillin counterselected in GM medium supplemented with phenylalanine , vitamin-B1 , and penicillin G ( 20,000 U/ml ) .
Surviving cells were plated on GM plates supplemented with phenylalanine , vitamin-B1 , and methionine and then scored for their ability to grow without the methionine supplement .
Methionine auxotrophs were single-colony purified on L-agar plates and again tested for the methionine requirement .
Methionine auxotrophs were tentatively classified as described previously ( 1 ) .
Cells were grown overnight in LB and washed twice in GM medium , and 0.1-ml samples were spread on GM plates supplemented with phenylalanine and vitamin-Bl .
Crystals of cystathionine , homocysteine , vitamin-B12 , and methionine were then placed on sectors of the plates .
Mutants that grew on cystathionine or homocysteine were classified as carrying metA or metB mutations , those that grew on homocysteine were classified as carrying metC mutations , those that grew on vitamin-B12 were classified as carrying metE mutations , and those that grew only on methionine were classified as carrying metF mutations .
One mutant tested grew well only with a methionine supplement , although slow-growth occurred with a vitamin-B12 supplement after 72 h of incubation .
This mutant , designated strain GS190 , was tentatively classified as a metF mutant .
To stabilize the mutation in strain GS190 , cells were grown in LB at 30 °C , and then 0.1-ml samples were spread on L-agar plates and incubated overnight at 42 °C ( 3 ) .
Tem-perature-resistant survivors were single-colony purified and tested for the methionine requirement .
One temperature-resistant survivor with a phenotype identical to that of strain GS190 was saved and designated as strain GS244 .
Cells were grown in the appropriate media and harvested in the mid-log phase of growth .
The vitamin-B12-dependent homocysteine transmethylase was assayed as previously described ( 21 ) .
The 5,10-methylenetetrahydrofo-late reductase was assayed as described by Kutzbach and Stokstad ( 7 ) .
Protein determinations were made by the method of Lowry et al. ( 8 ) .
, - Galactosidase levels were measured as described by Miller ( 10 ) by using the chloro-form-sodium dodecyl sulfate lysis procedure .
Construction of the met-lacZ fusion phages .
The ABlac fusion phage was described previously ( 20 ) .
The construction of the AElac , XFlac , and XHlac fusion phages was similar to that of ABlac and will be described in detail elsewhere ( manuscript in preparation ) .
Briefly , a DNA fragment encoding the amino terminus of the respective S. typhimurium methionine structural gene plus the upstream transcriptional and translational control regions was fused to amino-acid codon 8 of the lacZ gene in the plasmid fusion vector pMC1403 .
These constructs encode chimeric proteins , each consisting of an enzymatically active pgalactosidase moiety having an amino terminus derived from the respective met structural gene .
The fused gene systems , located on approximately 7-kilobase-pair ( kbp ) EcoRI-Sall fragments on the fusion plasmids , were ligated into phage Xgt2 by using the methods described previously for the XBlac phage ( 20 ) .
The resulting hybrid phages were used to lysogenize various strains in this study .
In phage XElac , the metE-lacZ fusion occurred at codon 22 of the metE gene and included approximately 280 base pairs ( bp ) of upstream DNA .
In phage XFlac , the metF-lacZ fusion occurred at codon 13 of the metF gene and included approximately 600 bp of upstream DNA .
The precise point of translation initiation has not yet been determined for the metH gene , and so , in the XHlac phage , the codon numbering has not been assigned in the open reading frame in which the metH-lacZ fusion occurs .
The XHlac phage includes at least 400 bp of upstream DNA .
Construction of the X lysogens .
Appropriate strains were lysogenized with XHlac , AElac , XFlac , and ABlac fusion phages by the procedure described previously ( 20 ) .
After purification , lysogens were tested for the presence of only a single copy of the A phage by comparing the relative levels of,-galactosidase enzyme produced in several isolates and by infecting the isolates with X c190 c17 phage ( 17 ) .
Construction of plasmid pGS191 .
The general procedures for plasmid DNA isolation , cloning , restriction enzyme cleavage , and transformation have been described previously ( 9 ) .
Plasmid pGS47 , which contains the S. typhi-murium chromosomal fragment carrying the metE and metR genes , was digested with restriction endonucleases HindIII and Sall ( Fig. 2 ) .
The digestion products were run on a 1 % low-melting-temperature agarose gel , and the approximately 2-kbp fragment deduced to carry the metR gene was isolated .
This fragment was then ligated into the HindIlI and Sall sites of plasmid pBR322 .
The ligation mixture was used to transform strain GS244 , and the cells were plated onto GM plates containing phenylalanine , vitamin-B1 , and ampicillin and incubated at 37 °C .
Several Met ' transform-ants were single-colony purified , and plasmid DNA was prepared .
The resulting plasmids were checked by restriction enzyme analysis ( data not shown ) to confirm the presence of the 2-kbp HindIII-SalI fragment .
One of these plasmids was again used to transform strain GS244 to methionine prototrophy on GM plates containing phenylal-anine , vitamin-B1 , and ampicillin to confirm the presence of the metR gene .
The plasmid designated pGS191 .
was RESULTS Transduction analysis .
During a routine screening procedure for methionine auxotrophs , we isolated one mutant with an unusual phenotype .
This mutant , strain GS244 , grew well only with a methionine supplement , although slow-growth occurred over 72 h with a vitamin-B12 supplement .
This phenotype is characteristic of either a metF mutant or a metE metH double mutant ( 1 ) .
Thus , an initial series of transductions was carried out to determine the status of the metE , metF , and metH genes in strain GS244 .
P1 cml clr-100 phage was grown on strains GS232 ( metF63 ) , GS470 ( metE70 ) , and GS472 ( metE70 metH ) and used in transduction with strain GS244 as the recipient .
Met + transductants were selected on GM plates supplemented with phenylalanine and vitamin-B1 with strains GS232 and GS470 as donors and on GM plates supplemented with phenylalanine , vitamin-B1 , and vitamin-B12 with strain GS472 as the donor .
Transductants and abortive transductants were observed in all three transductions , suggesting that the mutation in strain GS244 is not in metF , metE , or metH .
Since it is possible that the inability of strain GS244 to grow on either GM medium or GM medium plus vitamin-B12 is due to more than one mutation , transductants selected on the vitamin-B12-supplemented plates with GS472 as the donor were scored on GM plates supplemented with phenylalanine and vitamin-B1 .
All transductants selected on the vitamin-B12-supplemented plates could grow on GM plates without the vitamin-B12 supplement ( 50 of 50 scored ) , suggesting that a single mutation is responsible for the phenotype of strain GS244 .
We have designated this mutation as metR .
Reciprocal transductions were also performed with P1 cml clr-100 phage grown on strain GS244 and with strains GS232 ( metF63 ) , GS470 ( metE70 ) , and GS472 ( metE70 metH ) as recipients .
Met + transductants were selected on GM plates supplemented with proline with GS232 as the recipient , GM plates with GS470 as the recipient , and GM plates supplemented with vitamin-B12 with GS472 as the recipient .
Transductants and abortive transductants were observed with all three recipients .
Since either metE + or metH + transductants can grow on GM plates supplemented with vitamin-B12 when strain GS472 was used as the recipient , the transductants were scored on GM plates without the vitamin-B12 supplement .
metH + transductants can be distinguished from metE + transductants since the former would still require methionine for growth in the absence of vitamin-B12 .
Over 90 % of the transductants ( 97 of 100 scored ) still required a methionine supplement , verifying that they were metH + transductants .
All of these results indicate the presence of functional metF , metE , and metH genes in strain GS244 .
Furthermore , when GS472 was used as the recipient , we GS470 ( metE70 ) 45 64 30 GS243 ( metE ) 24 29 17 GS244 ( metR ) 28 46 39 a Only the relevant markers are given .
The mutation in strain GS244 was designated as metR .
See Table 1 for complete genotypes .
In each case , the donor was strain GS38 ( ilv ) .
b Met + transductants were selected on GM plates supplemented with isoleucine , leucine , and valine and then spotted on the scoring media .
All plates were also supplemented with phenylalanine and vitamin-B1 , since strains GS243 and GS244 carry the pheA9OS and thi markers .
c Percent cotransduction refers to the percentage of the Met + transductants that received the ilv marker from the donor .
expected about 50 % of the transductants to grow on GM plates without vitamin-B12 , since the metE and metH genes are not linked by P1 transduction ( 2 ) .
One explanation for the low yield of metE + transductants is that the new mutation leading to methionine auxotrophy is closely linked to the metE gene by P1 transduction .
To further test the possibility that metR is linked to metE , we mapped the new mutation with respect to another marker , ilv , known to be cotransducible with metE ( 2 ) .
P1 cml clr-100 phage was grown on strain GS38 ( ilv ) and used to transduce strains GS470 , GS243 , and GS244 to methionine independence on GM plates supplemented with phenylalanine , vitamin-B1 , isoleucine , leucine , and valine .
Met + transductants were then scored on GM plates supplemented with phenylalanine and vitamin-B1 .
Of the Met + transductants with strain GS244 as the recipient , 39 % are ilv , suggesting that the metR mutation in GS244 is linked to ilv and could lie near the metE gene ( Table 2 ) .
Measurement of metF , metE , and metH gene expression .
As mentioned above , either a metF mutant or a metE metH double mutant has a nutritional requirement satisfied only by a methionine supplement .
Although the transduction analysis indicated that all three genes are intact , we tested the possibility that the mutation in strain GS244 somehow prevents the expression of these genes and is therefore responsible for the phenotype of strain GS244 .
We measured the expression of these three genes in strain GS244 and the parent strain GS162 grown under conditions that derepress ( D-methionine ) the metF , metE , and metH genes .
Since the substrate for the vitamin-B12-independent transmethylase was unavailable , we measured metE gene expression indirectly by using a metE-lacZ gene fusion .
In this system , a lambda phage carrying the metE-lacZ fusion ( XElac ) was used to lysogenize strains GS244 and GS162 .
The production of P-galactosidase activity in these lysogens is directed by the S. typhimurium metE gene control region .
Expression of the metF gene was actually higher in strain GS244 than in the parent strain GS162 ( Table 3 ) .
However , expression of both the metH gene and the metE-lacZ fusion was reduced in strain GS244 compared with the parent strain GS162 .
These results suggest that the new locus defined by the metR mutation is involved with activation in trans of the metE and metH genes and that the methionine auxotrophy of strain GS244 is a result of insufficient production of both transmethylase enzymes .
Complementation in trans of the GS244 mutation .
Since the transduction analysis suggested that the metR mutation in GS244 lies near the metE gene , it was possible that one o a The growth media contained phenylalanine , vitamin-B1 , and D-methionine ( vitamin-B12 was added to cultures for the metH assay ) .
Units of specific activities are , for reductase , nanomoles of HCHO formed per minute per milligram of protein at 30 °C ; for B12-transmethylase , nanomoles of methionine formed per minute per milligram of protein at 37 °C ; and for P-galactosidase , nanomoles of O-nitrophenol formed per minute per milligram of protein at 28 °C .
b Substrate for the vitamin-B12-independent transmethylase ( metE ) was unavailable .
Thus , metE gene expression was determined by measuring l-galactosidase enzyme levels in XElac lysogens of GS162 and GS244 carrying the metE-lacZ gene fusion .
more of the plasmids previously constructed carrying the S. typhimurium metE gene ( 16 ) also carries the metR gene .
To test this possibility , we transformed GS244 with four metE plasmids .
Two of these plasmids , pGS47 and pGS69 , carry a functional S. typhimurium metE gene .
Plasmids pGS47metE : : TnS and pGS69metE : : TnS are derivatives of pGS47 and pGS69 , respectively , in which the metE gene on the plasmid has been inactivated by insertion of the transposable element TnS .
Transformants were selected on L-agar plates containing the appropriate antibiotics and were then scored on GM plates supplemented with phenylalanine and vitamin-B1 .
The metE strain GS243 was also transformed with these plasmids as a control .
Both of the metE + plasmids , pGS47 and pGS69 , complement the metR mutation in strain GS244 ( Table 4 ) .
In addition , one metE plasmid , pGS47metE : : TnS , also complements strain GS244 , supporting the transduction data indicating that the metE gene is not the site of the mutation in strain GS244 .
However , the other metE plasmid , pGS69metE : : TnS , fails to complement strain GS244 , suggesting that the metE gene on this plasmid is involved in the ability of the parent plasmid pGS69 to complement strain GS244 .
Therefore , we tested plasmids pGS47 and pGS69 for the presence of the transacting factor by measuring metE-lacZ gene expression .
When lysogen 244XElac was transformed with plasmid pGS69 , the P-galactosidase levels ( reflecting metE gene expression ) were 25-fold lower than when the same lysogen was transformed with plasmid pGS47 ( Table 5 ) .
Thus , plasmid pGS47 carries the metR gene necessary for the activation in trans of the metE-lacZ fusion .
We have recently shown that the metR gene encodes a polypeptide of Mr 31,000 ( L. Plamann and G. Stauffer , unpublished results ) .
Plasmid pGS69 does not carry the metR gene but probably complements the methionine auxotrophy of GS244 on the basis of the high copy number of the metE gene in the transformant .
In support of this view , high-copy plasmids which the metH also capable of complementcarry gene are ing GS244 when in the of vitamin-B12 ( data grown presence not shown ) .
As expected , plasmid pGS47metE : : TnS is able to activate the expression of the metE-lacZ fusion , indicating that this plasmid carries a functional copy of the metR gene .
In addition , since the expression of the is metH gene reduced in GS244 ( Table 3 ) , we tested whether the metR aThe status of the metE gene on the plasmid was determined by complementation tests with known metE mutants ( 16 ) .
b Test plates were GM supplemented with phenylalanine , vitamin-B1 , and , when indicated , vitamin-B12 or L-methionine .
Symbols : - , no growth after 72 hr ; + , slight growth after 72 h ; + + + , good growth after 24 h. Incubation was at 37 °C .
gene carried on the plasmids could increase expression of the metH gene .
We used a metH-lacZ fusion system carried on a lambda phage ( XHlac ) in which P-galactosidase production is directed by the S. typhimurium metH gene control region .
Strain GS244 was lysogenized with XHlac , and the lysogen was transformed with the same plasmids as above .
All plasmids except pGS69 activate the expression of the metH-lacZ fusion ( Table 5 ) .
Location of the trans-acting factor on pGS47 .
A comparison of the physical maps of plasmids pGS47 and pGS69 ( Fig. 2 ) ( 16 ) suggests that the region of DNA on pGS47 most likely to encode the trans-acting metR product includes an approximately 700-bp segment that is not present in pGS69 .
We therefore isolated a 2-kbp HindIII-SaIl DNA fragment from pGS47 containing the 700-bp segment and an inactive segment of the metE gene and ligated it into the HindIII-SalI sites of the plasmid vector pBR322 .
Transformation of GS244 with this ligation mixture resulted in Apr transform-ants that grew on GM plates supplemented with phenylalanine and vitamin-B1 without a methionine supplement .
One of these transformants was purified , and plasmid DNA was aUnits of specific activity are nanomoles of O-nitrophenol produced per minute per milligram or protein at 28 °C .
The growth medium was GM supplemented with phenylalanine and vitamin-B1 and with D-methionine for the 244XElac lysogens or with D-methionine plus vitamin-B12 for the 244XHlac lysogens isolated .
This new plasmid , designated pGS191 , was used to again transform GS244 to methionine independence to confirm that the metR gene is present on the cloned fragment ( Table 4 ) .
Since GS244 is an E. coli K-12 strain , the ability of the metR gene on plasmid pGS191 ( which was derived from S. typhimurium chromosomal DNA ) to complement the E. coli mutation indicates that both organisms have a similar activation system for the metE and metH genes .
Nature of regulation of methionine biosynthesis by metR .
The plasmid complementation tests described above suggest that the metR locus encodes a protein that is involved in the activation of the metE and metH genes .
As a first step in discerning the nature of this trans-activation by the metR gene product , the effects of the metR mutation in GS244 were analyzed by comparing the expression of several met genes in this strain with those in the Met + parent strain GS162 and the metE mutant strain GS243 .
These compari-sons were facilitated by lysogenizing these strains with AElac , XHlac , XFlac , and XBlac fusion phages ( see Materials and Methods ) , allowing 3-galactosidase levels to be assayed as an indication of expression of the respective met gene being examined .
The XBlac phage was used as a representative gene from the nonfolate branch of the methionine biosynthetic pathway ( Fig. 1 ) .
The results of these comparisons are shown in Table 6 .
The expression of the metE-1acZ , metH-lacZ , metF-lacZ , and metB-lacZ-fusions in response to methionine limitation ( D-methionine ) or methionine excess ( L-methionine ) in the GS162 lysogens ( Met + ) and GS243 lysogens ( metE ) generally followed the patterns reported previously for the respective met genes in E. coli ( 1 , 6 ) .
The addition of L-methionine to the GM growth media substantially repressed the expression of the metE , metF , and metB genes , especially when compared with the derepressed levels in the methionine auxotroph GS243 .
The addition of L-methionine marginally repressed the expression of the metH gene .
In addition , the metE and metF genes were repressed by the addition of vitamin-B12 to the GM media .
In contrast , regulation of the metH-lacZ , metF-lacZ , and , particularly , the metE-lacZ-fusions is altered in GS244 .
P-Galactosidase levels in lysogen 244XElac grown under methionine-limiting conditions was 200-fold lower than in lysogen 243XElac .
Since lysogens 244XFlac and 244XBlac both showed derepressed P-galactosidase levels under these growth-conditions , it is clear that GS244 is methionine limited in the D-methionine-supplemented GM media , and thus the low P-galactosidase levels seen in 244XElac and 244XHlac lysogens can not be due to repression by adequate internal pools of methionine .
The metR mutation in GS244 also disrupted the normal vitamin-B12-dependent repression of the metE-lacZ and metF-lacZ-fusions , although the effect was more apparent for the metF gene .
The vitamin-B12 repression seen in 243XFlac was nearly abolished in 244XFlac , and the Igalactosidase activity approached the level seen for 244XFlac grown under derepressing conditions ( Table 6 ) .
Interestingly , the release of the vitamin-B12-dependent repression in lysogens 244XElac and 244XFlac was paralleled by lower expression of the metH gene in 244XHlac under similar growth-conditions .
Since the metH gene product has been shown to be involved in the vitamin-B12-dependent repression of the metE and metF genes ( 6 , 11 , 22 ) , it is plausible that the decreased production of metH gene product in GS244 is responsible for the disruption of the vitamin-B12-dependent repression mechanism .
In contrast , it is clear that the addition of L-methionine to the growth medium 243XFlac 1,845 240-288-244 XFlac 1,034 97 800 162XBlac 227 95 NDb 243XBlac 2,940 194 ND 244XBlac 1,920 188 ND aUnits of specific activity are nanomoles of O-nitrophenol produced per minute per milligram of protein at 28 °C .
The growth medium was GM medium supplemented with phenylalanine , vitamin-B1 , and , when indicated , Dmethionine , L-methionine , or D-methionine plus vitamin-B12 .
b ND , Not done .
results in significant repression of all the met-lacZ-fusions tested , including the GS244 derivatives .
This suggests that the metJ-mediated repression system functions independently of metR and can override the stimulatory effect of metR .
DISCUSSION We have provided evidence for the existence of a new regulatory element , designated metR , that encodes a transacting protein required for expression of the metE and metH genes of E. coli and S. typhimurium .
Consistent with this interpretation , the metR mutation shares a number of characteristics with mutations in other positive activator genes ( 14 ) : ( i ) the mutation occurs outside the target genes ( metE and metH ) and thus acts in trans ; ( ii ) the mutation is recessive to the wild-type allele ; and ( iii ) the mutation affects the synthesis of gene products rather than their activity .
This last point would be crucial in the assignment of the metR gene product as an activator .
Since the two genes it controls ( metE and metH ) code for gene products having identical transmethylase functions , it is possible that the metR gene product functions as a subunit necessary for the formation of an active transmethylase complex .
However , our results show that the metR gene product increases the levels of activity of the unrelated enzyme P-galactosidase in metE-lacZ and metH-lacZ fusion strains ( Table 6 ) , demonstrating that it must function either directly or indirectly by increasing transcription or translation of the metE and metH genes .
Furthermore , the metE and metH genes in high copy can complement the metR mutant , suggesting that the metR gene product is not a necessary subunit for transmethylase activity .
Smith and Childs ( 18 ) previously reported that the metE locus in S. typhimurium could be divided into two complementation groups and suggested that the vitamin-B12-independent transmethylase might consist of two different polypeptide subunits .
The group I metE mutants were isolated seven times more frequently than were the group II mutants , suggesting a larger target site for the group I locus The metE gene of S. typhimurium encodes a polypeptide of approximate Mr 92,500 ( 16 ) , and the metR gene encodes a polypeptide of only Mr 31,000 ( Plamann and Stauffer , unpublished results ) .
In addition , the group II mutants had an extended growth lag compared with group I mutants when the strains were grown in GM medium supplemented with vitamin-B12 .
On the basis of our results , we predicated that group I nmutations inactivate the metE gene coding for the transmethylase , whereas group II mutations inactivate the metR gene coding for the activator .
We tested S. typhimurium strains carrying group I mutations ( metE205 , metE230 , and metE237 ) and strains carrying group II mutations ( metE197 , metE387 , and metE396 ) in complementation tests with the metE metR + plasmid pGS191 .
Consistent with the above interpretation , pGS191 complements group II but not group I mutations .
The level of P-galactosidase produced in 244XElac ( pGS191 ) is significantly higher than in 244XElac ( pGS47 ) ( Table 5 ) .
This elevation is probably not due simply to a higher copy of the metR gene in the pGS191 transformant , since a similar elevation was seen in 244XEiac ( pGS47 metE : : TnS ) .
It is unlikely that the TnS insertion into pGS47 would raise the production of the metR gene product the exact amount necessary to compensate for a difference in copy number between pGS47 and pGS191 .
A more likely explanation is that inactivation of the plasmid-borne metE gene is responsible for the higher P-galactosidase levels seen in the pGS191 and pGS47metE : : TnS transformants .
The metE gene product could act negatively in its own regulation in two ways .
First , it could function as an antagonist of the metR gene prQduct , either by interfering directly in the activation mechanism or by repressing metR gene expression .
Alternatively , activation probably depends not only on the presence of a functional metR gene product but also on a coactivator , e.g. , an intermediate in the methionine biosynthetic pathway .
Methionine intermediates have been implicated in the regulation of the metE gene in E. coli ( 5 ) and the metH and metF genes in S. typhimurium ( 22 ) .
It was concluded that cystathionine may function as an inducer for the metE gene and that O-succinylhomoserine may function as an inducer for the metH and metF genes .
In addition , in E. coli , a functional metF gene is required for vitamin-B12-mediated repression of the metF gene , ahd 5-methyltetrahy-drofolate may be involved in this repression ( 12 ) .
Inactivation of the plasmid-borne metE gene in 244XElac ( pGS191 ) and 244XElac ( pGS47 metE : : TnS ) could allow a greater accumulation of the methionine intermediates O-succinylhomo-serine , cystathionine , homocysteine , and 5-methyltetrahy-drofolate than would 244XElac ( pGS47 ) .
The pGS47 transformant has multiple copies of the metE gene and thus might drain off the intermediates more quickly , preventing their accumulation .
The level of p-galactosidase produced in 244XHlac ( pGS47 ) was not significantly lower than in 244XHlac ( pGS191 ) or 244XHlac ( pGS47 metE : : TnS ) ( Table 5 ) .
Thus , multiple cop-ies of the metE gene did not decrease expression from the metH gene , suggesting that different coactivators are required for expression of the metE and metH genes via the metR gene product .
We are currently examining the effects of combinations of mutations in other met genes to directly control the concentrations of methiohine intermediates , thereby allowing identification of the coactivator ( s ) .
A requirement for the metR gene product thus far is limited to the metE and metH genes .
We have tested whether the metR gene product affects the third gene in the folate branch of the pathway , metF , and a gene in the nonfolate branch , metB .
Only a moderate decrease in the expression of the metF-lacZ fusion or the metB-lacZ fusion in strain GS244 was seen compared with the decrease seen after the addition of L-methionine to the growth medium ( Table 6 ) .
The metE gene is activated to a much greater extent than is metH .
In E. coli cells grown under derepressing conditions , the metE gene product represents as much as 5 % of the total cellular protein ( 23 ) .
However , when cells were grown in media containing vitamin-B12 , synthesis of the non-B12 transmethylase ( metE gene product ) was repressed over 100-fold , and there was a more moderate repression of the metF gene product ( Table 6 ) .
It was suggested that the vitamin-B12-dependent transmethylase is a more efficient enzyme and thus the cell finds it more economical to use the B12-dependent pathway when vitamin-B12 is available ( 5 ) .
It is possible that the availability of vitamin-B12 to the organism is fairly reliable in its natural habitat , so that the B12-dependent ( metH ) pathway is normally used and induction of the synthesis of the less efficient metE gene product is necessary only when vitamin-B12 is unavailable .
This hypo-thesis predicts that the metH gene would be partially constitutive and would require only a low level of activation by metR , whereas the metE gene would require a high level of activation to obtain sufficient expression .
It is interesting in this respect that GS244 reverts much more frequently when grown on vitamih B12-containing medium than on non-B12-containing medium ( unpublished results ) .
Although we have not yet characterized these revertants , it appears that the metH gene can overcome its dependence on metR activation much more easily than can metE .
ACKN ) WLEDGMENTS This investigation was supported by Public Health Service grant GM26878 from the National Institute of General Medical Sciences .
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