Au In both Salmonella typhimurium and Escherichia coli the metJ gene codes for a protein that is involved in the regulation of the methionine pathway ( 5 , 13 , 17 ) .
This protein interacts with S-adenosylmethionine to form an active complex that presumably functions as a classical repressor ( 13 , 17 ) .
The metJ gene of S. typhimurium has been cloned and its nucleotide sequence has been determined ( 18 , 19 ) .
To facilitate studies on regulation of the metJ gene , we have fused the upstream control sequences for this gene to the E. coli lacZ gene .
Deletion derivatives of this fused gene have been constructed to test a model proposing that the metJ gene is transcribed from two distinct interacting promoters ( 19 ) .
MATERIALS AND METHODS Bacterial strains , phage , and plasmids .
Descriptions and origins of bacterial strains are given in Table 1 .
All bacterial strains are derivatives of E. coli K-12 .
Plasmid pGS107 was described previously ( 18 ) .
Plasmid pMC1403 ( 2 ) was from M. Casadaban .
Phage Xgt2 ( 10 ) was from R. Davis .
Other plasmids and phage were isolated during this investigation .
Luria broth and glucose minimal media have been described ( 16 ) .
Supplements were added at the following concentrations : phenylalanine , 50 , ug/ml ; vitamin-B1 , 1 pLg/ml ; D-methionine , 150 , ug/ml ; L-methionine , 50 , ug/ml ; ampicillin , 100 , ug/ml ; 5-bromo-4-chloro-3-indolyl-f3-D-galactoside ( X-gal ) , 40 , ug/ml .
The general procedures used for restriction enzyme cleavage , ligation , plasmid and phage DNA isolation , isolation of DNA fragments from polyacryl-amide gels , and transformation have been described ( 6 ) .
DNA sequence analysis was done by the method of Maxam and Gilbert ( 7 ) .
Construction of plasmids pBlac and pJlac .
A 480-base pair ( bp ) RsaI DNA fragment isolated from plasmid pGS107 that carries the promoters and the coding sequences for the amino-terminal ends of the S. typhimurium metB and metJ genes was ligated in both orientations into the SmaI site of plasmid pMC1403 ( Fig. 1 ) .
The RsaI cleavage sites occur between codons in the metB and metJ genes and therefore maintain the reading frame of the lacZ gene in plasmid pMC1403 .
The ligation products were then used to transform strain GS563 , the cells were plated on Luria broth-ampicillin plates containing X-gal , and blue colonies were isolated .
Plasmid DNA isolated from several blue transformants was analyzed by restriction enzyme digestion to determine the orientation of the 480-bp RsaI fragment .
Both metB-lacZ and metJ-IacZ fusion plasmids were isolated , and the DNA was sequenced to verify the fusion points ( not shown ) .
The resultant plasmids , designated pBlac and pJlac , code for two chimeric proteins .
The MetB-LacZ protein is a fusion of the first 31 amino-terminal amino-acids of the metB gene product to the eighth amino-acid of P-galactosidase .
The MetJ-LacZ protein is a fusion of the first 41 amino-terminal amino-acids of the metJ gene product to the eighth amino-acid of P-galactosidase .
Construction of plasmid pJlacA & Pjl .
The 490-bp EcoRI-BamHI DNA fragment from plasmid pJlac carrying the metB and metJ control regions was isolated and digested completely with restriction enzyme AhaII and digested partially with restriction enzyme HinPI .
The appropriate fragments were isolated from a polyacrylamide gel and religated , deleting the internal AhaII-HinPI fragment and resulting in a fusion of base -83 to +6 ( Fig. 2 ) .
This fused fragment was then religated into its original plasmid vector pMC1403 and used to transform strain GS563 .
The deletion endpoints were verified by DNA sequencing ( not shown ) .
Construction of plasmid pJlacAPJ2 .
The method of construction of pJlacAPJ2 was similar to that of pJlacAPjl except that an internal Sau96I DNA fragment was deleted , resulting in a fusion of base +36 to +72 ( Fig. 2 ) .
Construction of plasmid pBlacAPj2 .
Construction of pBlacAPJ2 deleted the same internal Sau96I fragment as was deleted in pJlacAPJ2 , but the parent plasmid used was pBlac and thus yielded a metB-lacZ fusion containing a deletion of the metJ promoter PJ2 .
Construction of the lacZ fusion phage .
Plasmids constructed above were cleaved at the unique Sall site in the pMC1403 vector , and the protruding ends were filled in with deoxynucleotide triphosphates and the Klenow fragment of E. coli DNA polymerase I. EcoRI linkers were ligated to the blunted ends , and the plasmids were digested with EcoRI .
The resultant DNA fragments were run on a low-melting-temperature agarose gel , and the 6,700-bp fragment that carries the lacZ fusion and the lac Y gene was isolated .
This 6,700-bp fragment was then ligated with EcoRI-cleaved Xgt2 phage DNA , and the mixture was packaged by using a commercial lambda-packaging system according to the manufacturer 's instructions .
Packaging mixtures were used to infect strain GS245 , and the cells were plated in tryptone soft agar plus X-gal .
Blue plaques were picked and the phage were plaque purified .
The phage are designated according to the plasmid DNA used in their construction ; e.g. , XJlac was constructed with plasmid pJlac DNA ; XBlac , with plasmid pBlac DNA , etc. .
Lysogens were isolated by putting a drop of phage suspension on a soft-agar overlay of an appropriate host and picking phage-resistant colonies from the center of the zone of lysis .
Lysogens were then single colony purified on Luria broth-X-gal plates .
The Si nuclease mapping procedur of Weaver and Weissmann ( 21 ) was used with modification as previously described ( 19 ) .
Essentially , the EcoRI-BamHI fragment carrying the metB and metJ control regions from plasmid pJlacAPjl or pJlacAPJ2 was labeled at the BamHI termini with 32p , the strands were separated , and the strand complementary to metJ mRNA from each was hybridized to total cellular RNA isolated from either GS243 ( pJlacAPji ) or GS243 ( pJlacAPJ2 ) , respectively , grown in glucose minimal-medium plus D-methionine .
Hybridization mixtures were then treated with Si nuclease , and the Si nuclease-resistant DNA products were electrophoresed alongside a sequencing ladder of the original DNA strand .
P-galactosidase activity was assayed as described by Miller ( 8 ) , using the chloroform-sodium dodecyl sulfate lysis procedure .
All chemicals and enzymes used are commercially available .
The lambda-packaging system was from Amersham Corp. , Arlington Heights , Ill. .
RESULTS Autoregulation of the metJ gene .
To test if the metJ gene product is involved in the control of the metJ gene itself , the XJlac phage was used to lysogenize a metJ + strain ( GS243 ) and a metJ strain ( GS597 ) .
The lysogens were then grown under conditions of methionine limitation and methionine and the levels of P-galactosidase excess , were measured .
Expression of the fused metJ-lacZ gene is sensitive to repression by methionine in GS243 ( AJlac ) ( Table 2 ) .
In contrast , expression is elevated and nonrepressible by methionine in GS597 ( XJlac ) .
These results suggest that the metJ gene product functions as a control element in its own regulation .
To be certain that the observed differences in 1Bgalactosidase levels reflect gene expression mediated by the metJ gene product , we used XBlac lysogens as positive controls .
Expression of the metB-lacZ gene is sensitive to repression by methionine in GS243 ( XBlac ) , but expression is elevated and nonrepressible by methionine in GS597 ( XBlac ) ( Table 2 ) .
Activity of promoters PJl and PJ2 .
Previous studies suggested that transcription of the metJ gene in S. typhimurium originates from two distinct start sites approximately 65 bp apart ( 19 ) .
It was proposed that two promoters , PJl and PJ2 , exist for the metJ gene , although the possibility remained that the shorter transcript is a processed form of the longer transcript .
To test the capability of both PJl and PJ2 to function as promoters , deletions were constructed in plasmid pilac which inactivated either promoter PJl ( pJlacAPjl ) or PJ2 ( pJlacAPJ2 ) .
Figure 2 shows the extent of the deletions in relation to the metJ gene control elements .
The deletion in pJlacAPjl includes not only the proposed -10 and -35 regions of promoter Pjl , but also the sequence proposed as a possible operator site for the metB gene in E. coli .
The GS243 ( XJlacAP3 , ) PJ2 23 24 GS597 ( XJlacAPjl ) PJ2 21 23 GS243 ( XJlacAPJ2 ) PJi 7.5 33 GS597 ( kJlacAPJ2 ) PJ1 81 79 GS243 ( ABlac ) PB 205 2,368 GS597 ( ABlac ) PB 7,529 7,634 GS243 ( XBlacAPJ2 ) PB 201 1,833 a Units of specific activity are nanomoles of O-nitrophenol produced per minute per milligram of protein at 28 °C .
For comparison , a fully induced culture that contains the wild-type lac operon and grown on glucose has about 1,000 U of activity ( 8 ) .
Growth medium was glucose minimal supplemented with phenylalanine , vitamin-B1 , and either D-methionine ( methionine limitation ) or L-methionine ( methionine excess ) deletion in pJlacAPJ2 includes the proposed -10 region of PJ2 , leaving PJl and the proposed operator sequence intact .
S1-nuclease-mapping of the 5 ' termini of the transcripts produced from these deletion plasmids indicates that transcription initiates at the expected transcription start sites for PJl and PJ2 ( Fig. 3 ) .
This suggests that both PJl and PJ2 function as promoters in-vivo and that the shorter transcript previously observed ( 19 ) results from transcription initiation at PJ2 rather than RNA processing .
Furthermore , when the lambda derivatives of pJlacAPj1 and pJlacAPJ2 ( XJlacAPj1 and XJlacAPJ2 , respectively ) are used to lysogenize the lac deletion strain GS243 , both lysogens produce significant levels of P-galactosidase ( Table 2 ) .
To test whether promoters Pi , and PJ2 repond to methionine repression mediated by the metJ repressor protein , strains GS243 ( metJ + ) and GS597 ( metJ ) were each lysogenized with XJlacAPjl and XJlacAPJ2 , and the Igalactosidase levels were measured in the lysogens grown under conditions of methionine limitation and methionine excess .
P-Galactosidase activity in the metJ + lysogen GS243 ( XJlacAPJ2 ) is sensitive to repression by methionine , exhibiting about a 4-fold range of control , whereas in the metJ lysogen GS597 ( XJlacAPJ2 ) the enzyme levels are elevated an additional 2.5-fold and are non-repressible by methionine ( Table 2 ) .
These results suggest that promoter pjl is under metJ repressor control .
In contrast , GS243 ( XJlacAPj1 ) shows no difference in P-galactosidase levels between repressing and derepressing growth-conditions , and the levels are not further elevated in GS597 ( XJlacAPjj ) ( Table 2 ) .
We used XBlacAPJ2 phage as a control to verify that the operator site had not been removed when promoter PJ2 was deleted .
, B-Galactosidase activity in the metJ + lysogen GS243 ( XBlacAPJ2 ) is sensitive to repression by methionine , exhibiting a ninefold range of control ( Table 2 ) .
DISCUSSION We have constructed a fused gene consisting of the S. typhimurium metJ control region and the E. coli lacZ gene to study regulation of expression of the metJ gene .
A lambda phage carrying the S. typhimurium metJ-lacZ fusion ( XJlac ) was used to lysogenize either a metJ + or a metJ E. coli strain and the lysogens grown under conditions of either methionine limitation or methionine excess .
The P-galactosidase levels are responsive over a 1.8-fold range to methionine addition to the growth medium in the metJ + lysogen , but are unresponsive to methionine and elevated 3 to 4-fold in the metJ lysogen ( Table 2 ) .
These results suggest that the S. typhimurium metJ gene is autoregulatory in E. coli .
A fusion of the E. coli metJ control region and the lacZ gene was recently reported ( 14 ) .
In this system , the E. coli metJ gene appears to also be regulated by its own gene product , but repression was observed to be independent of methionine supplementation to the growth medium .
The cells in the experiments with the E. coli metJ-lacZ lysogens were Met ' and therefore not limited for methionine .
However , we routinely see about a 1.4-fold repression by methionine supplementation with S. typhimurium XJlac lysogens even when the host is Met ' and thus not limited for methionine ( data not shown ) .
We previously proposed that metJ mRNA is transcribed from two distinct promoters , Pi , and PJ2 , separated by about 65 bp ( 19 ) .
Construction of the two deletion derivatives of plasmid pJlac ( pJlacAPjl and pJlacAPJ2 ) allowed us to look at the two promoters separately .
When promoters PJl and PJ2 were each tested for the ability to initiate transcription , both were found to produce their respective mRNA transcripts ( Fig. 3 ) .
In addition , when the lambda phage carrying these deletion derivatives ( XJlacAPj1 and XJlacAPJ2 ) were used to lysogenize E. coli hosts , both derivatives produced , Bgalactosidase ( Table 2 ) .
Thus , both Pi , and PJ2 function as true promoters in-vivo .
Promoter PJl is regulated by both the metJ gene product and methionine supplementation to the growth medium .
When the metJ + lysogen GS243 ( XJlacAPJ2 ) , in which only promoter PJl functions , is grown under conditions of methi-onine limitation , P-galactosidase levels increase four-t fivefold above the levels found when the lysogen is grown under conditions of methionine excess ( Table 2 ) .
Furthermore , when the host metJ gene product is inactivated , as in the metJ lysogen GS597 ( XJlac4PJ2 ) , enzyme levels are increased another two-to threefold and are no longer repressible by methionine addition to the growth medium .
Thus , in these experiments a > 10-fold range of regulation is seen for PJl when comparing fully repressed and fully derepressed conditions .
This 10-fold range of regulation for PJl alone is significantly greater than the 3-to 4-fold range of regulation seen when Pjl and PJ2 act in combination in the parent AJlac , as discussed above .
Promoter PJ2 did not respond to either methionine supplementation to the growth medium or the status of the metJ gene of the host .
It is difficult to draw any conclusions from these results regarding the regulatory mechanism for PJ2 , since the deletion in XJlacAPj1 removes both PJl and the proposed operator sequence ( Fig. 2 ) .
Thus , several explanations of the inability of promoter PJ2 to respond to either methionine or the metJ slatus of the host are possible and indistinguishable at this time .
( i ) PJ2 may be normally regulated by methionine and the metJ repressor much like Pjl , but in XJlacAPj1 the operator site is deleted .
However , it seems unlikely that PJ2 operates in the same manner as P-J ( i.e. , repressed by methionine ) , since it must temper the 10-fold range of activity of XJlacAPJ2 to the 3-to 4-fold range seen in the parent AJlac .
( ii ) pJ2 may be a constitutively expressed promoter .
However , the relative proportion of metJ mRNA initiating at PJ2 versus PJl has been shown to vary under conditions of methionine limitation versus methionine excess in the growth medium ( 19 ) .
( iii ) Transcription initiation at PT2 could be dependent on the amount of transcription initiating at PJl , which has been shown here to respond to methionine and the metJ gene product .
In support of this hypothesis , we have shown previously that the RNA polymerase binding site for PJl , determined by DNase I `` footprinting , '' overlaps not only the proposed operator sequence , but also the expected RNA polymerase binding site for PJ2 ( 19 ) .
Thus , transcription initiation at PJ2 may be inhibited by the binding of RNA polymerase at PJl .
Growth conditions which lead to formation of the operator-repressor complex would inhibit RNA polymerase binding at PJl and thus stimulate transcription from PJ2 .
We are attempting to construct mutations in promoter PJl which would leave the proposed operator sequence and its spacing from PJ2 intact to clarify the regulation of PJ2 * It is interesting that the sum of the,-galactosidase levels given in Table 2 for promoters PJi and PJ2 , when measured individually for a given host and growth-condition , do not add up to the level seen in the parent AJlac for that same host and growth-condition .
In fact , the levels seen with XJlac are 2.5-to 3-fold higher than the sum of the deletion derivatives .
The difference is probably due multiple copies of not to XJlac , since the bacterial strains have been lysogenized with the three phage in eight separate experiments , and the results are identical .
It would be very unlikely to consistently obtain multiple lysogens with the XJlac phage and yet not obtain multiple lysogens with XJlacAPJ1 and XJlacAPJ2 .
It is also unlikely that two or more copies of the metJ-lacZ fused gene have been inserted into the Xgt2 chromosome during the construction of XJlac .
Such a recombinant chromosome would have a size of 55 kbp and would show a greatly reduced plaque size ( 22 ) .
In addition , the tgalactosidase seen for the totally derepressed lysogen GS597 ( AJlac ) is very similar to the level reported for the E. coli metJ-lacZ fusion under similar conditions ( 14 ) .
It is possible that the regions deleted in XJlacAPj1 and XJlacAPJ2 are necessary for the most efficient expression of the metJ gene .
A number of regulatory genes have been shown to be autoregulatory .
Three of these for which the transcription initiation sites are known , trpR ( 4 ) , lexA ( 1 ) , and araC ( 20 ) , do not use a tandem promoter system for transcription like the S. typhimurium metJ gene reported here .
Tandem promoters , however , have been reported for genes coding for enzymatic proteins , e.g. , glnA ( 12 ) , carA ( 11 ) , and gal ( 9 ) .
Whether the tandem promoters for the metJ system are unique among regulatory genes remains to be seen .
We previously reported that in S. typhimurium the metJ gene appears to be more efficiently transcribed than the metB gene ( 19 ) .
However , 3-galactosidase levels produced in XBlac lysogens are always higher than the levels produced in AJlac lysogens under identical growth-conditions ( Table 2 ) .
It appears that metB mRNA is more efficiently translated than metJ mRNA .
This evidence suggests that translational efficiency plays an important role in maintaining the level of the metJ gene product .
The trpR gene was also found to have a moderately active promoter but a poorly translated transcript ( 4 ) .
In-addition , the response of the metB gene to the metJ repressor covers a range of 37-fold , whereas the response of the metJ gene to the metJ repressor covers a range of only 3-fold ( Table 2 ) .
Since the proposed operator region for the metJ gene is shared with the metB gene , the repressor must exert more stringent-control over the metB promoter than over the met .
Recently , in the E. coli metJ-metLB system , purified metJ repressor protein was shown to bind to the proposed operator region shared by these genes ( 15 ) .
The footprint pattern was interpreted as an asymmetric binding of repressor to the operator , with repressor preferentially binding to the meiB side of the operator .
Such an asymmetric interaction could explain the unequal response of the metJ and metB promoters to repression by the metJ gene product .
ACKNOWLEDGMENT This investigation was supported by Public Health Service grant GM26878 from the National Institute of General Medical Sciences .
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