169 , No. 1 Mutations Affecting the Regulation of the metB Gene of Salmonella typhimurium LT2 MARK L. URBANOWSKI , LYNDA S. PLAMANN , AND GEORGE V. STAUFFER * Department of Microbiology , University of Iowa , Iowa City , Iowa 52242 We isolated and characterized cis-acting mutations that affect the regulation of the metB gene of Salmonella typhimurium LT2 .
The mutations were isolated in an Escherichia coli lac deletion strain lysogenized with A bacteriophage carrying a metB-lacZ gene fusion ( AJBlac ) in which , l-galactosidase production is dependent upon metB gene expression .
The mutant lysogens show elevated , poorly regulated,3-galactosidase production .
The altered regulation is a result of disruption of the methionine control system mediated by the metJ repressor .
The mutations are located in a region of dyad symmetry centered near the -35 sequence of the metB promoter .
We propose that these mutations alter the repressor binding site and define the metB operator sequence .
In addition , we discuss a highly conserved , nonsymmetric DNA sequence of unknown function which occurs in the control regions of the metA , meiC , metE , metF , metG , and metjB genes of both S. typhimurium and E. coli .
The methionine biosynthesis genes in Salmonella typhimurium and Escherichia coli are scattered around the chromosome ( 1 , 19 ) .
The cell coordinates the expression of these genes for the efficient synthesis of methionine by means of a negative control system ( for a review , see reference 17 ) .
The current model for methionine regulation suggests that the met repressor ( encoded by the metJ gene ) binds to the control regions of the met genes and negatively regulates the amount of transcription from these genes .
The extent to which each met gene is repressed is thought to result from the different affinity of the repressor for each individual operator , the position of the operator with respect to the promoter , or a combination of the two .
Attempts have been made to discern the sites of action of the met repressor by analyzing and comparing DNA sequences of various E. coli met genes ( 3 , 13 ) and by studying in-vitro repressor DNA binding ( 21 ) .
In these investigations , sequences were found that possess dyad symmetry and show homology among the met genes , suggesting that the sequences could be repressor binding sites .
We report here the isolation and characterization of cis-acting mutations that genetically identify the operator site for the S. typhimurium metB gene .
MATERIALS AND METHODS Bacterial strains , plasmids , and bacteriophages .
JL781 ( wild type ) and JB672 ( metJ ) are S. typhimurium LT2 strains .
GS243 ( metE AlacU169 pheA905 araD129 rpsL thi ) and GS597 ( metJ97 AlacU169 pheA905 araD129 rpsL thi ) are E. coli K-12 strains .
Plasmids pGS107 ( 23 ) and pBR322 ( 10 ) were described previously .
Plasmid pMC1403 ( 5 ) was from M. Casadaban .
Phage Xgt2 ( 15 ) was from R. Davis .
Phage XcI90 c17 ( 20 ) was from M. Feiss .
Other plasmids and phages were isolated during this investigation .
L agar and glucose minimal ( GM ) medium have been described previously ( 22 ) .
Lactose minimal ( LM ) me-dium is identical to GM medium except that glucose is replaced by 0.4 % lactose .
Supplements were added at the following concentrations ( micrograms per milliliter ) : phenyl-alanine , 50 ; vitamin-B1 , 1 ; D-methionine , 150 ; L-methionine , 50 ; ampicillin , 100 ; 5-bromo-4-chloro-3-indoyl-p-D-galacto-side ( X-gal ) , 40 .
The general procedures used for restriction enzyme cleavage , ligation , plasmid isolation , and isolation of DNA fragments from polyacrylamide and agarose gels have been described before ( 10 ) .
Phage DNA was isolated by using a modification ( 27 ) of the rapid method of Benson and Taylor ( 4 ) .
DNA sequence analysis was done by the method of Maxam and Gilbert ( 12 ) .
Construction of X.JBlac phage .
The XJBlac phage construction was similar to that previously reported for the ABlac phage ( 25 ) , except that XJBlac carries a functional metJ gene .
Plasmid pGS107 , which carries the S. typhimurium metB and metJ genes , was partially digested with restriction enzyme RsaI , and a 732-base-pair ( bp ) fragment was isolated .
This fragment contains a functional metJ gene and a truncated metB gene .
The RsaI cleavage site within the metB gene occurs between codons 31 and 32 .
The fragment was ligated into the SmaI site of the lacZYA fusion vector pMC1403 , and the ligation mixture was used to transform the lac deletion strain GS243 .
Transformants were plated onto L agar plates containing ampicillin and X-gal , and blue colo-nies were isolated .
Plasmid DNA isolated from several blue transformants was analyzed by restriction enzyme digestion to determine the orientation of the 732-bp fragment .
One plasmid was chosen in which the truncated metB gene was fused to the lacZ gene in pMC1403 .
To construct the XJBlac phage from this plasmid , the metJ + gene , the metB-lacZ fusion , and the lacY and lacA genes carried on this plasmid were cloned into the single EcoRI site in phage Xgt2 by the method described previously ( 25 ) .
Selection of AJBlac mutant phage .
Strain GS243 was lysogenized with the XJBlac phage by putting 1 drop of the phage suspension onto a soft agar overlay of GS243 and incubating the plate at 30 °C overnight .
Phage-resistant cells were picked from the zone of lysis , and the presumed lysogens were single colony purified on L agar plates containing X-gal .
One lysogen , designated 243AJBlac , was again streaked for isolated colonies on an L agar plate containing X-gal .
Individual colonies were grown overnight in GM medium supplemented with phenylalanine , vitamin-B1 , and methionine .
Cells were then washed , centrifuged , and concentrated fivefold .
A 0.1-ml sample ( 2 x 109 cells ) of each culture was plated onto LM plates supplemented with phen-ylalanine , vitamin-B1 , and methionine and incubated at 300C .
Two colonies from each plate which arose within 24 h ( colonies numbered between 50 to 200 per plate ) were streaked for purity onto L agar plates containing X-gal and again tested for the Lac ' phenotype with LM plates supplemented with phenylalanine , vitamin-B1 , and methionine .
A phage stock was prepared from each mutant by temperature induction , and these phage were again used to lysogenize GS243 .
The new lysogens were single colony purified and then scored for the Lac ' phenotype as described above .
Lac ' lysogens were assumed to have a phage-associated mutation and were retained for further study .
Phage DNA was prepared from each Lac ' lysogen , and the metJB control region DNA was sequenced to locate the mutation .
Construction of XBlac mutant phage .
Phage DNA from selected XJBlac mutants was digested with restriction enzyme EcoRI , and from each mutant a 6,900-bp fragment that carries the metB-lacZ fusion , the metJ gene , and the lacY and lacA genes was isolated from low-melting-temperature agarose gels and ligated into the EcoRI site of plasmid pBR322 .
Each recombinant plasmid was then digested with restriction enzymes BamHI and RsaI , and a 483-bp fragment that carries the metB control region and a truncated metJ gene was isolated ( Fig. 1 ) .
This fragment was then ligated into the pMC1403 vector at the BamHI and SmaI sites .
The orientation of the fragment was confirmed by restriction enzyme analysis .
Each plasmid was then digested with EcoRI , and a 6,700-bp EcoRI fragment that carries the mutant metB-lacZ fusion ( now met !
) was ligated into Xgt2 as described before ( 25 ) .
A purified stock of each XBlac mutant phage was prepared and used to lysogenize GS243 and GS597 as described above .
, - Galactosidase activity was assayed as described by Miller ( 14 ) , using the chloroformsodium dodecyl sulfate lysis procedure .
All chemicals and enzymes used are commercially available .
The lambda packaging system was from Amersham Corp. ( Arlington Heights , Ill. ) .
Essential elements of the XJBlac phage used in the mutant selection .
The phage carries a functional metJ + gene , a metB-lacZ fusion and the lacY and lacA genes .
Details are given in the text .
Horizontal arrows indicate the start site and direction of transcription of the metB promoter , PB , and the three metJ promoters , Pjo , PJi , and PJ2 ( 24 ; also see the text ) .
The upper expanded part of the figure shows the nucleotide sequence of the region that contains the metB operator site for the metJ repressor .
Base changes for each mutant are indicated below the sequence .
The proposed -35 region for promoters PB and PJo is shown in bold letters .
The dyad symmetry of the proposed metB operator , which is disrupted in mutations Blacl , Blacl2 , Blac25 , and Blac5 , is shown as a broken line between the two strands .
Bases that are homologous to the 8-bp repeat unit of Belfaiza et al. ( 3 ) are indicated by dots above the sequence .
The two heavy arrows indicate the BamHI-RsaI fragment used to construct the ABlac derivatives of the mutant phage .
RESULTS Selection of mutations in the metJB control region .
Our primary interest in this study was to genetically define the metJ repressor binding site within the metJB shared control region .
To select cis-acting up mutations in this region , we used a metB-lacZ fusion phage ( XJBlac ) .
This phage encodes a MetB-LacZ fusion protein identical to that encoded by the XBlac phage reported earlier ( 25 ) , and also encodes a functional metJ repressor protein ( Fig. 1 ) .
Thus , in the 243XJBlac lysogen used in the mutant selection , there are two copies of the metJ gene , one at the normal chromosomal locus and the other at the site of the AJBlac insertion .
Although it is possible that hybrid E. coli-S .
typhimurium repressors will be formed , two experimental results indicate that such repressors should function normally .
( i ) The amino-acid sequences show .98 % homology .
( ii ) A single copy of either the E. coli or S. typhimurium metJ gene produces similar levels of repression of the metB-lacZ fusion ( unpublished data ) .
Strain 243XJBlac does not grow on LM plates supple mented with phenylalanine , vitamin-B1 , and methionine because of repression of the metB-lacZ fusion by the metJ repressor apd thus insufficient P-galactosidase production .
Mutations in this lysogen which lead to the ability to grow on the selection plates are likely to alter the metB operator or promoter site , rather than inactivate the repressor .
Although the operator is presumably a smaller target than the metJ gene , inactivation of both metJ genes would require two independent mutation events .
Another class of mutations that could lead to derepression of the metB-lacZ fusion are metK mutations .
The metK encodes Sgene adenosylmethionine synthetase , which is responsible for the synthesis of the corepressor S-adenosylmethionine ( 17 ) .
However , metK mutants normally grow very slowly even in GM media ( data not shown ) , and such mutants would not be evident on the selection plates within 24 h. With this Lac ' selection procedure , 19 out of 20 mutants isolated have mutations located near metB .
Locations of mutations in XJBlac mutant phage .
The location of the mutation in each of 13 Lac '' mutants was determined by DNA sequence analysis of the JBlac control region .
The base changes found are shown in Fig. 1 .
Three types of mutations were found : transitions , transversions , and one deletion ( not shown ) .
Five different base changes were identified , designated Blacl ( isolated seven times independently ) , Blac5 , Blacl2 , Blac24 , and Blac25 ( isolated three times independently ) .
All five changes are clustered in the region around the -35 sequence for the metB promoter ( PB ) .
In addition , this region overlaps the -35 sequence for the furthest upstream metJ promoter ( designated pJo ) .
Promoterpjo was recently discovered in the E. coli metJ gene ( 9 ) and subsequently found in S. typhimurium ( manuscript in preparation ) .
Analysis of the DNA sequence around the mutations revealed a region of dyad symmetry ( Fig. 1 ) that is disrupted by mutations Blacl , Blacl2 , Blac25 , and Blac5 .
Mutation Blac24 increases the symmetry beyond the sequence underlined , and also alters the -35 sequence of promoter PB , making this sequence a perfect match with the -35 consensus sequence for E. coli promoters ( 16 ) .
I-Galactosidase activity in wild-type AJBlac and mutant AJBlac lysogens .
, - Galactosidase levels were measured in GS243 lysogenized with wild-type XJBlac and AJBlac mutants grown under conditions which normally either repress ( L-methionine ) or derepress ( D-methionine ) the metB gene ( Table 1 ) .
The five mutants isolated all have P-galactosidase levels substantially higher than the level in the wild-type a Each lysogen used was shown to carry a single copy of the appropriate X phage by infection with A cI90 c17 ( 20 ) .
b Units of specific activity are nanomoles of 0-nitrophenol produced per minute per milligram of protein at 28 °C .
Growth medium GM medium was supplemented with phenylalanine , vitamin-B1 , and either D-methionine ( methionine limitation ) or L-methionine ( methionine excess ) .
a Each lysogen used was shown to carry a single copy of the appropriate X phage by infection with X cI90 c17 ( 20 ) .
bUnits of specific activity are nanomoles of 0-nitrophenol produced per minute per milligram of protein at 28 °C .
Growth medium was GM medium supplemented with phenylalanine , vitamin-B1 , and either D-methionine ( methionine limitation ) or L-methionine ( methionine excess ) .
lysogen , especially when grown with L-methionine .
These elevated values are sufficient to explain the Lac ' phenotype shown on the LM selection plates .
In addition , regulation of,-galactosidase synthesis is altered in the mutant lysogens .
A comparison of the derepressed and repressed levels in the wild-type XJBlac lysogen shows that regulation varies over a 38-fold range , whereas in the mutant lysogens , regulation varies over only a 1.6-to 10.7-fold range .
Evidence for metJ repressor involvement .
The altered regulation of,-galactosidase levels in the XJBlac mutants ( Table 1 ) and the disruption of a region of dyad symmetry in the coritrol regions of these mutants ( Fig. 1 ) suggest that the mutations have altered the metJ repressor binding site for the metB gene .
To show that the altered regulation was due to an altered metJ repressor binding site and not some other independent system , we measured the level and the regulation of 3-galactosidase production in cells in which the metJ repressor was inactivated .
To construct such a test system , it was nacessary to inactivate the metJ gene carried on each of the mutant XJBlac phage and lysogenize a metJ mutant with each reconstructed mutant phage .
For this reconstruction , only the wild-type AJBlac and mutant XJBlacl , XJBlacl2 , and XJBlac25 phage were used ( see above ) .
, - Galactosi4ase levels were measured in strain GS597 ( metJ ) lysogenized with wild-type ABlac phage and the XBlacl , XBlacl2 , and XBlac25 mutant phage .
As a control , 0-galactosidase levels were also measured in strain GS243 ( metJ + ) lysogenized with each phage ( Table 2 ) .
In the GS243 lysogens , the regulation of , B-galactosidase production follows the same pattern seen with the XJBlac lysogens except that the enzyme levels are somewhat higher and the range of regulation is not as great ( cf. Tables 1 and 2 ) .
This difference most likely reflects the presence of two copies of the metJ repressor gene in the XJBlac lysogens .
In contrast , the GS597 lysogens , which do not produce an active repressor , show elevated , nonrepressible levels of,-galactosidase , which are not significantly higher in the mutants when compared with levels in the wild type .
This result suggests that the altered regulation of P-galactosidase synthesis seen in 243ABlacl , 243B131ac12 , and 243ABlac25 is due to disruption of the metJ .
repressor system , rather than a mutation in some other independent system that increases metB expression .
Mutations in an independent metB regulatory system would have shown an additive effect in the GS597 lysogens 12 25 5 U a BamHI I II ` hR1 PJ2 PJo PJi EcoRI I bacZ metB 4-P PB If2 Rsal I Rsal sI 100 200 400 500 700 300 Basepairs FIG. 1 .
Essential elements of the XJBlac phage used in the mutant selection .
The phage carries a functional metJ + gene , a metB-lacZ fusion and the lacY and lacA genes .
Details are given in the text .
Horizontal arrows indicate the start site and direction of transcription of the metB promoter , PB , and the three metJ promoters , Pjo , PJi , and PJ2 ( 24 ; also see the text ) .
The upper expanded part of the figure shows the nucleotide sequence of the region that contains the metB operator site for the metJ repressor .
Base changes for each mutant are indicated below the sequence .
The proposed -35 region for promoters PB and PJo is shown in bold letters .
The dyad symmetry of the proposed metB operator , which is disrupted in mutations Blacl , Blacl2 , Blac25 , and Blac5 , is shown as a broken line between the two strands .
Bases that are homologous to the 8-bp repeat unit of Belfaiza et al. ( 3 ) are indicated by dots above the sequence .
The two heavy arrows indicate the BamHI-RsaI fragment used to construct the ABlac derivatives of the mutant phage .
Comparison of P-galactosidase levels in GS243 and GS597 lysogenized with XBlac wild type and ABlac mutants metJ , B-Galactosidase activityb D-Methionine/genot e L-methionine genotYPe D-Methionine L-Methionine ratio Lysogena 243XBlac 243ABlacl 243XBlac12 243ABlac25 + + + 3,101 4,494 1,893 4,123 221 1,500 14.0 3.0 1.7 5.5 1,127 751 + ¬ 597ABlac 597XBlacl 597XBlacl2 597XBlac25 4,794 5,553 1,928 5,470 4,510 5,276 1,796 4,771 1.0 1.0 1.1 1.1 ¬ ¬ ¬ TABLE 1 .
Comparison of,-galactosidase levels in GS243 lysogenized with XJBlac wild type and XJBlac mutants , B-Galactosidase activityb D-Methionine/L-methionine D-Methionine L-Methionine ratio Lysogena 243AJBlac 243XJBlacl 1,434 4,163 5,590 2,212 4,541 3,490 37 38.7 3.8 1.6 2.2 10.7 8.2 1,082 3,503 1,022 424 424 243XJBlacS 243XJBlac12 243XJB1ac24 243XJBlac25 DISCUSSION We isolated mutations in-vivo which affect the expression of a metB-lacZ gene fusion .
3-Galactosidase assays of these mutants showed elevated , partially regulated enzyme synthesis , typical of repressor binding site mutations ( Table 1 ) .
A DNA sequence analysis located the mutations within or near the -35 region of the metB promoter PB , and four out of five of the mutations disrupt a region of dyad symmetry ( Fig. 1 ) .
The fifth mutation alters the -35 sequence of promoter PB so that it perfectly matches the consensus sequence 5 ' - TTGACA-3 ' ( 16 ) .
For three of the mutants , we showed that the altered regulation of promoter PB is due to disruption of the metJ repressor system , most likely by alterations in the repressor binding site ( Table 2 ) .
At least two DNA regions have been proposed as metJ repressor binding sites in E. coli .
The first , suggested by Duchange et al. ( 6 ) and analyzed for homologies by Michaeli et al. ( 13 ) , is a short palindromic sequence , 5 ' - ATCTA-AC ------- GT-TAGAT-3 ' .
For the S. typhimurium metB gene , this sequence begins 1 base to the left of the Blac5 mutation ( Fig. 1 ) , and includes the Blac5 mutation .
However , three other mutations affecting metB regulation , Blacl , Blacl2 , and Blac25 , map well outside this symmetry region .
A second possibility , suggested by Belfaiza et al. ( 3 ) , is that the repressor binding region may be composed of tandemly repeating 8-bp palindromes which vary in their frequency of repetition and degree of homology to the consensus palindromne 5 ' - AGACGTCT-3 ' .
The region of repetition proposed by these investigators for the E. coli metB gene encompasses most of the sequence given in Fig. 1 and includes all of our mutations .
Interestingly , most of the sequence forming the dyad symmetry defined by our mutations Blacl , Blacl2 , Blac25 and Blac5 ( Fig. 1 ) can be generated by three repetitions of the above consensus sequence .
The Blacl2 mutation requires further comment .
It was isolated as an up mutation , which is reflected in the 27-fold increase of,-galactosidase activity in the 243XJBlac12 lysogen over that of the wild-type 243XJBlac lysogen under the repressing growth-conditions used on the original selection plate ( Table 1 ) .
However , when the Blacl2 mutation is compared with wild-type Blac in a system which does not have a functional metJ repressor , it appears as a down mutation ( cf. the 597XBlacl2 and 597XBlac lysogens [ Table 2 ] ) .
In the GS597 lysogens , RNA polymerase does not have to compete with repressor for promoter binding , and thus the P-galactosidase activity in these strains probably reflects the intrinsic efficiency of the promoters involved .
Hawley and McClure ( 7 ) have shown that bases the -35 near consensus sequence 5 ' - TTGACA-3 ' are also conserved to some extent , including the T residue 2 bases upstream of the conserved sequence .
The Blacl2 mutation changes this T residue of the consensus sequence to an A residue .
A similar change ( T -- G ) at this position in the ant gene of bacteriophage P22 results in a mild down mutation ( 26 ) .
The Blac24 mutation changes the -35 sequence of promoter PB to a perfect match of the -35 consensus sequence .
This change also increases the dyad symmetry region ( Fig. 1 ) , as well as changes a base in the repressor consensus sequence of Belfaiza et al. ( 3 ) .
It will be interesting to see if this mutation alters expression of metB solely by increasing the homology to the -35 promoter generating consensus , a promoter up mutation , whether it also alters the affinity of or this sequence for the repressor .
It will be necessary to measure the affinity in-vitro of the Blac24 DNA with purified repressor and RNA polymerase to resolve this question .
Binding of purified E. coli metJ repressor to the DNA of the E. coli metJB control region has been demonstrated in-vitro by Smith et al. ( 21 ) .
Using a DNase protection assay , they found that at low concentrations of repressor protein in the presence of the corepressor S-adenosylmethionine , the repressor preferentially protects a region of DNA encompassing the -35 sequences of promoter PB and the nearest metJ promoter ( our PJo ) .
This is the same region in which our mutations are located , suggesting that these mutations are affecting the metJ repressor binding site for the metB gene .
Although this region is very highly homologous between S. typhimurium and E. coli , an important exception is the A T base pair immediately to the left of the Blacl mutation ( Fig. 1 ) , which in E. coli has been reported as a G. C base pair ( 6 , 9 ) .
This substitution in E. coli would disrupt both the symmetry defined by our mutation and the consensus sequence proposed by Belfaiza et al. discussed above ( 3 ) .
The metJB control region contains not only promoters PB and PJo , but also two other metJ promoters , PJi and PJ2 ( Fig. 1 ) .
We have shown previously that in a metJ + host , promoter PJl is subject to repression by methionine ( 25 ) .
Since the RNA polymerase binding sites for promoters PJo ( defined by inspection of the sequence ) and PB ( 24 ) both overlap the region of dyad symmetry defined by the mutations as an operator site , it is of interest to question whether the metJ repressor binding site involved in metB regulation also plays a role in the autoregulation of the metJ gene .
To answer this question , we fused the mutant metJB control fragments to the lacZ gene in which B-galactosidase production is directed by the three metJ promoters .
Our results indicate that the mutations do affect metJ gene expression , but that the mechanism is complex and involves ihteractions between the overlapping promoters and the shared operator region ( manuscript in preparation ) .
GENE ORGANISM REFERENCE 5-GACGTCAATACATC T66A CATC TAAAC TTCTTTGCGT 5-5 A6C TI G G-ACGGTACAC-T AITAG6TA C ATC T A C TATACCBAAC-CAACITACAAAITTTCTTTGCG-T179 5-TGATTTTAACCGTC TGGA TGTC TAAAC TAGCATGAAT 331 5-TGTTITTTATACGTC TGGA TGTC TAC TGCATGAAT 5-GTACCTTTACATTAITGGAITGTGITAAAC ATCCAGACGT 5-TTCATTTTTACATC TGGA CGTC TAAAC GGATAGATGT 65 5-TTCATCTTTACATC TGGA CGTC TAAAC GGATAGATGT 5-TATCTTCAGCTATC TGGA  GTATAAGCGT ITGTC ITAAAC -95 5-CCCGACTCCTTTTT TGTA TAGA TAAAC CATCAGCTGA 5-ACCTAAAGAGAATT TGGT TAGC TAAAC TGTTGTGTGG ( 23 ) ( 9 ) S.T. METJB METJB E.c. S.T. E.c. T. S. S.T. E.c , THIS LAB ( 3 ) ME , [ MET , MEiE mETE METE META MEIG MEIK THIS LAB THIS LAB ( 18 ) ( 13 ) E , c. ECc , E.c. ( 2 ) ( 11 ) CONSENSUS SEQUENCE : Tc ITGGA YRICc ITAAAC FIG. 2 .
DNA sequences of S. typhimurium and E. coli met genes aligned to show a highly conserved 15-bp sequence within the various promoters .
The most highly conserved bases , except for the first T residue , are boxed for clarity , and a consensus sequence is given at the bottom of the figure .
The numbers above the rightmost bases correspond to the positions of those bases in the DNA sequences published in the respective references .
Asterisks indicate the start sites of transcription .
The proposed -35 (--) and -10 ( - ) regions , if known , are overlined for genes transcribed to the right metE , metF , and metA ) or underlined for genes transcribed ( metJ , to the left ( metB and metC ) .
The metK and metG genes show less homology to the consensus sequence , but are included for completeness .
St. , S. typhimurium LT2 ; E.c. , E. coli K-12 ; y , pyrimidine ; r , purine The dyad symmetry found in the S. typhimurium metJB operator region shows only partial homology with the control regions of other S. typhimurium met genes ( unpublished data ) .
This is similar to the results found in E. coli , as discussed above ( 3 , 12 ) .
The imperfect homologies may reflect the various responses of the different met genes to repression by methionine .
In Fig. 2 the DNA sequences of various met gene control regions from both S. typhimurium and E. coli are aligned to emphasize a highly conserved , nonsymmetric sequence , 5 ` T-TGGA -- TAAAC-3 ' .
This sequence is located near the point of transcription initiation , except for the metJB control region .
Note that the orientation of the sequence in the metB and metC genes to the direction of transcription is opposite that of the other genes .
The occurrence of this sequence at a position between the metB and metJ genes , instead of near the transcription initiation points , may indicate that this sequence , like that of the repressor binding site , is shared by both genes .
We have not determined a role for this sequence , but it is probably not the operator site for the metJ repressor .
The mutations reported here which affect the regulation of metB expression by.the repressor fall outside this sequence , except for the Blac25 mutation .
The effect of the Blac25 mutation on the function of this highly conserved sequence is difficult to discern , since the particular G C base pair changed by the Blac25 mutation is also part of the dyad symmetry and the -35 sequence of promoter PJo .
We examined the DNA sequences of the Genbank library for the occurrence of the conserved sequence .
Except for the E. coli lexA gene ( 8 ) , this sequence has not been found in any other genes .
Whether or not this highly conserved sequence is involved in another level of control of the met regulon is currently being investigated .
We thank Young Mok Park for performing the computer search of the Genbank library .
This investigation was supported by Public Health Service grant GM26878 from the National Institute of General Medical Sciences .
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Cloning and initial characterization of the metJ and metB genes from Salmonella typhimurium LT2 .
Urbanowski , M. L. , and G. V. Stauffer .
Nucleotide sequence and biochemical characterization of the metJ gene from Salmonella typhimurium LT2 .
Urbanowski , M. L. , and G. V. Stauffer .
Autoregulation by tandem promoters of the Salmonella typhimurium LT2 metJ gene .
Youderian , P. , S. Bouvier , and M. M. Susskind .
Sequence determinants of promoter activity .
Zagursky , R. J. , K. Baumeister , N. Lomax , and M. L. Berman .
Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA .