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Mol Gen Genet (1989) 216:164-169

© Springer-Verlag 1989

Short communications

DNA sequence of the metC gene and its flanking regions from Salmonella typhimurium LT2 and homology with the corresponding sequence of Escherichia coil Young M. Park and George V. Stauffer Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA

Summary. The D N A sequence of the Salmonella typhimurium metC gene and its flanking regions was determined. The metC gene contains an open reading frame of 1185 nucleotides encoding a polypeptide of 395 amino acids with a predicted molecular weight of 42874 daltons. SI nuclease mapping experiments located the transcription start site of the metC gene. The nucleotide sequence and the deduced amino acid sequence for the metC genes of S. typhimurium and Escheriehia eoli were compared. Although there are 279 nucleotide replacements, most do not change the amino acid sequence. Nucleotide sequence analysis of the flanking regions of the S. typhimurium metC gene shows that there is an open reading frame upstream and an open reading frame downstream of the gene. The existence of the divergently transcribed upstream open reading frame (designated ORF1) was confirmed by the construction of an ORFI-lacZ fusion. The transcription start site of ORF1 was determined by $1 nuclease mapping. Key words: metC Cystathionine-fl-lyase - Nucleotide sequence - Recombinant D N A

and Stauffer 1987). In this paper we present the nucleotide sequence of the metC gene. This sequence and the deduced amino acid sequence were compared with the corresponding sequences of E. coli (Belfaiza et al. 1986).

Materials and methods Strains and media. GS243 is pheA905 thi AlacU169 AmetE: : Mu and GS597 is pheA905 thi AlacU169 met J97 (Urbanowski et al. 1987). Strains were grown to the mid-log phase of growth in G M medium supplemented with 50 gg/ml phenylalanine, 1 ~tg/ml vitamin B1, and either 50 gg/ml D-methionine (derepressing growth conditions) or 750 rtg/ml L-methionine (repressing growth conditions). Ampicillin (150 ~tg/ml) was also added for the plasmid carrying strains. Assay of fl-galactosidase, fl-galactosidase activity was assayed as described by Miller (1972), using the chloroformsodium dodecyl sulfate lysis procedure. DNA sequencing. D N A sequences were determined using the procedure of Maxam and Gilbert (1980).

Introduction Cystathionine-fl-lyase (EC 4.4.1.8), encoded by the metC gene, is responsible for the conversion of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. The metC gene is one of ten genes involved in methionine biosynthesis. The ten methionine genes are scattered throughout the chromosome in both Salmonella typhimurium (Sanderson and Roth 1983) and Escherichia coli (Bachmann 1983) and are controlled in a similar but noncoordinated manner (for review, see Rowbury 1983). The current model for regulation of the met genes is that S-adenosylmethionine interacts with the metJ gene product to form the active repressor. The active repressor then acts on each of the met genes (except metH), each of which has its own operator site. In order to understand the molecular mechanism(s) involved in the regulation of the metC gene, we cloned this gene from S. typhimurium into a multicopy plasmid (Park Offprint requests to: G. Stauffer Abbreviations: Ap, ampicillin; bp, basepairs; GM, glucose minimal; kb, 1000 bp; L agar, Luria agar; L broth, Luria broth; ORF, open reading frame; [], designates plasmid-carrier state

S1 nuclease mapping. The S1 nuclease mapping procedure of Weaver and Weissmann (1979) was used to determine the transcription initiation site for metC and ORF1 mRNA. A 363 bp Sau3AI-Hinfl D N A fragment, which includes the metC and ORF1 promoter regions, was labeled with 3zp at the 5' termini of either the HinfI site (for metC) or the Sau3AI site (for ORF1) using procedures described in Maniatis et al. (1982). The labeled D N A was precipitated along with 50 ~tg of total cellular R N A isolated by the method of Baker and Yanofsky (1968) from the metJ mutant strain GS552 transformed with the metC plasmid pGS141 (Park and Stauffer 1987) and grown in GM medium plus 150 gg/ ml ampicillin. The mixture was resuspended in 20 gl of hybridization buffer (80% formamide, 0.4 M NaC1, 0.04 M Pipes, pH 6.4, 1 mM EDTA), heated to 90 ° C for 2 rain, and allowed to hybridize for 4 h at Tm of D N A - D N A +20-4 ° C (Berk and Sharp 1977). $1 nuclease was added (300 units in 100 gl of cold $1 nuclease buffer containing 0.25 M NaC1, 0.03 M sodium acetate, pH 4.6, 0.001 M ZnSO, and 20 gg/ml calf thymus DNA), and digestion was carried out at 20 ° C for 1 h. The $1 nuclease-resistant D N A was ethanol precipitated and the transcription start sites were determined by comparison with a G and purine (Pu)

165

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r, 1.0

.... 1.5

I II 2.0

met C Fig. 1. DNA fragment used to determine the nucleotide sequence of the Salmonella typhimurium metC gene and its flanking regions. The DNA sequence of the entire 2.2 kb RsaI-MluI DNA fragment carrying the metC gene was determined using the procedure of Maxam and Gilbert (1980). The location and orientation of the metC gene is indicated by the large arrow. Numbers indicate increments in kb

sequencing ladder of the original full length D N A fragments. Results and discussion Sequencing strategy

The metC gene of S. typhimurium was initially isolated on a 9.1 kb EcoRI D N A fragment and subsequently localized to a 1.4 kb region of this fragment (Park and Stauffer 1987). A 2.2 kb RsaI-MluI D N A fragment, which contains this 1.4 kb D N A region, was used for D N A sequencing. A physical map of this fragment is presented in Fig. 1. The D N A sequence of both strands was determined for the entire region and is shown in Fig. 2. Analysis of the nucleotide sequence of the metC gene

The nucleotide sequence and deduced amino acid sequence of the S. typhimurium metC gene were compared with the corresponding E. coli sequences (Belfaiza etal. 1986) (Fig. 2). The metC gene of each organism is 1185 nucleotides long and codes for a polypeptide 395 amino acid residues in length. The predicted A U G l:ranslation initiation codon, which was mapped by N-terminal amino acid sequence analysis for the E. coli MetC protein (Belfaiza et al. 1986), is preceded by a good ribosome binding site (Shine and Dalgarno 1975). The frequency of nucleotide base pair changes and deduced amino acid codon changes in the coding region of the metC gene of S. typhimurium and E. coli were analyzed. Although there are a significant number of nucleotide replacements (279/1185), 179 of the resulting codon changes are synonymous codon replacements. Thus there are only 54 actual amino acid replacements out of 395 residues. Of these replacements, 20 result in substitutions that are not likely to alter protein structure significantly (Dayhoff et al. 1978). A comparison of the codon usage of the S. typhimurium metC gene with the codon usage of strongly and moderately to weakly expressed genes in E. coli suggests that metC is a moderately to weakly expressed gene (Ikemura 1985). Presence of open reading frames in the regions flanking the metC gene

Analysis of the nucleotide sequences flanking the metC gene revealed two additional open reading frames in the up-

stream and downstream regions (Fig. 2). The upstream open reading frame (designated ORF1) begins at position - 2 2 3 and continues for at least 330 bp. Although there are several A T G codons in phase with the ORF1 reading frame, we tentatively picked the G T G codon at position - 2 2 3 as the most likely translation start site since it is preceded by a good ribosome binding site 6 bp upstream, and because this region is conserved between the E. coli and S. typhimurium sequences. In the E. coli nucleotide sequence (Belfaiza et al. 1986) there is 1 bp deletion at position - 3 8 8 compared with the S. typhimurium nucleotide sequence. This deletion results in a frameshift mutation and a translation termination codon at position - 4 2 7 in the E. coli sequence (Fig. 2). To test whether the ORFI open reading frame encodes a protein, we constructed an ORFl-lacZ fusion. In this fusion, designated pORFl-lac, the 86th amino acid codon of the predicted ORFl-encoded polypeptide was fused in the correct reading frame to the 8th codon of the lacZ gene in the plasmid fusion vector pMC1403 (Casadaban et al. 1980). This fusion resulted in lacZ expression (Table 1), indicating that the fragment contains a translation initiation site. The downstream open reading frame (designated ORF2) begins at position + 1357 and continues for at least 276 bp. This open reading frame is preceded by a good ribosome binding site. The ORF2 open reading frame is well conserved (greater than 90%) between S. typhimurium and E. coli through the 54 bp reported for the E. coli sequence. Transcription start sites for the metC gene and O R F I

The transcription start site of the metC gene was determined by S1 nuclease mapping (Fig. 3A), and is indicated as + 1 in Fig. 2. The same transcription initiation site was observed whether the R N A was isolated from a metJ + or metJ mutant strain. In the - 1 0 region of the metC gene, which is conserved between S. typhimurium and E. coli, three of six residues are identical to the consensus sequence 5'-TATAAT-3' (Rosenberg and Court, 1979). The - 3 5 region of the metC promoter in both organisms lacks homology to the consensus sequence 5'-TTGACA-3' (Rosenberg and Court 1979), suggesting that the metC gene may be poorly transcribed. To test whether metC is a poorly transcribed gene, we constructed a metC-lacZ fusion. In this fusion, designated pClac, the 77th amino acid codon of metC was fused in the correct reading frame to the 8th codon of the lacZ gene in the plasmid fusion vector pMC1403 (Casadaban et al. 1980). A 6920 bp EcoRI-SalI D N A fragment that carries the metC-lacZ fusion (and the lacy and lacA genes) was then isolated and inserted into the phage vector 2gt2 (Panasenko et al. 1977) by the method of Urbanowski et al. (1987). E. coli metJ + and met J - strains lysogenized with this phage, designated 2Clac, produce low levels offl-galactosidase (Table 1), in support of this hypothesis. The SI nuclease mapping procedure was also used to determine the transcription start site of ORF1. Three major S1 nuclease resistant D N A bands were observed for ORF1 (Fig. 3 B). It is possible that multiple transcription initiation sites exist for ORF1, or that the multiple bands are generated by S1 nuclease nibbling at the ends of the D N A - R N A hybrid. Within the ORF1 regulatory region, there are two possible overlapping - 10 regions (Fig. 2), with five of six

#T 3'-CA TGC AAG AAA TTA GGG AGG CAA TAG AAG CGA CGG GAC TCT CTC AAG GTC AAG TAA GAC GCG AAG TAA CTA CTC GAC ACG CGA CTC CGA Ar8 Glu Lys lle Gly Gly Asn Asp Glu Set Gly Gin Set Leu Glu Leu Glu Asn Gin Ala Glu Ash lle Leu Gin AIa Set Leu Set

-so6 Ser CGA AAA ACG Lys AIa

END PRO VAL ARG LYS LEU TRP CYS ASN SER SER ALA SER Gly Asp Asn Asn Glu Leu Gin Pro T G T A T C C T AA C C A C T A T G C GT AA G G ~ C T CGA CTT TGG ACG GCG TTA CAG CCT TCG GAC TAG ATT TCT CGC GCG TAG CCG ATT AAC GTC AAC AAG CGC GAA ATT CGC CGC GAA Set Phe Giy Ala Ala Ile Asp Set Ale Gin Asp Leu Set Arg Ale Asp Ale Leu Gln Leu Gln Glu Ar8 Lys Leu Ar 8 Arg Lys

-406 Ash G TA C G A G A A C C T T C G T AAC GCA CTT TTT AAG TTG CGA AAA TGA CTT CTT CTA TCG GGT CCA CTG CTG ACT GCG GTC TTA GTT TGG TTA GTA GTG CGT GAA GTG CTG Gin Thr Fhe Phe Glu Val Ser Lys Set Phe Phe Ile Ala Trp Thr Val Val Set Ala Leu lle Leu Gly lle Mat Val Cys Lys Val Val -300 T C A C C TTA TAG CCG CAC GAC TAT GTA CGG GGT CTG CCT TTC TAG GCA GAC GTA GTT TAA TAA TGG GTG AGACATAGAGGTCCTGCGTTTAGTGTTTTAGAC lle Asp Ala His Gin Tyr Meb Gly Trp Vel Set Leu Asp Thr Gin Met Leu Ash Asn Gly Met RBS -200 ** * -I00 G T G TTC C T" G T A CACGCCA 5'-ACTATTTACATACTGCCGCACCTTTACTGCTTTTTCTTTGCGCTTACGCATTAAAAAAGCCACCTGTTTTTAT TCGTATTATTATAGTTTTGTTGCAGCTTAACTATCAGCAAGAGTAATGATAAATGTATGACGGCGTGGAAATGACGAAAAAGAAACGCGAATGCGTAATTTTTTCGGT-5• G ATAAT -i0 C -35 C A C AAG G A C A

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-35 -I0 "+I RBS TTTGGCAAAATTTT•ATCTGTATCA•ACGT•GCCAGGGTGCAGATGGTTATATTCATGCTAGTTTAGACAT•CAGACGGTTAAAATCAGGAAACGCAA• CG TG T GC AT TT A GCCC CGA TA A T CCG

Mat Thr Asp Lys Gln ATG ACG GAT AAA CAG G C A Ala Lys

io.o Leu Asp Thr Lys Leu Val Ash Ala GIy ArK Set Lys Lys Tyr Thr Leu Gly Set Val Asn Set Val lle Gin Ars Ala Set Set Leu Val TTG GAT ACC AAA CTG GTA AAC GCA GGA CGC AGC AAA AAA TAC ACG CTC GGC TCA GTG AAT AGT GTG ATT CAA CGC GCT TCT TCA CTG GTG CT TC G T T GG A C G G C Gin Ala 200 Phe Asp Thr Val Glu Ala Lys Lys His Ala Thr Arg Asn Leu Aia Asn GIy Glu Leu Phe Tyr Gly Ar8 Arg Gly Thr Leu Thr His Phe TTT GAC ACC GTC GAG GCC AAA AAA CAC GCT ACC CGC AAT CTC GCC AAC GGC GAA TTA TTT TAC GGA CGC CGG GGA ACG CTG ACC CAC TTT GT A A G A T G T A G G C T G C T A T C Ser Arg 30o Ser Leu Gin Glu AIa Met Cys Glu Leu Glu Gly Gly Ale Gly Cys Ala Leu Phe Pro Cys Gly Ale AIa Ala Ve]. Ala Asn Thr Ile Leu TCG CTC CAG GAA GCC ATG" TGT GAA CTG GAA GGC GGC GCC GGA TGC GCG CTC TTC CCG TGC GGC GCG GCG GCG GTC GCC AAC ACC ATT CTG CTA AC G T A C T A T C G A T T TT T Gin Val Set

400 Ala Phe Val Glu Gln Gly Asp His Iie Leu Met Thr Asn Thr Ala Tyr Glu Pro Set Gln Asp Phe Cys Set Lys lie Leu GIy Lys Leu GCA TTC GTT GAG CAG GGC GAC CAC ATC CTG ATG ACC AAC ACA GCT TAT GAA CCC AGC CAG GAT TTC TGC AGC AAA ATT CTC GGC AAA CTG T TAC A T TGGT C C G T T C A lle Val Set GIy Val Thr Thr Ser Trp Phe Asp Fro Leu Ile GIy Ale Asp Ile Thr GGC GTG ACG ACA AGC TGG TTC GAC CCG CTC AYC GGC GCT GAT ATC ACG A TCA T T G T T C GTT Val

Gin His Ile Gin Pro ASh Thr Lys Val Val Fhe Leu Glu Ser CAA CAT ATT CAG CCG AAC ACG AAA GTC GTA TTT CTG GAA TCC A G C G A T A G G Lys Leu lie

500 Pro Gly Set lle Thr Meb Glu Val His Asp lle Pro Set lie Val Set AIa Val Arg CCC GGT TCT ATT ACC ATG GAA GTA CAT GAT ATT CCG AGC ATT GTT AGT GCG GTC AGA A C C C C C C G GCG GCC C A C C Val Ala Ala

Arg Val AIm Pro Glu Ala Val lie Met lle Asp CGC GTC GCG CCA GAA GCC GTC ATC ATG ATT GAY A T G T G T A T C C Ser Val Asp Ile

Fig. 2. Nucleotide sequence and deduced amino acid sequence of the Salmonella typhimurium metC gene and its flanking regions. The corresponding Eseheriehia eoli sequences are compared, with only the Eseherichia coli sequence differences presented. The heavy vertical arrows at positions - 4 7 0 and + 1411 indicate the ends of the reported Escheriehia eoli sequence (Belfaiza et al. 1986). The transcription initiation sites determined by $1 nuclease mapping are indicated by + 1 for the metC gene and by asterisks at positions - 1 9 1 , - 1 9 6 , - 1 9 7 for ORFI. The most likely --10 and - 3 5 regions for the metC promoter and the ORF1 promoter are indicated. A possible operator site for the MetJ protein that is composed of two repeating 8 bp palindromes that vary in their degree of homology to the consensus sequence 5'-AGACGTCT-3' (Belfaiza et al. 1986) is indicated by the dots above the sequence between positions - 5 and + 11. The ribosome binding site (RBS) and deduced N-terminal amino acid sequence for the metC gene, ORF1, and a downstream open reading frame (ORF2) are indicated. Arrows at the end of the metC gene indicate two regions of dyad symmetry that could form stem and loop structures once transcribed. The open triangle at position - 3 8 8 indicates a 1 bp deletion in the Escherichia eoli sequence (Belfaiza et al. 1986). The amino acid sequence in capital letters starting at position - 3 8 8 shows the truncated peptide and translation termination site in Eseherichia eoli caused by this 1 bp deletion

167

so9 Ash Thr Trp Ala AHo Gly Val L,u Pho Glu Ala Lou Glu Phe His Ilo Asp Ile Set Ile Gin Ala GHy Thr Lys Tyr Leu IHe Gly His AAT ACT TGG GCG GCT GGC GTT CTG TTC GAA GCT CTG GAA TTC CAT ATT GAT ATC TCT ATC CAG GCG GGC ACT AAG TAT CTG ATT GGT CAT G T T GGC C G T T A C C C A G G C C A C T G T A G ~fa Aap Giy Val Ala Val 700 Sot Aap AHa Mot Vai GHy Thr Aia Vll Aia Aan Aia Arg Cya Trp Glu Gln Leu Ar8 Glu Asn Ala Tyr Leu Met Gly Gln Met Leu Asp TCT GAT GCG ATG GTC GGG ACT GCC GTT GZA AAT GCG CGC TGC TGG GAA CAA TTG CGG GAA AAC GCC TAT CTG ATG GGG CAA ATG CTC GAe C T G G C A T C G G T A AT C G TGC Cya VaH Ilo 8O0 AHa Asp Thr AIm Tyr Met Thr Sot Ar8 Gly Lou Ar~ Thr Leu Giy Val Ar8 Leu Arg Gln His Gin Glu Ser Set Leu Lya lle.Aia Ala GCC GAC ACG GCT TAT ATG ACC AGC CGC GGT CTG CGC ACA CTC GGT GTC CGA CTG CGC CAG CAT CAA GAA AGC AGC CTC AAA ATC GCC GCA C TA G TT T A T T G GG T A T C C A T HIs Val Glu Ilo 900

Trp Leu AIa Ash His Pro Gln Val Ala Ar& Vsl Asn Hls Pro AIa Leu Pro Gly Ser Lys Gly His Ala Phe Trp Lys Arg Asp Phe Thr TGG CTG GCG AAC CAT CCT CAG GTC GCG CGC GTT AAT CAT CCG GCC CTG CCG GGT AGC AAA GGT CAT GCG TTC TGG AAA CGA GAC TTT ACA AGA G A T A C C T T T C T C AA GIu Glu 1000

G1y Set Sor Gly Leu Phe Set Pho Val Lou Asn Lya Lys Leu Thr Glu Ala Glu Liu GGT AGC AGC GGC CTG TTC TCC TTT GTG CTC AAT AAA AAG CTG ACC GAG GCA GAA TTA C G A T T G A C AT A T A G C G Lys Ash A s h Glu

Set TCA G G Ala

Ala Tyr Leu Asp Asn Phe Set Leu Phe Set GCC TAT CTG GAT AAC TTT TCG CTG TTC AGT AA C AGT T A C Asn

1100 Met Ala TyE Set Trp Gly GIy Tyr G1u Ser Lou Ils IHe Ale Asn Gln Pro GHu GHn Ile AHa Ala IHe Arg Pro AHa Gly Gly Val Asp ATG GCC TAC TCC TGG GG& GGC TAC GAA TCG CTG ATT ATT GCT AAT CAG CCG GAA CAG ATC GCC GCG ATT CGT CCC GCA GGC GGC GTA G^T G C G T T C C G A A A T C C A CA AG A C Leu His Gln Glu Ile 12o0

Phe Thr Gly Thr Lou VaH Ars Val His Ilo Gly Lou Giu Sor Vai Asp Asp Leu Ils Ala Asp Leu Ala Ala Gly Phe Ala Ar8 Ilo Val TTT ACC GGT ACG CTG GTT CGG GTG CAT ATT GGT TTA GAM AGT GTT GAT GAT TTG ATC GCT GAT TTA GCC GCC GGC TTC GCC AGA ATT GTG G G CT A CC CG AGA C C C T C CG A T T GC A Sot Ilo Leu Asp Asp End 1300 TAA AGTTGCCGGGGATGGACATATATGCAGACAAT T TC~GT~GAAAAG TCT GC GTT TGT TGCGTCCGGGA~CAAGGC~TCCCGGACGAT TCAGGAGTACAATAGGCGAATAAAAGCATA CA ACTTT AT T T ATTGT TAAA G AG G T 1400

RBS Met Ala Val llo Gln Asp Ile Ile Ale Ala Leu Trp Gln His Asp Phe Ale Ala Leu Ale Asn Pro His Val AATGCTGTTCCACAGGAAAGTTC ATG GCT GTC ATT CAA GAT ATT ATC GCT GCG CTC TGG CAA CAT GAT TTT GCC GCG CTG GCG AAT CCA CAC GTT C C T C C C 1500 Vail SeE Val Vail Tyr Fhe Val Met Pho Ala Thr Leu Phe Leu Glu Asn Gly Leu Leu Pro Ale Set Phe Leu Pro Gly Asp Set Leu Leu GTT AGC GTC GTC TAC TTT GTC ATG TTC GCC ACG CTA TTT TTA GAA AAC GGT CTG CTG CCA GCG TCA TTC TTA CCT GGC GAT AGT CTG CTG 1600

Leu Leu Ala Gly Ala Lou Ilo Ala Gln Asp Val Met His Phe Leu Pro Thr Ile Gly Ile Leu Thr Ala Ala Ala Ser Leu Gly Cy~ Trp CTA CTG GCA GGC GCG TTG ATC GCC CAG GAT GTG ATG CAT TTT TTG CCG ACG ATT GGC ATT CTC ACC GCC GCG GCC AGT CTC GGC TGC TGG Leu Sot Tyr Ilo GHn GHy Ar& Trp CTA AGT TAT ATC CAG GGA CGC TGG C-3'

Fig. 2 (continuation)

residues identical to the E. coli consensus sequence 5'-TATAAT-3', including the three most conserved residues at positions 1, 2, and 6 (Rosenberg and Court 1979). Only the most likely - 10 region is indicated since it is an appropriate distance from a possible - 3 5 consensus sequence, with three residues homologous to the E. eoli consensus sequence 5'-TTGACA-3'. Since the transcription initiation site for the divergently transcribed ORF1 gene is only 191 nucleotides from the transcription start site for the metC gene, we tested the possibility that it might be regulated like other met genes.

Expression of an O R F l - l a c Z fusion is not controlled by methionine supplementation to the growth medium or by the MetJ repressor protein (Table 1). These results indicate that ORF1 is not controlled as part of the met regulon. Potential M e t J repressor binding site

The MetJ repressor binding sites in S. typhimurium and E. coli are composed of tandemly repeating 8 bp palindromes that vary in their frequency of repetition and degree of homology to the consensus sequence 5'-AGACGTCT-3'

168 Table 1. Effect of methionine supplementation on the expression

of the metC-lacZ and ORFl-laeZ gene fusions Strain

GS243 [pClac] GS597 [pClac] GS243 (2Clac) GS597 (2Clac) GS243 [pORFl-lac] GS597 [pORFl-lac]

fl-galactosidase activity" D-methionine

L-methionine

1620 5475

590 4496

95 b 275 3513 3675

23 192 3559 3693

Specific activities are expressed as nanomoles of o-nitrophenol produced per minute per milligram protein at 28 ° C b For comparison, Escherichia coli metJ + and metJ - strains lysogenized with 2Blac phage show approximately ten fold higher levels of activity under the same growth conditions (Urbanowski et al. 1987) a

(Belfaiza et al. 1986; U r b a n o w s k i et al. 1987; Belfaiza et al. 1987). Two tandem repeats of the 8 bp sequence are found in the metC control region, spanning the transcription initiation site (Fig. 2). In this region 14 o f 16 bp are conserved between S. typhimurium and E. coli. One o f the 2 bp differences seen in the E. coli sequence (position + 9) represents a closer match of the repeating 8 bp palindromes to the consensus sequence, and m a y be responsible for the greater range o f regulation o f the metC gene by the MetJ repressor in this organism (Park and Stauffer 1987; A h m e d 1973; Chater 1970; Greene et al. 1973; Lawrence et al. 1968). A genetic analysis o f the S. typhimurium metC o p e r a t o r region will provide additional evidence as to the precise nature and extent of the MetJ repressor binding site. Distal region o f the metC gene

Fig. 3A and B. $1 nuclease mapping of the transcription initiation site for metC and ORF2 mRNA using a 362 bp Sau3AI-Hinfl DNA fragment which includes the metC and ORF1 promoter regions. A Identification of the 5' terminus of metC mRNA. The HinfI 5" 32-labeled DNA fragment that was protected from $1 nuclease digestion by hybridization to metC mRNA is shown in the lane marked $1. B Identification of the 5" termini of the ORF1 mRNA. The Sau3AI 5' 32p-labeled DNA fragments that were protected from S1 nuclease digestion by hybridization to ORF1 mRNA are shown in the lane marked S1. The transcription start sites were determined by comparison with a G and purine (Pu) sequencing ladder of the original full length DNA fragments.

Following the 3' end of the metC gene there are two regions of dyad symmetry that could form stable stem and loop structures once transcribed (Fig. 2). Both structures resemble known transcription terminators. $1 nuclease m a p p i n g was used to determine whether metC transcription terminates at either of these structures. The procedure used to m a p the 3' terminus of the metC transcript was similar to the 5' $1 nuclease mapping, with the following modification. A 567 bp DdeI-AccI D N A fragment, which extends 323 bp beyond the end o f the metC structural gene, was labeled with 32p at the 3' terminus of the DdeI end (Maniatis et al. 1982). The labeled D N A fragment was then treated as described in Fig. 3 for the 5' S1 nuclease mapping. Transcription termination, however, was not detected at either structure (not shown). This result suggests that the end of the metC transcript m a y extend b e y o n d these two structures. Since the DdeI-AeeI D N A fragment extends 85 bp into ORF2, it is possible that the metC gene is part o f an o p e r o n containing at least one other gene. Acknowledgements. This investigation was supported by Public Health Service grant GM26878 from the National Institute of General Medical Sciences. References

Ahmed A (1973) Mechanism of repression of methionine biosynthesis in Escherichia coli. I, The role of methionine, S-adenosyl-

169 methionine, and methionyl-transfer ribonucleic acid in repression. Mol Gen Genet 123:299-324 Bachmann BJ (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180-230 Baker RF, Yanofsky C (1968) The periodicity of RNA polymerase initiations: A new regulatory feature of transcription. Proc Natl Acad Sci USA 60 : 313-320 Belfaiza J, Parsot C, Martel A, Bouthier de la Tour C, Margarita D, Cohen GN, Saint-Girons I (1986) Evolution in biosynthetic pathways : Two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region. Proc Natl Acad Sci USA 83:867-871 Belfaiza J, Guillou Y, Margarita D, Perrin D, Saint-Girons I (1987) Operator-constitutive mutations of Escherichia coli metF gene. J Bacteriol 169:670-674 Berk AJ, Sharp PA (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721-732 Casadaban M J, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active fl-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971-980 Chater KF (1970) Dominance of the wild-type alleles ofmethionine regulatory genes on Salmonella typhimurium. J Gen Microbiol 63 : 95-109 Dayhoff MO, Schwartz RM, Orcutt BC (19'78) A model of evolutionary change in proteins. In: Dayhoff lvIO (ed) Atlas of protein sequence and structure. National Biomedical Research Foundation, Washington, pp 345-352 Greene RC, Williams RD, Kung H-F, Spears C, Weissbach H (1973) Effects of methionine and vitamin B12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12. Arch Biochem Biophys 158 : 249-256 Ikemura T (1985) Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol 2:13-34 Lawrence DA, Smith DA, Rowbury RJ (1968) Regulation of methionine synthesis in Salmonella typhimurium : mutants resistant to inhibition by analogues of methionine. Genetics 58:473492

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Press Cold Spring Harbor, New York Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymo165 :499-560 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Press Cold Spring Harbor, New York, p 352-355 Panasenko SM, Cameron JR, Davis RW, Lehman IR (1977) Five hundred-fold overproduction of DNA ligase after induction of a hybrid lambda lysogen constructed in vitro. Science 196:188 189 Park YM, Stauffer GV (1987) Cloning and characterization of the metC gene from Salmonella typhimurium LT2. Gene 60: 291-297 Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13: 319-353 Rowbury RJ (1983) Methionine biosynthesis and its regulation. In: Herrmann KM, Somerville RL (eds) Amino acids: biosynthesis and genetic regulation. Addison-Wesley Publishing Co, Reading, Mass, pp 191-211 Sanderson KE, Roth JR (1983) Linkage map of Salmonella typhimurium, edition, VI. Microbiol Rev 47:410-453 Shine J, Dalgarno L (1975) Determinant of cistron specificity in bacterial ribosomes. Nature 254:34-38 Urbanowski ML, Plamann LS, Stauffer GV (1987) Mutations affecting the regulation of the metB gene of Salmonella typhimurium LT2. J Bacteriol 169:126-130 Weaver RF, Weissmann C (1979) Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini 15S/?-globulin mRNA precursor and mature 10S /%globulin mRNA have identical map coordinates. Nucleic Acids Res 7 : 1175-1193

Communicated by H. Hennecke

Received August 16, 1988