Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258


The mercurial-resistance determinant from Staphylococcus aureus plasmid pI2S8 is located on a 6.4- kilobase-pairBgl H fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA se- quence of the Bgl II fragment was determined. The mer DNA Abbreviation: ORF, open reading frame. 

sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and mern (organomerculrial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage iden- tity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mor sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons. 
The mercury-resistance determinants from Gram-negative bacteria consist of a regulatory gene (merR), an operator/ promoter region, and at least three structural genes (1-7). The three structural genes merT, merP, and merA code for a membrane transport protein, a periplasmic Hg2+-binding protein, and the mercuric reductase enzyme subunit, respectively. The reductase functions to detoxify mercury by reducing Hg2+ and Hg+ ions to elemental Hg0 (8). Some plasmid mercurial-resistance determinants also contain an additional gene, merB, whose product is the enzyme orga- nomercurial lyage. The lyase cleaves the carbon-mercury bond of organomercurials such as phenylmercuric acetate (9). One product is Hg2+, which is subsequently detoxified by the mercuric reductase. Mercurial-resistance determinants that contain the organomercurial Iyase gene are designated broad-spectrum determinants; those without the Iyase gene are designated narrow-spectrum determinants (6,10). 
Less is known about mercurial resistance in Gram-positive bacteria. The most thoroughly studied determinant from a Gram-positive bacterium is the broad-spectrum mer operon of the Staphylococcus aureus plasmid p1258. This mer operon was mapped by both deletion mutants (11,12) and transposon (Tn) insertion mutants (13). These studies estab- lished the gene order merR merA merB. 
Mercury resistance is widely distributed among genera of Gram-negative and -positive bacteria (6,7,10). The mercury- resistance determinants from most Gram-negative bacteria are highly homologous (2-4, 6, 14). Conversely, the deter- minants from Gram-negative sources are less homologous with those from Gram-positive organisms (11,14). 
The DNA of two narrow-spectrum mercurial-resistance determinants from Gram-negative bacteria, those from trans- posons Tn2J and Tn501, have been sequenced. The se- quences were 85% identical at the nucleotide level (2-4). We now have completed the DNA sequence for the broadspectrum mercury-resistance determinant from plasmid pI258, which was originally isolated from S. aureus. Seven large open reading frames (ORFs) were identified and com- pared with the genes from Tn2J and TnSOO. The ORF for the p1258 mercuric reductase was identified. The organomercu- rial lyase ORF merB was identified by comparison with the merB Iyase gene sequence recently obtained from still an- other Gram-negative bacterial plasmid (15).t MATERIALS AND METHODS Media and Chemicals. Bacterial cultures for plasmid prep- arations and Escherichia coli strains containing M13 phage were grown in 2XNY broth (16) or on 5-bromo-4-chloro-3indolyl f3-D-galactopyranoside (X-Gal) agar (16). Ahtibiotics and lysozyme were purchased from Sigma. 203Hg(NO3)2 and T4 phage DNA ligase were from New England Nuclear. DNA polymerase I (Klenow fragment) was from United States Biochemical (Cleveland). Restriction endonucleases and BAL-31 exonuclease, from New England Biolabs or Inter- national Biotechnologies (New Haven, CT), were used ac- cording to the vendors' recommendations. 
Mercury-Volatilization Assays. Cells were grown in broth containing tryptone (Difco, 8 g/liter), Casamino acids (Sig- ma, 2 g/liter), and NaCl (5 g/liter) to the mid-logarithmic phase of growth and harvested by centrifugation, and then the 203Hg-volatilization assays were performed (11). 
Bacterial Transformation and DNA Sequenicing. Transforma- tion of E. coli (16) and Bacillus subtilis (17) was done as described. Large-scale isolation of plasmid DNA from E. coli (18), Bacillus strains (17), or S. aureus (12,19) was performed as described. Replicative forms of M13 and M13 with cloned inserts were prepared by the same procedure used to isolate plasmid DNA (18). The 6.4-kilobase-pair (kbp) Bgl II fragment from plasmid pRAL1 (19) was cloned in both orientations into the Bgl II site of M13 bacteriophage mWB2348 (20), and then the hybrid phage DNA (mRAL1 and mRAL2) was introduced into E. coli WB373 by transformation (20). For both hybrid phages, ordered deletions were generated (16, 2 sequenced by the dideoxy method as mc 

RESULTS

Cloning the Mercury-Resistance Detern p1258 with restriction endonuclease Xbc fragments (12), one of which (a 12.5-kbp f the mercury-resistance determinant (12, Xba I fragment from plasmid p1258 was c I site of pUC12 (ref. 21; the resulting r pRAL1 and introduced by transformation JM83; ref. 21). The mercury-resistance d4 aureus plasmid p1258 did not confer mercu coli JM83(pRAL1) (data not shown). Ti fragment from pRAL1 was cleaved wit fragments were produced, with the merc terminant located on the 6.4-kbp Bgl II frz was cloned into cloning vector pBD64 (2: After introduction of the pBD64-derived into B. subtilis strain BD170 (22) by transfc resistant colonies were isolated. Plasmi tained from one of these colonies. The 6.4-kbp BgI II fragment on plasmid pRAI restriction enzyme mapping (data not sho Expression of the Mercury-Resistance D shows the results of mercury-volatilization BD170(pRAL2) and control strains. B. (pRAL2) volatilized mercury 10 times more cells had been preinduced with 2.5 IiM HI rate of mercury volatilization by E. coli was greater than that for the mercury-resist bacteria in this experiment (Fig. 1A). Al resistant bacteria had an inducible phen4 BD170(pRAL2) was resistant to Hg2+, ph( tate and thimerosal, while strain BD170(p tive to these mercurials (data not shown). 
DNA Sequencing. Fig. 2 shows the DNS 6404-bp Bgl II fragment and the amino acid major ORFs present on the DNA sequence. 13) presented evidence for the direction agreement with the orientation deduced sequence. Seven ORFs were identified on ti An eighth ORF was identified, on the oppo translated from right to left (as shown in Fii (pRAL2) and control strains. Cells were grown turbidity reading of 50 (filter 54) and then grown in the presence (induced cells, solid symbols) or at open symbols) of 2.5 ,u M Hg2+ The cells w centrifugation and the rate of volatilization of m 203Hg2+ was determined as described (10,11). (, HB1O1(pDU1003) (ref. . 

0). Deletions were

The first ORF from left to right (Fig. 2) is probably the )dified (16,20). regulatory gene merR. The first ORF runs off the end of the Bgl II fragment to the left in Fig. 2, but since the p1258 mer operon was inducible as cloned (Fig. 1), it is probable that the iinant. Digestion of initiation site for the merR ORF occurs within the Bgl II 7 I resulted in four fragment at the position shown in Fig. 2. Possible ribosome ragment) contained binding sites are also indicated in Fig. 2. After the merR ORF 19). The 12.5-kbp comes the potential operator/promoter region with dyad :loned into the Xba symmetries (indicated by arrows in Fig. 2) and an additional plasmid was called set of potential -35 and -10 transcriptional initiation signals, into E. coli strain followed by six ORFs (Fig. 2) and a possible transcriptional eterminant from S. 
termination site starting at base 6151. 
iry resistance on E. 
Analysis of mer ORFs. The amino acid sequences of each of he 12.5-kbp Xba I the predicted polypeptides were analyzed by the hydropathy h Bgl II and four method of Kyte and Doolittle (25). ORF3 contained a :ury-resistance de- canonical "leader sequence" (26) with 2 lysines plus an agment (12), which arginine following the N-terminal methionine, and then a 2) and phage M13. 
series of 19 hydrophobic amino acids (ending with an alanine) Iplasmid (pRAL2) 
prior to the next charged amino acid. ORF4 and ORF5 might irmation, mercury-encode very hydrophobic polypeptides. The amino acid I pRAL2 was obsequence translated from ORF4 contained several hydropho-> presence of the bic stretches, indicative of four to seven membrane-spanning L2 was verified by helices; that of ORF5 contained a central hydrophobic iwn). 
sequence bordered by more hydrophilic polypeptide se- eterminant. Fig. 1 quences at both the amino and carboxyl ends. 
iexperiments with There is a possibly significant homology between the ORF5 subtilis BD170amino acid sequence of p1258 and the amino acid sequence of e rapidly when the the MerT membrane transport protein of Tn2l and Tn501 of g2+ (Fig. 1B). The Gram-negative systems. The residues Cys-47, Cys-48, Gly- HB101(pDU1003) 50, Pro-51, Leu-54, Val-55, Ala-56, Leu-57, Gly-58, and tant Gram-positive Gly-61 (numbered relative to the initiator methionine) of the of the mercury-ORF5 product correspond to the identical amino acids and otype. B. subtilis spacings at positions 24, 25, 27, 28, 31, 32, 33, 34, 35, and 38 enylmercuric ace- of Tn2l MerT. Nine of these 10 amino acids are identical in oBD64) was sensi-the MerT sequence of Tn501 as well (2). 
The last two ORFs translated left to right (as indicated in k sequence of the Fig. 2) are merA (mercuric reductase) and merB (organomer- translations of the curial lyase). The approximate locations of the merA and Novick etal. (12, merB genes on the 6.4-kbp Bgl II fragment had been deter- )f transcription in mined (12,13). The sequences were compared using the from the DNA program of Wilbur and Lipman (27), which maximizes the his strand (Fig. 2). 
pairing of identical bases (or amino acids). When the DNA ,itstrand, that if sequence of each ORF from the p1258 mer operon was 2site from ta if compared with the genes from Tn2J and TnS01 mercury- g. 2) from bp 6266 resistance operons (2-5), a clearly significant homology was )eptide. 
found only for merA (data not shown). The merA ORF was 44% identical at the DNA level to the merA genes from TnSOJ and Tn2J. Transposons Tn2l and TnSOJ lack merB genes, but o-the merB sequence from a different plasmid from a Gramnegative bacterium became available (15). The DNA se- quence of the merB ORF of Serratia plasmid pDU1358 (15) showed 40% base-pair identities when optimally aligned with the merB ORF sequence from p1258 (data not shown). 
There were significant homologies between the amino acid sequences of mercuric reductase and organomercurial lyase enzymes from S. aureus plasmid p1258 and the comparable amino acid sequences from Tn2J and pDU1358, respectively 15 30 (Figs. 3 and4). The mercuric reductase from p1258 was 40% identical (220/547 residues) to the mercuric reductases from Gram-negative bacteria, and the organomercurial lyase from t cells of BD170-pI258 was 39% identical (83/212 residues) at the amino acid in broth to a Klett level to the lyase from pDU1358. The homology between the an additional hour two organomercurial lyase sequences was strongest in the sence (uninduced, middle 100 amino acids of the two proteins. Little homology iere farvesteoi y found between the N-and C-terminal (Fig. 4). 
A) Circles, E. coli ing ORFs of pI258 and the previously identified genes of the plasmids of Gram-negative bacteria. 2. DNA sequence of the mercury-resistance determinant contained on the 6404-bp BgI II fragment from p1258. The amino acid translation of the identified ORFs is given in standard one-letter amino acid symbols, and ribosome binding sites (RBS) and stop codons (*) are shown. Dyads of symmetrical sequences in the predicted operator/promoter region are indicated by arrows. Only the anticoding strand is shown. DISCUSSION Mercuric reductase belongs to a class of flavin-containing, NAD(P)H-dependent oxidoreductases including glutathione re- ductase (8, 28, 29) and lipoamide dehydrogenase (30). Comparisons ofthe amino acid sequences ofthese enzymes showed that while the p1258 mercuric reductase is 40%o identical to both of the mercuric reductases from Gram-negative bacteria (Fig. 3 and data not shown), it is only 30%o identical to both glutathione reductase (from human erythrocytes) and lipoamide dehydrogenase (from E. coli; data not shown). 
A b BgII AGaATCTTGTA63ATATCCTTTTAGARAAAGTCGAAAG2AGATTSBCATACAGGTACATCT TCAACATTCCGTTGAATCCATTGAAAAAGAACAAGGAAAGTTTCATGTGTACGCTCGAAAAAA I120 AGAGGATATAACGCGG3TTTG3AAG3CAGACATTGTTATTCATGGTGCTGGACGGTCCCTGCCTTAGATATGAATCTTGAAAAAG6GAATATAGAAAGGAAAAAACATGGTGTCCATGTTAA RBS start merR M N L E K G N I E R K K H G V H V N TGATATTTGCAAAGiTGTAAGTAACCCG.AAT(TCTAT.C'AGCTGGAGATGCTGCAGCAACGGATGGCTTGCCCCTCACACCTGTAGCCAGTGCAGATTCTCATGTCGTAGCATCTAATTT E Y L Q S V S N P N V Y A A G D A A A T D G L P L T P V A S A D S H V V A S N L ATTGAAAG66AACAATTGAAT'ATCCCGTGATTCCATCTGCTGTATTTACCGTACCTAAAATGGCATCGGTAGGTATGAGCGAGAGGAAGCCAAAAACTCTGGCCGGAATAT L K G N S K K I E Y P V I P S A v F T V P K M A S v G M S E E E A K N S G R N I TATAAAGTACAAAGCPAATCTCCGATGGTTTACGTATAAACGGACAAATGAGACTTTGCTGCGTTTAAAGTGCTGATTGACGAAGATCATGATCAAATTGTTGGTGCTCATTTGAT K V K Q K N I S D W F T Y K R T N E D F A A F K V L I D E D H D QI V G A H L I TAGTAATGAAGCC6ATGAATG.ATTAATC'ATTTTGCAACAGCCATTCGTTTTGGGTTTCAACCAAAGAATTGAAACAAATGATATTTGCCTATCCAACGGCAGCTTCGGACATTGCACA S N E A D E L I N H F A T A I R F G I S T K E L K G M I F A Y P T A A S D I A H CATGTTGTAAGTTTGCGTTTTGTGAG.ATGTTTATAAACC'ACTTTATCGT'CTAATCTTGTTATATGATCTTAGAATCAAAAAGAAAATAACTTTTTTGACAGCCGCATTTTTCAACG3GAAG M L * end merR < * -35 -10 AAT6TGGCTTCTTGGCGTGAAGAGcAG3ATATcTTTATTCTTAAACTTcTr AAAAGCTTATGTGAACAcTTGATAAATAAGGTTTTTTATCTGAccTATTTTATAAGATTATTCTATAAA - 0 -p* - 0. AGAAAAAATAATGTATGATTG3ACCGTGTACTATGGTACAGGGTTTATACTTTTTATTGAGGTGACAATATGGGGATGAAAATCAGTGAATTGG3CTAAAGCGTGTGATGTGAATAAAGAA start ORF2 RBS M G M K I S E L A K A C D V N K E ACCGTTCGG6TATTACGAGCGGAAAGGATTGATAGCCGGGCCTCCCAGAAACGAATCAGGGTATCGAATATATTCAGAGGAAACAGCAGATCGGGTACGGTTTATTAAACGAATGAAGGAA T V R Y Y E R K G L I A G P P R N E S G Y R I Y S E E T A D R V R F I K R M K E TTGGATTTCTCGCTAAAGGAAATCCACCTGTTGTTTGGTGTGGTTGATCAAGATGGGGAGAGATGTAAAGATATGTACGCCTTTACCGTTCAAAAAACCAAAGAAATCGAGCGGAAAGTG L D F S L K E I H L L F G V V D Q D G E R C K D M Y A F T V Q K T K E I E R K V CAGGGTTTGTTACGAATCCAACGGTTATTAGAGGAATTA'AAAGAAAAGTGTCCAGATGAAAAGGCGATGTATACCTGTCCTATTATTGAAACGTTAATGGGAGGGCCTGATAAATAAATG Q G L L R I Q R L L E E L K E K C P D E K A M Y T C P I I E T L M G G P D K * end ORF2 GGGGAAGAT;TTGATGAAA'AAACGCATTTCCTTCACCGCTATAATGACG6TGTTGTTAATCGGTTTAACAGCTTGTGGAG3CAGAATCTGATACCGCGAATGAATCAAAGGTTCAAGACAT R85 K K R I S F T A I M T V L L I G L T A C G A E S D T A N E S K V 0 D I start ORF3 TCAAGGAAATCCAGTCAGCCTACCTAATG'AGAAACCCACACTCATTTATTTTATGGCAACCTGGTGTCCATCTTGTATATACAATGAGGAAATCTTTAAGGAAATGCATCAACTAAACCC Q G N P V S L P N E K P T L I Y F M A T W C P S C I Y N E E I F K E M H Q L N P GAACGATG3TTCAATTG3ATCACAG3TTAGTTTAG3AcCCTAACACAGATACAAAAGMAAACCTTGCGAAATTTAAACAGG3ATTATGGCGGGGATTGGcCCCATGTTTTAAAGAATGG3TAAAGA N D V Q L I T V S L D P N T D T K E S L A K F K Q D Y G G D W P H V L K N G K E AATTGCGG.ATACCTATGGCGSTCAAAcAATTGGAAGAGATCGTAcTGGTcAATTcTGAaAATGAGGTATTcTATcGTTcTGTTc6AccGTccTTTGATGATTTAAAAGAAGcGTTGAcTcA I A D T Y G V K Q L E E I V L V N S E N E V F Y R S V R P S F D D L K E A L T 0 AATAG3GAG3TGGAATTATGAGTTTTTCGTTTCTCTTCAT TCTGACCGCTGGAAT GGTTGCGGCCTT TAATCCTTGTGGTATCGCCT TGCTTCCTTCTTATATCTCCTATTTAATTGGAGGC I G V E L * R8S M S F S F L F I L T A G M V A A F N P C G I A L L P s Y I S Y L I G G end ORF3; start ORF4 GAAACAAAGG6ATCATTCGTTCCGCTATGCAATTTTTAAAGGATTAGGiGCTTG6TGG3AGCGATGACCACGGGGTTTTTAACGATTTTTGTATTGGCTG6TTTAT TGATAGGAGGATTGGGA E T K D H S F R Y A I F K G L G L G G A M T T G F L T I F V L A G L L I G G L G AGCOCACTAacAG8GATTTT;TCCGATTCTT~CATTGG3TTATGGGarTATACTCATTG.CTTT'ATTGGGG.TT.GG~CATGCTATTCGGGnAGCATTTGCCGATTAAAATAGGATCTTTTCAAGTC S A L T G I F P I L S L V M G I L I A L L G L G M L F G X H L P I K I G S F Q V AAGaCCAGGGAAATGGTCTATCTATTTTTACGG3AATAGCCTATGCCGTGACATCACTTGGTTGTACCTTGCCAGCCTTTATGTTGGTGGTCTCTGCATCGCTGAATGACAATAGCGTAACG K P G K W S I Y F Y G I A Y A V T S L G C T L P A F M L V V S A S L N D N S V T GCCTGATCATCAAGTTCA'TCATCTACTC'CCTTGGTATG'GGAAT TGTGGTGAAGC(3ATCACGATGGTCTCATTGATTTCACGACAATTGGTACAGAAGTTTCTGCACAACTATATGGGT A V I I K F I I Y S L G M G I V V T A I T M V S L I S R Q L V Q K F L H N Y M G TCTATCCAAAAAATAGCAGCTGTTGTGATTTTCCTCTCCGTTTTGTACATGGCTTATTACTGGTATTTCGGTTCTGGTGGCATTTGTACATTTTAAAATCCAACCGATCAGGTGTAACAA S I Q K I A A V V I F L S V L Y M A Y Y W Y F G S G G I C T F * end ORF4 AAAATAG3ATeAAAGATGTAAAAAAGCTAAACGAACAAATTTCAAGTTAGGCTATAGGCCATGTATGTACCTTAACCAAAGAATAAAATTTTTTTTAGAAAGAGTGGTGAGAATGATGGAT start ORF5 RBS VY L N t R I K F F L E R V V R M M D WAAATCGCTC63AAAGGTA'ACC.CTiGGGGTGTTTGGGrccTTTTTCGGAA'TATTATTG3GTTCCTTTATTG'GTTCCTTTAC'TTTGTTGCGC'AGGACCGATT'CTCCTTGTTG'CATTAGGGAG3C E N R S KG N R W G v W A F F G I L L V P L L V P L L C C A G P I L L V A L G s ACAGGTATTGGTGCCCTCT'TTGCTGGTGCGACAGGAAATTGGTGGTTGACCGGCATTTTTGCAGCATTGGCCATTGTTATGATTGCTCTGATCCTCAGCAAGTTGTTGAAGAACAAATAC T G I G A L F A G A T G N W W L T G I F A A L A I V M I A L I L S K L L K N K Y AATTCTCCTGAAGGCAATGGAAAAAaCAAAAAATA\AAACGGATTGCTGTACGccTCCAGAGAGCGTGGAT cGGAAAcATGAGACAAGATAAAT ATGAGATAAATGT ACAGAACTGTCTATC N S P E 8 N G K T K N K T D C C T P P E S V D R K H E T R *end ORF5 AAACTTTPAATAGAAAGAGGGATTCAAATGACTCAAAATTCATATAAAATACCCATTCAAGGCATGACATGCACAGGCTGTGAAGAACATGTAACCGAAGCATTGGAACAAGCCGGAGCT start erA RBS N T Q N S Y K I P I Q G M T C T G C E E H V T E A L E Q A G A AAAGATG.TTTCGG3CCGATTTccGaAcGcGG(iTGAGGrCCATT'TTTG.AACTCA'GCGATGATCAGATCGAAAAGGCTAAGCAAAATATTTCGGCAGCCGGCTATCAACCCGGAGAGGAAGAAAGC K D V S A D F R R G E A I F E L S D D QI E K A K Q N I S A A G Y Q P G E E E S * . . .pa . . . ... CAGCCCTCTGAAAACAGTGTAGATTTCAATCGGGATGGCGATTACGATCTTCTGATTATTGGTTCCGGCGGTGCGGCGTTTTCTGCAGCTATCAAGGCCAATGAAAACGGGGCGAAAGTG A P S E N S V D F N R D G D Y D L L I I G s G G A A F S A A I K A N E N G A K V . . ~~~Hpal GCCATG'GTAGACGGGGAACCGTCGGGGGGACCTGCGTTAACATCGGTTGTGTACCGTCAAAAACCATGCTTCGTGCCGGTGAAATAAACGGTCTCGCCCAAAACAATCCGTT TACCGGA A M V E R G T V G G T C V N I G C V P S K T M L R A G E I N G L A Q N N P F T G * . . . . . . S a CTTCAACATACCGGTGCTGCGGACCTTGc CCAATTGA CCGAACAAAAaAGATGGATTAGTCAGTCAAATGCGTCAAGAAAAATATATAGACTTGATCGAAGAATATGGATTTGATCTC L Q T S T G A A D L A Q L T E Q K D G L V S Q M R Q E K Y I D L I E E Y G F D L Xmal ATTCG3TGGCGAGGCCTCGTTTATCG3ACG3ATAAGACGATACAAGTGAATGGACAAAACATCACGT CTAAAAGCTTTTTAATCGCAACGGGGGC TTCTCCGGCTGTTCCGGAAATCCCGGGA I R G E A S F I D D K T I Q V N G Q N I T S K S F L I A T G A S P A V P E I P G ATGAATGAGGTTGATTATTTAACAAGTACATCCG3CACTCGAAT TAAAAG'AGGTTCCACAACGATTGGCAGTGATCGGTTCTGGCTATATCGCAGCGGAATTAGGTCAAATGTTTCACAAC M N E V D Y L T S T S A L E L K E V P Q R L A V I G S G Y I A A E L G Q M F H N CTCGGAA4CAGAAGTGATCTCATGCAAAGAAGCGACGTCTGTTTAAAACCTACGATCCTGAAATTTCCGAAGCCATCGATGAATCCTTAACTGAGCAAGGACTTAACCTGATCACTGGG L G T E V T L MQ R S E R L F K T Y D P E I S E A I D E S L T E Q G L N L I T G GTCACTTATCAAAACGTTGAGCAAAACGGTAAGTCGACA'AGCATTTATATTGAAGTGAACGGTCAAGAACAAGTCATCGAAGCCGATCAAGTCCTCGTGGCAACAGGAAGAAAGCCGAAC V T Y Q K V E Q N G K S T S I Y I E V N G Q E Q V I E A D Q V L V A T G R K P N ACAGAGATTTAAACCTTG'AATCAGcAGGTGTGAAAACAGGGAAAAnAAGGcGAAGTGCTGACCAATGAATATTTGCAAACGTCGAATAACCGAATATATGCCGCGGGcGATGTGWCCTC T E T L N L E S A G V K T 6 K K G E V L T N E Y L Q T S N N R I Y A A G D V T L GGTCCVCAATTCTTTATGTTGCAGCTTAATGAAGGCGG ITTGTGGCAAATAATGCGTTGGGTCTAGCGKAACGCAAAATCGATCTTCGCTTTGTTCCCGGCGTAACCTTCACCAATCCA G P Q F V Y V A A Y E G G I V A N N A L G L A K R K I D L R F V P G v T F T N P
S I A T V G L T E Q Q A K E K G Y D V K T S V L P L D A V P R A L V N H E T T G YTCTATAAACTTSTAGTCAACGCCCAAACCCAGAAATTGATCGGAGCGCACATTHTGAGTGIV EATGCTGGAGATGTGATTTATGCGGCAACGTTAGCGGTTCAATTTGGATTGACCATT 4680 V Y K L V V N A Q T Q K L I G A H I V S E N A G D V I Y A A T L A V 6t F G L T I GiAAGACCTTACGGSATAGCTTTG3CGCCTTATTTAACl;ATGsr TGPACGATTAAAGCTTGCA6CCTTGACGTTTGATAAAGACGTATCGAAATTATCTT6TTGTGCAGGCTAATGGTTCCTT E D L T D S F A P Y L T M A E G L K L A A L T F D K D V S K L S C C A * end merA EcoRI TT6ATTCTAATG3CTATGTT(AGCeAGcATTAAAACAATGAAGTGG CGAAMAATATAA66qGAW3ATAAGcTTATG3AAAAATATTTCAGAATTCTCAGCCCAACTTGATCAAACTTTTGAT start merE RBS M K N I S E F S A Q L D Q T F D C _A CACGTCTCTATGGmAGTGGTTATTCCGaTCCGTTGCTAAAAATGCTGGCGG;AGGGCGATCCAGTCCCCGTTGAGGACATCGCGGCGGAGACCGGGAAGCCCGTCGAGGAAGTT Q G E A V S M E W L F R P L L K M L A E G D P V P V E DI AA E T G X P V E E V AAGCAAGTCCTACA6ACTCTACCTAST6TGGAACTTGATGA6CAGGGVCCGTGTCGTCGTTATGGCCTCACACTGTTCCCTACCCCCCATCGCTTCGAGGTTGATGGGAAGCAACTATAT K Q V L O T L P S V E L D E Q G R V V G Y G L T L F P T P H R F E V D G K Q L Y A W C A L D T L M F P A L I G R T V H I A S P C H G T G K S V R L T V E P D R V G3TAAGCG3TCGAG3CCTTCAACAGCCGTT6TCTCGBATTGTTACACCAG3ATGAAATGGCCTCG6TTCGGTCGG3CCTTCTGTAACGACGTTCACTTTTTCAGTTCACCGA&TGCAGCCCAAGAC V S V E P S T A V V S I V T P D E M A S V R S A F C N D V H F F S S P S A A Q D T HLCTTAACCAACAC ESTSATC VGACPCVTTTT ETAAGAT FELCTTGGTCGCCATTTGGGAGCGCGTTATGAGGARTCAGGACCTACTAATGGGTCCTGTTGTAACATT W L N Q H P E S S V L P V E D A F E L G R H L G A R Y E E S G P T N G S C C N I
The three-dimensional structure of the human glutathione reductase is known (28,29). The amino acid residues in- 3. Alignment of the mercuric reductase amino acid se- quences from plasmid p1258 (top lines) and Tn21 (bottom lines). The sequences were aligned with the program of Wilbur and Lipman (27) using parameters K-tuple = 2; window = 20; gap penalty = 5. Standard one-letter symbols for amino acids are used; colons indicate identical residues in both sequences; dashes (indicating gaps) are used to optimize the pairing of the two sequences. volved in the active site and the binding of NADPH and FAD have been determined (29,31). If we compare the amino acid sequences of the mercuric reductases from p1258 and Tn21 (Fig. 3), it is evident that the amino acid matches are not evenly dispersed throughout the sequences but rather are clustered at those positions where previous studies with glutathione reductase (28, 29, 31) have shown that the amino acid residues are involved in the enzymatic activity of this protein (Table 1). There are three pairs of cysteine residues conserved in all three mercuric reductase enzymes: one pair is at the active site (residues 123 and 128 in Fig. 3) and one pair each at the N-and C-termini (residues 15 and 18 and residues 544 and 545, respectively). 
The amino acid residues comprising the active site of the p1258 mercuric reductase (residues 118-132, see Fig. 3) are identical to those of Tn2J and Tn5OI except for the conserv- ative substitutions of a valine for an isoleucine at position 119 and an isoleucine for a valine at position 126 in the p1258 sequence. The active sites for glutathione reductase and lipoamide dehydrogenase (3, 8, 30) are closely related. 
Table 1 lists the glutathione reductase amino acid residues involved in making contact with FAD (30, 31), as well as the residues in the equivalent positions in lipoamide dehydrogenase and mercuric reductase. For each comparison, protein sequences were aligned (27). The amino acid sequence of (bottom lines) (see Fig. 3). (28,29,31). tE. coli lipoamide dehydrogenase sequence (30) glutathione reductase is overall only about 25-27% identical to each of the three mercuric reductase sequences. However, a comparison of the (3 x 28 = 84) mercuric reductase amino acid residues listed in Table 1 to the glutathione reductase FAD contact positions shows that 60% (50/84) of the amino acid residues are identical. For the three mercuric reductases, the residues listed in Table 1 are identical in 89% (25/28) of the positions as compared with only 40% for the entire amino acid sequences of the p1258 and Tn2J proteins. For glutathione reductase vs. p1258 mercuric reductase, 64% (18/28) of the positions shown are identical, compared with the 26% match for the overall sequence. Similarly, the lipoamide dehydrogenase sequence and the mercuric reductase sequences were identical in 51% (43/84) of the possible matches in Table 1, much higher than the 26% overall identity. A similar analysis of glutathione reductase residues involved with binding of NADPH (28-30) and the comparable positions in lipoamide dehydrogenase and mercuric reductase (Table 1) showed similar higher frequencies of identities than did the overall sequences. 
When the amino acid sequences from the p1258 ORFs were compared with the National Biomedical Research Founda- tion data base of amino acid sequences §, only the p1258 merA sequence had a strong significant homology with sequences from the data base. The p1258 mercuric reductase was related to the mercuric reductase from TnSOJ and plasmid R100 and to glutathione reductase and lipoamide dehydrogenase. §Protein Identification Resource (1987) Protein Sequence Database (Natl. Biomed. Res. Found., Washington, DC), Release 12.0. Wolfgang Witte participated in the initial cloning experiments and David Dubnau, Saleem Khan, Ellen Murphy, and Richard Novick provided advice and bacterial strains. This work was supported by National Institutes of Health Grants AI15672 and A124795. 

FIG. 1 .

 1 FIG. 1. Volatilization of mercury by intacl 




  1); triangles, Bacillus st squares, Bacillus strain 473 (ref.24). (B) Circles, (pRAL2); triangles, S. aureus RN23(pI258) (ref subtilis BD170(pBD64)(ref. 22) . 




  Aside from a weak homology between the first ORF, gluta- train I-5 (ref. 23); thione reductase and lipoamide dehydrogenase, no homology B. subtilis BD170was found between the amino acid sequences of the remain- 11); squares, B. 


FIG. 2 .

 2 FIG. 2. (Continued on opposite page.) 




  FIG.2. DNA sequence of the mercury-resistance determinant contained on the 6404-bp BgI II fragment from p1258. The amino acid 




  FIG.3. Alignment of the mercuric reductase amino acid se- quences from plasmid p1258 (top lines) and Tn21 (bottom lines). The sequences were aligned with the program of Wilbur and Lipman (27) using parameters K-tuple = 2; window = 20; gap penalty = 5. Standard one-letter symbols for amino acids are used; colons indicate identical residues in both sequences; dashes (indicating gaps) are used to optimize the pairing of the two sequences. 


FIG. 4 .

 4 FIG.4. Alignment of the organomercurial lyase amino acid sequences from plasmid p1258 (top lines) with that from pDU1358 


Table 1 .

 1 Comparison of mercuric reductase, glutathione reductase, and lipoamide dehydrogenase amino acids that may make contact with FAD or NAD(P)H 


	Glutathione	Lipoamide	Mercuric reductase
	reductase*	dehydrogenaset	p1258	Tn21	Tn5OI t
		FAD contacts		
	I 26	L 11	I 91	1 103	
	G 27	G 12	G 92	G 104	
	G 28	A 13	S 93	S 105	
	G 29	G 14	G 94	G 106	
	S 30	P 15	G 95	G 107	
	G 31	A 16	A 96	A 108	
	G 32	G 17	A 97	A 109	
	V 49	V 34	V 114	1 126	
	E 50	E 35	E 115	E 127	
	S 51	R 36	R 116	R 128	
	T 57	V 43	T 122	T 134	
	C 58	C 44	C 123	C 135	
	C 63	C 49	C 128	C 140	
	K 66	S 52	S 131	S 143	
	K 67	K 53	K 132	K 144	
	H 129	L 115	E 195	S 209	
	A 130	G 116	A 196	A 210	
	A 155	A 143	A 219	A 238	
	T 156	A 144	T 220	T 239	
	Y 197	1184	Y 260	V 279	
	E 201	E 188	E 264	E 283	
	R 291	R 275	R 348	R 366	
	V 329	I 310	A 386	A 404	
	D 331	D 312	D 388	D 406	
	T 339	A 320	V 396	V 414	
	F 372	Y 351	F 428	F 445	
	H 467	H 444	Y 521	Y 538	
	E 472	E 449	E 526	E 543	
		NAD(P)H contacts		
	G 194	G 181	G 257	G 276	
	A 195	G 182	S 258	S 277	
	G 196	G 183	G 259	S 278	
	Y 197	I 184	Y 260	V 279	
	1198	1185	I 261	V 280	
	E 201	E 188	E 264	E 283	
	I 217	E 204	Q 280	A 299	
	R 218	M 205	R 281	R 300	
	H 219	F 206	S 282	S 301	
	R 224	P 211	K 287	R 306	
	L 285	V 269	V 342	L 360	
	L 286	L 270	L 343	L 361	
	W 287	V 271	V 344	V 362	
	A 288	A 272	A 345	A 363	
	I 289	I 273	T 346	T 364	
	G 290	G 274	G 347	G 365	
	R 291	R 275	R 348	R 366	
	D 331	D 312	D 388	D 406	
	V 370	I 349	V 426	V 443	
	Amino acid residues (standard one-letter symbo	
	position numbers are given.			
	*Human glutathione reductase sequence		



  . 


		I 104
		G 105
		S 106
		G 107
		G 108
		A 109
		A 110
		I 127
		E 128
		R 129
		T 135
		C 136
		C 141
		S 144
		K 145
		E 210
		A 211
		A 239
		T 240
		V 280
		E 284
		R 363
		A 401
		D 403
		V 411
		F 442
		Y 535
		E 540
		G 277
		S 278
		S 279
		V 280
		V 281
		E 284
		A 300
		R 301
		N 302
		R 307
		L 357
		L 358
		V 359
		A 360
		T 361
		G 362
		R 363
		D 403
		V 440
	ls)	and their
	tFrom ref. 3.	

Proc. Natl. Acad. Sci. USA 84(1987)
Richard A Laddaga, Lien Chut, AND T K Misrat, Simon Silvert
Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258