Vol. 172, No. 4

JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 2194-2198

0021-9193/90/042194-05$02.00/0 Copyright © 1990, American Society for Microbiology

Cloning and Nucleotide Sequence of bisC, the Structural Gene for Biotin Sulfoxide Reductase in Escherichia coli DOROTHY E. PIERSONt AND ALLAN CAMPBELL* Department of Biological Sciences, Stanford University, Stanford, California 94305 Received 11 October 1989/Accepted 8 January 1990

Clones of the Escherichia coli bisC locus have been isolated by complementing a bisC mutant for growth with d-biotin d-sulfoxide as a biotin source. The complementation properties of deletions and TnS insertions located the bisC gene to a 3.7-kilobase-pair (kbp) segment, 3.3 kbp of which has been sequenced. A single open reading frame of 2,178 bp, capable of encoding a polypeptide of molecular weight 80,905, was found. In vitro transcription of plasmids carrying the wild-type sequence and deletion and insertion mutants showed that BisC complementation correlated perfectly with production of a polypeptide whose measured molecular weight (79,000) does not differ significantly from 80,905.

The Escherichia coli protein biotin d-sulfoxide (BDS) reductase reduces a spontaneous oxidation product of biotin, BDS, back to biotin (8). Mutants that are unable to synthesize biotin can use BDS as their sole source of biotin (25). The BDS reductase protein, which is encoded by the bisC gene, requires the presence of both a small thioredoxinlike protein and a molybdenum cofactor for activity (8, 9). The thioredoxinlike protein has not been well characterized. On the other hand, the genes required for molybdenum cofactor production have been studied intensively. The cofactor is a pterin ring structure which is extremely oxygen labile (6, 19). The production of the pterin cofactor, the addition of molybdenum to this molecule, and the insertion of the molybdopterin into apomolybdoenzymes require the products of eight genes in E. coli: chlA, B, D, E, F, G, M, and N (10, 20, 36, 37). Cells with a mutation in any one of these genes have a pleiotropic phenotype. In addition to being unable to reduce BDS, they cannot utilize nitrate, trimethylamine-N-oxide, or dimethyl sulfoxide anaerobically as a terminal electron acceptor (2, 14, 38), they cannot use formate as an electron donor (5, 16), and, as their name implies, they are resistant to chlorate (14, 28, 29). Chlorate reduction appears to be catalyzed by several molybdoenzymes of E. coli, and only when all are inactivated do cells become resistant to chlorate (37). The function of BDS reductase in E. coli is unknown. It may serve as a scavenger, allowing the cell to utilize BDS as a biotin source. Another possible role for this protein is to protect the cell from oxidation damage, as do superoxide dismutase and methionine sulfoxide reductase (3, 11, 13). BDS reductase may restore activity to enzymes that have been inactivated by the spontaneous oxidation of the biotin cofactor covalently bound to them. A recombinant phage containing the bisC gene was isolated by M. Martin in our laboratory by selecting from a clone bank clones that allowed strains with a particular bisC point mutation to grow on minimal media containing BDS as the sole biotin source. A clone bank of the E. coli chromosome from the bisC+ bio deletion strain KS302 (Table 1) was prepared by inserting a Sau3A partial digest of bacterial chromosomal DNA prepared by the method of Redfield and * Corresponding author. t Present address: Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305-5402.

Campbell (30) into a BamHI digest of the A cloning vector X1059 (21). bisC+ clones were isolated from the bank by lytic complementation of the bisC32 point mutation found in strain S1187 (Table 1) as follows. A 0.1-ml volume of a stationary culture of S1187 was mixed with the E. coli clone bank and plated in minimal A (26) soft agar on glucose minimal A plates containing 4 x 10-3 ,ug of BDS per ml and 0.007% triphenyltetrazolium chloride as an indicator dye. Feeding plaques were identified by a red halo that was formed by the growth of cells around the plaque due to release of biotin by cells lysed by bacteriophage containing bisC32-complementing sequences. Feeding plaques were picked and repurified twice. These phage were tested further for lytic complementation of another bisC allele, in strain S1130 (Table 1), which they complemented, and for the complementation of two chl gene mutations, chlG21 and chlE4J, which they failed to complement. A single positive clone containing an insert of 19.5 kilobase pairs (kbp) was used for all further studies. A partial restriction map of this clone, XbisC, is shown in Fig. 1A. A 5.8-kbp internal EcoRI-PstI fragment of XbisC was subcloned into plasmid pUC9 (39). This subclone, pBISC352, complemented all bisC alleles tested, including spontaneous temperature-sensitive mutations, nitrosoguanidine-induced mutations, TnS insertions, and Mu insertions. To define the position of the bisC gene on pBISC352 and to determine its orientation, the clone was subjected to deletion and insertion analysis (Fig. 1B and C) Deletions constructed by subcloning different restriction fragments of pBISC352 into pUC9 were tested for complementation of the bisC32 mutation. This deletion analysis places the gene on a 3.2-kbp HincII-HindIII fragment (Fig. 1A). The position of the bisC gene was further restricted by insertional mutagenesis with the two transposable elements Tn5 and Mu d11681 (7, 12). Cells harboring pBISC352 were infected with the TnS-containing phage XNK467 (Table 1) at a multiplicity of 5 and incubated at 30°C for 20 min to allow TnS to transpose from the phage to the plasmid and the chromosome. Plasmid DNA isolated from these cells by the boiling lysis method of Holmes and Quigley (18) was used to transform strain S1187 to ampicillin and kanamycin resistance by the calcium chloride transformation method (24), thus selecting for plasmids into which TnS had transposed. These transformants were then tested for growth on BDS. Cells that were lysogenic for Mu dI1681 phage (POI1681 [Table 1]) were 2194

VOL. 172, 1990

NOTES

TABLE 1. Bacterial strains, plasmids, and phages used Strain

S1130 S1134 S1187 S1196 S1312

S1316

KS302 POI1681

pUC9 pDEPO M13mpl8, mp19 X1059 ANK467

h bisC

Origin or reference

Genotype

F- araDJ39 AlacU169 thi rpsL 't(bioA-lacZ)301 bisC F- araD139 AlacU169 thi rpsL 'D(bioA-lacZ)301 chlE41 F+ bioA24 bisC32 (A imm434) F+ bioA24 chlG21 F- araD139 AlacUl69 thi rpsL bioA24 (X imm434) zbh428::TnJO bisC8::Mu cts F- araDJ39 A1acUJ69 thi rpsL bioA24 zbh428::TnlO bisC9::Mu cts HfrH A(gal-bio) araD139 araB::Mu cts Alac rpsL (Mu d11681 [Km] ABamHIcts) lac+ Apr

A.

P

H

I

II

P

E

2195

PHE

I _II

4 4 pBISC352

I 1

|

I

li

8 Curing of S1177 (8) Transduction to Tcr of bisC::Mu cts mutant of A. del Campillo-Campbell Same as for S1312

I

CS V

P IU M

I I

I

11 I

TR TUB

I kbp

B.

+

i

I

i

33 12

I I I

I

oI

-

c1857 b221 rex::TnS Oam

21 N. Kleckner

_

C.

Pam I

I

*1

I

jI

II

'll III

transformed (24) with pBISC352, and a Mu-transducing lysate was prepared from these cells by inducing the Mu prophage in a logarithmically growing culture at 43°C for 2.5 h. A bisC::Mu strain, S1312 (Table 1), was transduced (26) to resistance to ampicillin and kanamycin with this lysate. These transductants, which must harbor pBISC352 into which Mu dI1681 has transposed, were screened for growth on BDS. The positions of these insertions (Fig. 1C) suggest that the bisC gene occupies a maximum of 2.8 kbp of the original clone. There is a small region of overlap of complementing and noncomplementing insertions into the bisC clone. Since both of the noncomplementing Mu dI1681 insertions are in the same orientation relative to the bisC gene but the noncomplementing insertion is in the opposite orientation, this overlap is probably due to polarity effects of the insertion element in one orientation but not the other. This result suggests that these insertions are between the bisC promoter and the start of the bisC coding region. The polypeptides produced by the original bisC gene clone, pBISC352, were identified both in vivo by maxicell analysis (31) of cells containing the clone (data not shown) and in vitro by transcription and translation (41) of plasmid DNA. These polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (23) of [35S] methionine (Amersham)-labeled proteins prepared in an S30 extract (Amersham) used according to the instructions of the manufacturer with 3 ,ug of cesium chloride gradient-purified (24) plasmid DNA. Three polypeptides with molecular weights of 79,000, 34,000, and 22,000 (79K, 34K, and 22K polypeptides) were produced by this clone (Fig. 2). The 79K polypeptide was the sole polypeptide required for the BisC+ phenotype (Fig. 2). In experiments in which the products of the three TnS insertion mutants and the six deletion mutants which fail to complement bisC32 were produced by in vitro transcription or translation of purified DNA, the 34K and 22K polypeptides were produced but the 79K polypeptide was not (Fig. 2, lanes 3, 5, 8, 9, 10, 11, and 14). More

-

I

I

Apr

11

1

i

39 27 40

I

H I VRE

III I III

Tn5 Mini-Mu

FIG. 1. Location of the bisC gene on the bisC clone. (A) Restriction maps of the 19.5-kbp bacterial DNA insert in XbisC and of the 5.8-kbp subclone, pBISC352. The restriction enzymes used were PstI (P), HinclI (I), PvuI (U), MluI (M), ClaI (C), StuI (S), PvuII (V), BstEII (T), EcoRV (R), BglII (B), HindlIl (H), and EcoRI (E). The 1-kbp scale is for the pBISC352 map and the maps in panels B and C. (B) The fragments of bisC DNA present in several subclones of pBISC352 are aligned with the map of pBISC352 in panel A. The abilities of these subclones to complement the bisC32 allele are shown on the right. Some clones (+) complement the mutant for growth on BDS minimal plates, and other (-) do not. (C) Positions of Tn5 insertion are indicated above the line, and positions of Mu dI1681 insertions are indicated below the line; the map is aligned with the map of pBISC352 in panel A. Tn5 insertion clones were tested for the ability to complement the bisC32 mutation. Mu d11681 insertion clones were tested for the ability to complement the bisC8: :Mu mutation. Insertion clones that complement are indicated by a short line; those that do not are indicated by a long line.

important, the 34K polypeptide was not produced by one of the TnS insertion mutants that can complement the bisC32 mutation (lane 15), and the 22K polypeptide was not produced by a different complementing TnS insertion mutant (lane 4). The 79K polypeptide was produced, however, in these two insertion mutants and in two others that can complement the bisC32 mutation (lanes 12 and 13). The fact that our cloned segment complements bisC mutations suggests (but does not prove) that the gene responsible for the complementation is really the bisC locus. To prove that it is, one of the bisC temperature-sensitive structural gene mutations isolated by del Campillo-Campbell and Campbell (8), bisCJJ7(Ts) was cloned by using its ability to hybridize to the wild-type clone. The region containing the mutation was mapped to the interval between bp 1180 and 1780 on the wild-type bisC sequence map (see below and Fig. 3), which is within the open reading frame proposed here to be the BDS reductase coding region. The DNA sequence of this mutant from bp 1290 to 2050 was deter-

2196

J. BACTERIOL.

NOTES

...

p

FIG. 2. Analysis of polypeptide products of deletion and TnS insertion mutations in the bisC clone expressed in a coupled in vitro transcription-translation system. Polypeptides labeled with [35S]methionine were produced in an S30 extract of E. coli (41). These were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiograms of two gels are shown. The bisC fragments and insertion mutants tested in this system are indicated below the autoradiograms. Those marked with a plus sign complement the bisC32 mutation and produce the 79K polypeptide. Those marked with a minus sign do not complement the bisC32 mutation and do not produce the 79K polypeptide, although several do produce polypeptides of altered sizes. Lanes: -, no DNA; 1, pDEPO (27); 2, 5-kbp PstI-HindIII bisC fragment cloned into pDEPO; 3, 4.6-kbp PstI-BgllI bisC fragment cloned into pDEPO; 4 and 5, TnS insertions in plasmid used in lane 2; 6, pUC9 (39); 7, 5-kbp PstI-HindIII bisC fragment cloned into pUC9; 8, EcoRV-PvuII internal deletion of plasmnid used in lane 7; 9, StuI-EcoRV internal deletion of plasmid used in lane 7; 10, StuI-PvuII internal deletion of plasmid used in lane 7; 11, 3.9-kbp PstI-EcoRV fragment into pUC9; 12 through 15, TnS insertions in plasmid used in lane 7.

mined by the dideoxy sequencing method, and the sole change from the wild-type sequence was shown to be a G-to-T transversion at bp 1443 (data not shown). This base change results in a change from a methionine to an isoleucine codon in the BDS reductase protein. Additional evidence comes from Southern analysis of a Mu insertion (in S1316 [Table 1]) that has been mapped to the bisC locus by genetic means. By using probes representing different regions of the cloned gene, we have mapped the Mu insertion to the 0.3-kbp BstEII-EcoRV fragment in the center of the gene (Fig. 1). Sequences on either side of this region are still present in this strain (data not shown). Thus, two known bisC mutations map within the region proposed to be the bisC coding region. The sequence of the 3.3 kbp of the 3.7-kbp HincII-EcoRI fragment containing the bisC gene was determined by the dideoxy chain termination method (32). M13mpl8 and M13mpl9 clones of this fragment were subjected to exonuclease III deletions from either end (17). This produced a complete sequence, including every base of 3.3 kbp of the 3.7-kbp fragment, a minimum of two times in each direction. The sequence of this 3.3 kbp is shown in Fig. 3. An open reading frame thought to correspond to the bisC coding region and capable of encoding a polypeptide with a predicted molecular weight of 80,905 is also shown in Fig. 3. The three noncomplementing TnS insertions and the six deletion mutants which do not produce the 79K polypeptide

in the in vitro transcription-translation system all map to this open reading frame. The sequence places the 5'-to-3' orientation of the bisC gene from left to right on the map of Fig. 1A. An examination of the hydropathicity of the predicted BisC open reading frame by the method of Kyte and Doolittle (22) suggests that the product is a soluble protein, which is consistent with the location of the enzyme in partially purified preparations (9; unpublished observations). The codon usage in the predicted open reading frame (data not shown) is typical of weakly expressed E. coli proteins (15). The 700-bp sequence upstream of the bisC coding region includes no sequences with appreciable homology to the consensus ribosome-binding site (34) and promoter of E. coli (35). Because of this, we have no reason to assume that the major initiation site for translation is at nucleotide 577 (as shown) rather than at the downstream ATG at nucleotide 643. The bisC gene appears to be expressed at a low rate in vivo. The bisC product, the 79K polypeptide, is expressed in vitro at rates much lower than those of the vector-borne gene product, the P-lactamase polypeptide, and indeed lower than those of the other two polypeptides produced by the cloned DNA (Fig. 2). This low level of expression may explain the difficulty in identifying sequences that match consensus sequences important for gene expression in E. coli. The sequences of two other molybdoenzymes from E. coli show some similarities to that of bisC (1).

VOL. 172, 1990

NOTES i5 =O 103 Cho AMC TOO COM 195 MA" GMC CMO MC 26 LOT CAC TMC GCC 375 CAT TMT CCC Gee 465 CM TOT TCC CT? 555

ATM Coc CO TO GM

CM CCC

CCL CMG

i TGC

TGC CCC TG

TOO

AMCCCCOT? TMGCM CCT GTA CCM TM CCA Val LAr pta pro TOO CAT CM GCC Tip Asp Clu Ala

645

ATO Not 735 CTC Lou

30 CC TM CMCA 120 TGC TGC GCC MAT 210 CCC CGT MC MA 300 LAT AMG ATA MAC 390 AMA TCA MAG T?A 460 CCL AOT CAT CCT 570 CCC CCC T?A CCC

060 OT CCA AM val LAV LIe CAT CT? AT? Lsp Lou I1e

CCC TTT 750 CMC CMA Nis Clni

Cly Pto

45 TO CC CC fl TO CC CL CTA 135 CMG CCC GCC TNT ACC GMC ATC TNT 225 TT AMC TIC T?C GCC TIC GCC COT 315 ACC TNT CIT? CAT TOT TMA TCC TTA 405. TIC CTG CCL ATA CIC CC AMT TIC 495 CMC CAT AT? CCC TIC TCA CTC CCC 565 MAC CCA ATO CMA AC TCC TIC CMG NET Glu Asn Sar Lou Clni 075 CTT CCC TCA CCC GCA MAC CCC CM Lou Ala Sor Pro Clu LOU Pro Clni 765 CAL CAT AMA CCC AT? CCT GAM GCT ClniHis Lys Ary I1e Ar; 0lu Ala

75 00 CC TI CT cAc AT CA OTA 150 105 ACC CCC CT? CGA MA MGA 240 255 OTA MOGOTT ACT TOT CCA GCC 30345 ATA ACC TTT TTA TIC TTT GCC 420 435 TMA CTA ATA TNT MAC TCC TCA 525 510 CCA TIC CCCG CCC CAT CGM CC! 000 615 MCC CCC C CCC CAC CMG CTI Sor Ala Vol kr; Asp Clni Val 705 0690 CCC ATT COT CCC CMG OAT CMA Cly I1a Lrgayly Clni Lsp Clu 760 79S TAT GOCT CCC GCA TCC ATI TNT Tyr 01y Pro Ala Oar I1. Pb.

GM

ATML

2197

90

MC

WCC GCCC Ig0

MAT ACC CCC CCC

CAC 270 MAG TMC CCC CM ACC 360

ACC; COOT TCC CT GCCC 450 ATA CAT CTA ACC TTT 540 TCA MAC CCA CCCG CGA 630 CAC MGC MAT ACO COG His Sor Len Thrl Ar; 720 TNT CT? CC GTM MCT Pbs Vol LAr Vol Sar 610 CCI GOCT TCC TMC GCC Ala Cly Sor Tyr 017

ClaN 70 65900 640 65 625 TOO CC! TC AMC CCC CTC CIC CAT AMG GCCC TCC ACh TTA T?A CMA CCC TAT ATO GCCC CI CCL CCC GOCT TAT ACC COO CAT CTG GCG CAT TMp An Saw Lon 01y Vol Lou Nis Lys Ala SOar Tbr Lou Lou Clni Lip Tyr Not Ala Lum Ala 01y 017 Tyr Tb? 01y His Lou 017 Lop

615 CCL CMG CCC TAT TCC MCCCCC WG Tyr Sor Tb? 01y Ala Ala Clni Ala 1005 CMa CAT MCe CAT CTC OTO OTO CIO Clu 31s SOr Lop Vol Vol Vol Lou

StuN 960 630 645 ATC ATC CCG TAT OTC CTC COT GOT MT GCM CT? TAT I1e Not Pro Tyr Vol Vol 017 01y Oar Glu Vol Tyr 1035 1020 1050 Too MaT GCCI MC CCL CIC MAT ACC 010 AMA AT? CCC Tip Sar Ala LOU Pro Lou Lon Tb? Lou Lys I1e Ala

675 CM CMO CMG ACC MGT Clii Clni Cln Tb? Sar 1005 TOO M&T CCA TCC CAT Tip LOii Ala Sor Asp

Tip Pro Lou Vol Lou 1060 CMa CMa CCC CT? TOT Glu Clni 01y Lou Oar

960 Too CCC CIC aTn CIC

1095

1110

1125

1140

1155

1170

1165

1200

1215

1230

1245

1200

1275 TNT C10 0CC CC! TGC Pta Lou Ala LAvg Cyo 1365 OCA CMG AT? TOT GOT Ala Clu I1e Cyo 017 1455 ATO CMG CCC CMA CMG not ClIs LAy Clni Clin 1545 TNT CC! C TOT TMC Pta 017 Lou Oar Tyr

MCC MAC COT TAT GCC Tb? TSr 017 Tyr Ala 1360 CT? GCC GCCL GCCC AMA Vol 01y Ala Ala Lyo 1470 TNT COT CMG CM AMA Pteal0 Glu Clii Lys 1560 CAT TNT CCC M&T CC! Nis Pta Ala LOU Cly

1335 GAC CCL ATA GCC MLA 1425 ALT ACC ACC ATO CIC LOU Thr TSr Not Lou 1515 TIC GOO CMA ATC CCC Lou 01y Clii Ile 017 1005 1560 1575 OCT MC CCC MA CC COGCT TOT GCC OTG CTC TOT TCC ATO CMG CCC Cly Aon Pro Thr An An; Oar Ala Vol Lou SOar Oar Hat Clni 01y

1350 oCC GCC GMA TOO GCCA

TMC TTT TCI CCL CIC COT CAC MGC COO AM AM CTC ATC TOC AT? CAT CCL ATO CCL TOO CMk ACC CTC CAT TIC TNT GGC CAT AMA ATO Tyr Pta Oar Ala Lou LAr Lop Oar 01y Lys Lys Lou I1e Cy0 I1e Lop Pro, not Ar; Oar Glu TSr Vol Asp Ph. Pbs 01y Asp Lys not

CMG TOO GTM CCL CCC CAC kTO CCC ACC CAT CT? GCC CIG ATO CIC GCC ATC CCC CAT ACC CTC GTO CMA MAT COT TOO CAC GCM CM QCCC Glu Tip Vol Ala Pro His Not Gly Tbr ksp Vol Ala Lum Hat Lou Cly I1e Ala His TSr Lou Vol Olu LOn 017 Tip His LOp GlU Ala

1035

TOO OAT a09 aTO OAT

1290

1320 1305 GTC TIC CCC TCI TAT TIC CTG CCC CMG ACT Vol Pb. Ala Oar Tyr Lou Lou 01y Glu oar 1410 1395 CCIG AT? TIC CAC CMA LTC CCC CMA01CTO I1e Ar; Glu Lou Ala Ala I1e Pta Nis Clii 1500 1465 CAC TOC ATO ATC CTC ACO CTO GCCL GCCL ATO His Tip Nat I1e Vol Thr Lou Ala Ala Hat

1660 1005 1095 1050 CT? CCC CCC AT? CT? CMA GCC CIC CM MAC CCT OCT GGC GCCL TAT CMA CMC MAC A±LO n LAr 21e Vol Glu Ala Lou Olu AOin Pro 017 017 Ala Tyr Cln is 1765 1770 1755 CC M&C TNT MCT CAT CAT CMG CAT ACC MAT CCC CIO ATC GCCC 0GGCC GO Ala 017 Cly Ala ken Pha Tb? His Nis Clii LOp Tbr LOU Arg Lou I1.

Vol Ala 1740 TCC TOC Tip Tip 1030 CMA TOC Glu Cys

TIC

Pha

1605 1520 CC! MT CA! CTC MCC ATO MCC OCT CAT TAC MGT Lou Tb? Not Tb? 017 LOp Tyr Sar

Lop CMA Glu AT?

1995

CT TNT Pta Lop Vol Pta 2065 MC TNT TAT M&C Thr Ito Tyr Ami 2175

CMO ATO X1. Glu Not CCC AMG AT? LArg Lys I1.

CCC MT CMG 017 Los

Clni

CCC CMG TTA Ala Glu Lou CT? CCC CCA

2010

MGT CMA

SOar Glu 2100

CMG CCC

1600 1645 TOO AC GCCC CCC CCL AMA CMCCCC CAT Tip Tb? Ala Ala Ala Lys Hio Ala LOp Eco3v 1935 1950 MAT CMG CAT CTC GTO CCC ATO AMG CM Not Pro Clii Vol Lou Lys Clni AOn 3±s 2040 2025 TOO CM AMO CCC OCT TAT GCCL COT TNT Tip Glu Lys 017 017 Tyr Ala LAr Pb.

2445

1600 CCC TOO CM Ala Tip Glni 1690

TCI TNT CMa SOar Pb. Clu

1660 1905 Vol Pro Pro Ar; Tyr Glu Ala Ar; LOin 2070 2055 CM CCL AMA MGT GAMCTO CMA TOO CTO Olu 017 Lye Oar Clu Lou Clii Tip Lou 2145 21600 GCCI CM TIC TOO CMA CCC MAC CMO TTL Ala Glu Pb. Tip Clni Ala Lsn Clni Lou

OTM OT CCC CCL CCC TAT CM GCC COT M&T

2235 TCC CCI CCC CAT CCC SOar Alb Gly ksp Pro 2310 2325 CCC CAT TOC CCT CCG CAT CCA ATG TOO CTC GAC Pro Asp Cys Pro 017 3±a Pro Not Tip Lou Glu

2430

2415

2400

2250

TIA AM ACO CMG Lou Lys Thr Clni 2340 CCC CAC CM TOO Pro Lsp Glu Tip

CCG CCC CAC CCC CTC, CAC AGC CLO CTC MAT TAC ACT TOT CTC CGCC Pro Ala H±s Arq Lou His Sar Glii Lou Lsn Tyr Oar Oar Lou Ar;

2400 2475 CAT CCTIAGAOCT Ala LOU Ar; Clu Pro Vol Thr I1e His Pro Asp LOp 2550 2505 GGC CMA AT? CT? CCC CCL GCCC CTC AT? MOC CMG CCL Gly Oln 21. Lou Ala 01y Ala Vol I1e Oar Glu 017 2040 2055 ACC GCT CAC COT AT? TOT AMA MC OC GCCL GTO MC Tb? Ala LOp 01y 21. Cys Lys LOU 01y Ala Vol LOU

GCCL MT COT CMG CCT GTC ACC ATI

1710 CC! ATG MAC CCL l01 Not LOn Lrg

CC! Arg 1675 ATC CT? CTG CCI GCC ACI ACC 210 Vol Lou Pro Ala Thr Tbr

Vol ACO CGC TSr Avg 2130 2115 GGCCCCL C CMa CMa CT? CM TIC CCC CC& TNT 017 Ala Bar Clni Cln Vol Glu Lou Pro Pro Ph. 2220 2205 COG TNT LT? CGC TTC GCOT CCL TNT TIC CCC CCL Ar; Phe 21. Ar; Pb. Ala Ala Ph. Lou Pro Ar

Vol Ala An; Gln Ar; 2190 CCC CM MAC CCC GAC AC GCM Pro Glu AOin Pro LOp Oar Glu 2205 2260 2295 CMA ATC TIC TCA CMG COT AT? GCC CLT TAC COT TAC Olu 21e Pta Oar Clni Arg 21e Ala Asp Tyr 017 Tyr sot!!! 2355 2370 2385 CCC CMk CCL CMA CMG TIC CM CTA CNT TCI CCC CAT Ala Clu Pro Glu Clii Lou Glii Vol Loeu Oar Ala His

CC GM TTO TM OM TG Glu Loeu Tyr ALla Vol 2535 TIC TOO MC GCh CCC Lou Tip LOss Ala Ar; 2025 CCC CAT 010 CAT TTA Pro Lop Lou LOp Lou

1440

ATO GCCL CC TOO CCL Not Ala 017 Tip 017 1530 ACA CCC CCC CCC COT TSr Pro 017 017 Gly 1020 MC TIC CCO OCT GCC Oar Lou Pro 017 01y

astOZI

AMA LTC CC! Pro ATC Ile TCT Oar

CyO Lop Ala Vol Lop Lys I1e 1725 CAT T!C CCC CAT AT? CC! TNT 3±6 nta pro Lop I1a Ar; Pta 1615 TC AMA CCC &CM 1 TO OTO L210 LYa Pro Glu Lou Vol Vol

ACV LAnAsLp CAT mT CAT

Lop 017 I1. Ala Lyo Tb? Ala Glu Trp Ala

Pv'jI! 2490

CCC CMC CMG CGC GGC ATA Ala Clni Clu Arg 017 Ile 2560 AT? AM COT CCC OTO AT? I1e Lys Pro 017 Vol Ile 2070 MTO CTG ACC AMA GAT CTC Vol Lou Thr Lye Asp Lou

2505

CMA CAT GGC CAT LOT Clni Lop Gly Lop Thr 2595 TGC AT? CAC CM CGG Cys I1e His Clu 01y

2520

CT? CCC. Vol Arq 2010 GCCL TOO

Ala Tip 2700 2005 CCC AGC TCC CGG CTO CCL ATO Pro Sar Oar Ar; Lou 01y Hot

Sql!!

2775 2700 2730 2745 2715 GCT OTO COG OTA ATA COG CCC TOO CAT CCC TOO AAM MT ACA ACO OTC CCC MAC TOA CAC TTA CAC COT TIC MAC Ala Val Lrg Vol I1e Lrp Am; Tip His oly Tip Lys Anni Thu Thr Vol kip LOU 2605 2605 2550 2520 2635 MhT CCL TOT CCC TMG TIC OCT TIC ATC CTOe CCL TOC GCCL ATC CLC MT aTO AMA ACC CTO TOC OTG GTA AMA AT? 2695 2940 2910 2925 2955 TN TIC ATA AMC CTC CMG CAT CMG GTO COO ATO G CCOTG CTC CAC ATA CTC CAT CAG CGC CTT ACC MAT ACC GCG 3030 3015 3000 2965 3045 TOO "C AM CAT CGC TOO CMG AMA TCC GCC TIC CAT MAT GCOT GAC AMA ACC GAO MOG CT? ACC GTC TIC TTC CCL

GT? CCC CMC MT CAT TCA CCC TIC

3075

3090

3105

Hindlll 3120

3135

2790 CAC CGC CCL GCOT CAT

2660 TAT CGC COG TIC AT? 2970 CCT GAC GGC CTT CGG

30600

GAC CCL CTT TTG CGC 3150

AMG ATA CCC ATC CCC ACC AMC CCL LTC CMG TCA CGC CMG TM TIC GCOT TTT ATA MCr CCL TOO CCC CAG CT? CIA CT? TCC MOC 3195 3210 3240 3100 3225 3105 TOG MOG ATC 0CC 000 MGT TOT CM COT TOO COT TCC CCL LTC ATO OTT TAT TIC CCC CAT MCC MC MCC AGC CMA CCL CAT CAT 3300 3205 3330 3255 3270 3315 CCL GCC CMC ATO COT CCL TM MO CMG TM CMG AT? GTO GTG CCCa ACA MAC TIA AM CCL COT TNT TIrC MGT GCC TrA CAT AGG 0

3345

FIG. 3. Sequence of the bisC gyene. The sequence of 3,336 bp within the 3.5-kbp Hincll fragment containingz the bisC gene is shown. The position and sequence of an open reading frame capable of encoding a polypeptide of 80,905 molecular weight are also shown.

2198

NOTES

We thank A. del Campillo-Campbell and M. Martin for strains and unpublished information. This work was supported by Public Health Service grant A108573 from the National Institute of Allergy and Infectious Diseases. D. E. Pierson was a recipient of a National Science Foundation graduate fellowship.

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