Proc. Natl. Acad. Sci. USAVol. 89, pp. 643-647, January 1992Biochemistry

Opine catabolism and conjugal transfer Qf the nopaline Ti plasmidpTiC58 are coordinately regulated by a single repressor

(Agrobcterium tumefaciens/gene regujation/AccR)

SUSANNE BECK VON BODMANtt, G. THOMAS HAYMANt§, AND STEPHEN K. FARRANDt¶Departments of tPlant Pathology and IMicrobiology, University of Illinois, Urbana, IL 61801

Communicated by Luis Sequeira, September 16, 1991

ABSTRACT The Ti plasmids of Agrobacterium tume-faciens are conjugal elements whose transfer is strongly re-pressed. Transfer is induced by the conjugal opines, a group ofunique carbon compounds synthesized in crown gail tumors.The opines also induce Ti plasmid-encoded genes required bythe bacteria for' opine catabolism. We have cloned and se-quenced a gene from the Ti plasmid pTiC58, whose productmediates the opine-dependent regulation of conjugal transferand catabolism of the conjugal opines, agrocinopines A and B.The gene, accR, is closely linked to the agrocinopine cataboliclocus. A spontaneous mutant Ti plasmid, pTiC58Trac, whichconstitutively expresses conjugal transfer and opine catabo-lism, was complemented in trans by a clone of wild-type accR.Comparative sequence analysis identified a 5-base-pair dele-tion close to the 5' end of the mutant accR allele frompTiC58Trac. Analysis of lacZ fusions in conjugal transfer andopine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR canencode a 28-kDa protein. This protein is related to a class ofrepressor proteins that includes LacR, GutR, DeoR, FucR, andGlpR that regulate sugar catabolic systems in several bacterialgenera.

During interactions between Agrobacterium tumefaciens andits plant hosts, the bacterium senses and responds to severalplant-produced signals. For example, virulent agrobacteriause a two-component signal transduction system to sensesmall phenolic compounds released from wounded planttissue. In response, the bacteria express Ti plasmid-encodedVir functions, which, in turn, facilitate T-strand excision andtransfer to susceptible plant cells (1). The resulting plantneoplasias synthesize unique low molecular weight carboncompounds, called opines, which are thought to provide asource of carbon for tumor-colonizing agrobacteria (2).The opines also act as signals. For example, the Ti plasmid-

encoded functions required for opine catabolism are specif-ically induced by their cognate substrates (3, 4). In addition,conjugal transfer of the Ti plasmid, which is normally re-pressed, is induced by a subclass of opines, the conjugalopines (5, 6). A model proposing that conjugation and catab-olism of the conjugal opines are coregulated emerged fromthe observation that mutants constitutive for conjugation areoften derepressed for conjugal opine catabolism. Similarly,mutants selected for constitutive expression of these opinecatabolic functions are generally transfer-constitutive (5,7-9).For nopaline-type Ti plasmids such as pTiC58, conjugation

is induced by the sugar phosphate opines, agrocinopines Aand B (6). We report here that the coregulation of agrocino-pine catabolism (Acc) and conjugal transfer (Tra) of pTiC58is mediated by a repressor, AccR. This repressor is related to

negative regulatory proteins that control sugar catabolicoperons in several unrelated bacteria (10). Our findingsprovide a molecular framework for the coregulation modeland illustrate the diversity of regulatory mechanisms thatgovern the interaction between Agrobacterium and its planthosts. 11

MATERIALS AND METHODSStrains and Plasmids. A. tumefaciens strains used were

C58 (11), NT1(pTiC58Trac) (12), C58ClCE(pWI1003) (8),NT1(pAgK84-A1) (13), and C58C1RS (rifampin resistant,streptomycin resistant) (8); Escherichia coli strains wereDH5a, RR1, and S17-1 (14). Broad host range vectorspRK415 (15) and pLAFR6 (16) were used for subcloning andcomplementation analysis; pCM1 was the source of the catcartridge (17); and pUC18 served as cloning vector for DNAsequencing.

Reagents. Antibiotics, 5-bromo-4-chloro-3-indolyl 8-D-galactoside, o-nitrophenyl 13-galactoside, and nopaline werepurchased from Sigma.Mutant Construction. Plasmids pTHB58 (18) and pSVB20

(12) were mutagenized with Tn3HoHo1 (19), and the muta-tions were homogenotized into Ti plasmids by standardmethods (20).Agrocin 84 Sensitivity Assays. Because amounts of purified

agrocinopine A and B sufficient for catabolic studies are notavailable, sensitivity to agrocin $4, assayed on Stonier me-dium plates as described by Hayman and Farrand (18), wasused to monitor expression of acc. Uptake of agrocin 84depends upon the acc-encoded opine transport system andtherefore serves as a measure of acc expression (18). Strainsto be tested for sensitivity were grown in LB medium andwashed in 20 mM phosphate buffer (pH 7.0) before additionto the soft agar overlays. Induction by agrocinopine wasassessed by placing paper strips impregnated with -20 nmolof agrocinopine onto the overlay agar (18).

Conjugal Transfer Assays. The quantitative spot matingtechnique used in these studies has been described (12). TheTi plasmidless recipient strain C58C1RS was spread over thesurface of an AT minimal medium (6) selection plate con-taining streptomycin, rifampin, and a mixture of nopaline (1mM) and arginine (9 mM). Ten-microliter volumes of serialdilutions of donors were spotted onto the recipient plate. Foropine induction, donors were grown overnight on small agarblocks containing -100 nmol of agrocinopine A and B. Thecells were resuspended in 0.5 ml ofAT minimal medium anddiluted, and 10-.lI sam,3les were spotted onto recipient plates.Plates were incubated at 280C for 2-3 days and colonies

Abbreviations: ORF, open reading frame; CAT, chloramphenicolacetyltransferase; RB, ribosomal binding.§Present address: U.S. Department of Agriculture, Peoria, IL 61604.tTo whom reprint requests should be addressed."The sequence reported in this paper has been deposited in theGenBank data base (accession np. M81646).

643

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644 Biochemistry: Beck von Bodman et al.

within the spots were counted. Conjugation frequencies areexpressed as number of transconjugants per input donor.

,f-Galactosidase Assays. Strains were grown in AT minimalmedium to an OD6w of 0.2. The cultures were divided intotwo tubes, a solution of agrocinopines (final concentration,200 ttM) was added to one set, and incubation was continuedfor 3 hr. Levels of 8-galactosidase were measured as de-scribed by Stachel et al. (19).DNA Sequencing. EcoRI fragments 26 from the wild-type

and mutant Ti plasmids were cloned into pUC18 and bothstrands were sequenced using the Sequenase kit, version 2.0(United States Biochemical). The DNA sequences wereanalyzed using the DNA* computer program (DNASTAR,Madison, WI).Chloramphenicol Acetyltransferase (CAT) Assay. Cells

were grown in LB broth to late exponential phase, andcell-free extracts were obtained by sonication. CAT proteinwas detected using the CAT ELISA kit marketed by 5 Prime

3 Prime, Inc., following the recommended procedure.

RESULTSMutations and Genetic Complementations Identify a

pTiC58-Encoded Function that Regulates tra and acc. Twocosmid clones, pTHB112 and pTHB55, repressed conjugaltransfer and acc-encoded supersensitivity to agrocin 84 whenin trans to the spontaneous Trac/Accc mutant Ti plasmidpTiC58Trac. These two cosmid clones contain DNA insertsfrom pTiC58 that overlap the agrocinopine catabolic locus(acc) and Tra region I (Fig. 1). pSVB20, a similar cosmidclone from pTiC58Trac, showed no effect on either pheno-type (data not shown). We then subcloned regions of overlapfrom the wild-type and mutant cosmid clones into pRK415.pRKW26, containing EcoRI fragment 26 from wild-typepTiC58, repressed conjugal transfer to levels below 10-8 perinput donor. This repression was largely overcome by addi-tion of agrocinopines (Fig. 2A). Similarly, pRKW26 re-pressed the Accc phenotype; merodiploid strains containingthis clone were nearly resistant to agrocin 84 (no zone ofgrowth inhibition) (Fig. 2B). pRKC26, containing EcoRIfragment 26 from pTiC58TraC, had no effect on the Trac orAccc phenotypes (Fig. 2).

T-REGION

DNA Sequence Analysis of the EcoRI 26 Region. Fig. 3Ashows a 1190-base-pair (bp) DNA sequence of the wild-typeEcoRI fragment 26 starting from the left EcoRI site. Com-puter analysis identified four open reading frames (ORFs).ORFI, which could encode a 28-kDa protein, begins with aTTG (bp 322) or a GTG (bp 325) and terminates with a TAGat position 1096. The two potential initiation codons arepreceded by sequences that match the E. coli ribosomalbinding (RB) consensus sequence. Upstream from the po-tential initiation codons are sequences similar to the virE -10region (22) and with nearly perfect identity to the E. coli -35consensus sequence (23). In addition, the potential -10region is part of a 17-bp palindrome (CGCTCAIAGTAT-GiAGC~G). A similar repeat (GCCTCATTCAACAiA C) ispresent in the 3' noncoding region that separates ORFi fromORF4. ORF2 begins with an ATG at bp 595 and is in framewith ORFi. It is preceded by a potential RB site and - 10 and-35 sequences that resemble those of the virC promoterregion (22). ORF2 could encode an 18-kDa protein. ORF3proceeds in the opposite direction from an ATG at bp 960 andlargely overlaps ORFs 1 and 2 (Fig. 3B). There are noidentifiable 5' regulatory features associated with this ORF.ORF4 reads in the same direction as ORFi and ORF2,beginning with an ATG at bp 1156 (Fig. 3B) and extendsbeyond the right EcoRI site (data not shown).The sequence from pTiC58Trac is identical to that of the

wild-type Ti plasmid except for a 5-bp deletion (bp 336-340)in the ORFi (ORF3) coding region (Fig. 3). This deletioncreates a frame shift in ORFi that results in a new stop codon22 bp downstream from the site of the mutation (Fig. 3A). Thedeletion is outside of and 5' to ORF2.ORF1 Encodes a Protein Homologous to Regulators of Other

Sugar Catabolic Operon Systems. In a search of the NationalBiomedical Research Foundation Protein Information Re-sources data base, the regulatory proteins GutR (10), FucR(10), and GlpR (24) from E. coli showed between 26% and34% amino acid identity with the ORF1-encoded protein. Inaddition, the ORFi amino acid sequence aligns well withthose of the LacR proteins from Lactococcus lactis andStaphylococcus aureus and with DeoR, FucR, and GutRfrom E. coli (10) (Fig. 4). Although there are stringentlyconserved amino acids throughout the sequences, regions of

749 1Traill

1 289

Tral acc

['oIs 20 1 4 2_33_l2i 61-6 -

I 1-- I I---- - -I i I I I +

15 120 '25 .t

pTHB58

pTHB55A* pWI 1003

pSVB20

pTHB1 12

pTHH26--

FIG. 1. Organization of the pTiC58tra/acc region. The positions of theTn3HoHol lacZ fusions in acc (289) andTraIl (749) are shown as flagstaffs withthe transcription polarity indicated by thedirection of the flag. "R" denotes thecoding region for the tra/acc repressor.The lines below the map represent clonedsegments of the Ti plasmid: pTHB55,pTHB58, and pTHB112 are cosmidclones derived from wild-type pTiC58(18), whereas pSVB20 contains an insertfrom pTiC58Trac (12). Plasmid pTHH206is the smallest subclone encoding a func-tional acc locus (18). Arrowheads indi-cate that the clones extend beyond themap. The deletion in pWI1003 is indi-cated by a hatched box. Coordinates(kilobases) are from Depicker et al. (21).

EcoRl

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 645

A

NT1(pTiC5BTra )

C58

NT1 (pTiC58Tra )

+ pRKC26 -<

+ pRKW26 -<

Agrocinopine

B

NT1(pTiC58TraC) iNT1(pTiC8TraC? NT1 (pTiC58TraC(pRKW26) (pRKC26)l

FIG. 2. Regulation of Tra and Acc phenotypes by a cloneencoding the tra/acc repressor. (A) The top plate shows conjugaltransfer of pTiC58Trac and its wild-type parent, pTiC58 (strain C58),grown in the presence (+) or absence (-) of agrocinopines A and B.The bottom plate shows the in trans effects on constitutive transferof pTiC58TraC by pRKW26 (wild-type) and pRKC26 (mutant). (B)Sensitivity to agrocin 84, as determined by a zone of growthinhibition around a colony that produces the antibiotic, was used tomonitor the activity of the acc locus. The uptake of agrocin 84 intothe bacterium requires the acc-encoded transport system (18) andtherefore serves as a measure of acc expression. Each agrocin 84plate, prepared as described in the text, was overlaid with strain C58(upper row) or strain NT1(pTiC58TraC) (lower row) with or withouta clone encoding the wild-type (pRKW26) or the mutant (pRKC26)repressor function. Induction ofacc is indicated by an enhanced zoneof growth inhibition surrounding the agrocinopine-impregnated pa-per strip (18).

greatest identity are within a 22-amino acid sequence at theN terminus and an 11-residue sequence in the C-terminaldomain.

Repressor Activity Is Contained on a 920-bp Fragment. A920-bp HindIII/Xma III fragment containing ORF1, ORF2,and ORF3 from pRKW26 was cloned into pLAFR6 as pOW1.This fragment repressed both constitutive phenotypes con-ferred by pTiC58TraC. As with pRKW26, conjugal transferwas inducible by addition of agrocinopines A and B (data notshown). The homologous fragment from pTiC58Trac, clonedas pOC1, had no effect on the Trac/Accc phenotypes asso-

ciated with the mutant Ti plasmid.To determine which of the ORFs in pOW1 are transcribed,

we inserted a promoterless cat cassette in both orientationsinto the Sal I site located within the coding regions of all threeORFs (Fig. 3). Strain NT1 transformants showed resistanceto chloramphenicol only when the orientation of the cat gene

coincided with the transcriptional direction of ORF1 andORF2 (data not shown). Strains containing the cassettecloned downstream from the deletion in pOC1 were asresistant to chloramphenicol as those strains harboringpOWl::cat. These results were confirmed by direct ELISAsof cell-free extracts using antibody directed against thecat-encoded protein (data not shown).These results are consistent with the hypothesis that ORF1

encodes a repressor that negatively regulates conjugal trans-fer and agrocinopine catabolism. We propose that the gene becalled accR and its product, AccR.pOWi Regulates Transcription of lacZ Fusions in Ira and

acc. We constructed lacZ reporter fusions in acc (acc289) andTra region II (tra749) (Fig. 1). tra749 was homogenotized intopTiC58Trac, whereas acc289 was marker exchanged intopTiC58 and pTiC58TraC. The homogenotized acc::lacZ fu-sion in both plasmids abolishes sensitivity to agrocin 84,whereas tra749 confers a Tra- phenotype on pTiC58Trac. Inthe absence of opine, a strain harboring pTiC58TraCacc289produced levels of 8-galactosidase 500-fold higher than thosepresent in a strain containing pTiC58acc289 (Table 1). Intro-duction of pOW1 reduced the f8-galactosidase levels in theconstitutive mutant almost 300-fold (Table 1). No such effectwas seen in merodiploids containing pOC1. Repression me-diated by pOW1 was not reversed by the addition of agro-cinopines. This was expected since insertions in acc abolishtransport of the opine; such mutants cannot take up theinducer (18). The strain harboring pTiC58TraCtra749 pro-duced high levels of p-galactosidase activity when grown inthe presence or absence of agrocinopines (Table 1). Intro-duction of pOW1 repressed /3-galactosidase levels 400-fold,but in this case the effect was reversible by addition ofagrocinopines. As with the acc::lacZ fusion, pOC1 had noeffect on the constitutive expression of the tra::lacZ fusion.A Spontaneous Trac/Acc- Mutant of pTiC58 Contains a

Deletion in EcoRI Fragment 26. Most spontaneous Tracmutations in pTiC58 also confer constitutive expression ofacc (8). However, Ellis et al. (8) described a Trac mutant thatconcomitantly became Acc-. Southern hybridization analy-sis of pWI1003, the Ti plasmid in this mutant, identified an= 1.1-kilobase (kb) deletion located within EcoRI fragment 26and extending slightly into EcoRI fragment 16 (Fig. 1). Thisdeletion removes a portion of accR as well as the 5' end of theacc operon.

DISCUSSIONWe have localized, determined the DNA sequence, and ge-netically evaluated a region of pTiC58 encoding a regulatorygene whose product coordinately represses conjugal transferand conjugal opine catabolism. The gene, called accR, islocated between acc and Tra region I (Fig. 1). Optimalexpression of both phenotypes requires the cognate sugarphosphate opines, agrocinopines A and B. Several lines ofevidence indicate that the repressor is a protein encoded byORF1 (Fig. 3). (i) A spontaneous 5-bp deletion mutation in the5' end ofthe coding sequence results in constitutive expressionof tra and acc (Fig. 3). This deletion introduces a translationtermination codonjust downstream from the mutation. (ii) catfusions within this coding sequence express only when thetranscriptional polarity coincides with that of ORF1/ORF2.This eliminates the possibility that ORF3, which reads in thereverse orientation, is active. However, the fact that the catgene expressed from pOCi1::cat indicates that the deletionmutation does not affect transcription of the region. (iii) Thepredicted protein encoded by ORF1 shows overall structuralsimilarities to proteins that regulate expression of other sugarand nucleoside metabolic pathways (Fig. 4).The amino acid sequence identities between AccR and the

five regulatory proteins of the LacR family extend over theentire lengths of the proteins (Fig. 4). One conserved region

Biochemistry: Beck von Bodman et al.

646 Biochemistry: Beck von Bodman et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A

EaoRIM . . .a A A T T C C A A C T G G T T T C G T A T T G T G G T C G T G C 0 G A G T A GC G C T G A T C C G C G C C G G T T T T G T T 0 A C G G C A G

T T C GA C A T T GA A T GA G T CA T CA CT C CT C CT A GA A G C T T AC GA T T T T G G T G C G G T G C GA A A G T GA G C GA T T

. HIndd I I I *lT C GA T C A C A C T A A C A A A G G C T T C C G C T T A G C T A C C C GA AG C T T C T T GA T C C C C A A C A A T G C G T A A T C G T T

0 0 . 0 .- 3S5T G T A A T G T A T T G T T G C G T T G C A A C A A A A CT G T C A C C A A ACT T T C A T TTLAC AA G A C GA T T A C G CT C A T

____ *. . Rn Rs. . D 0

AOT AT GA 0 C GGTATAGACCATTTCTTGTTAA AAOCGTT OOT OT T CAACT CAACT CAGGA CCGCCAL V F N S T a D R 0

GGC G A A AT C GT T GA GC T T T T GC GC GA T GA GC A GT T T T TGGC TAT C GG C A GA T T GA C GGA G C ACT T C C AGA K V E L L R D E 0 F L A G R L T E H F 0

A T T T C G G T G G C C A C T G C G A G G C G C GA C T T GA G C GAA C T CC A C GA G G C C G G A C T C T T G C G T C GA A C T C A C GS V A T A R R D L S E L H E A G L L R R T H G

* . . -3 . . -10GCGGTGCGGTCAGCGTCACGCAGGTTACACAGOACAA0CCGAACGCTGCCCGCGCCGTCTGGAATCGGGC

G A V S V T a V T a D K P N A A R A V W N R A

GGAGAAGGCGGCCATAGCCaOAsTCGTTGCCGGTATOATCGTCGAGGGTGACACGGTTCT.CTGGACGCCE K A A A G V V A G U V E G D T V L L D A

GGT ACAACT GCGCT GGAAGTTGCCAAGAAGCT CGCT GACCGCAGAAACCT CACCT T CAT CT CGAAT GGT CG T T A L E V A K K L A D R R N L T F S N G L

T CGACAT T GT CGAGGA A T T GA CGAGA GGGGAGGGCAAAAGCAT CT A T T CGGT CGGGGGCGAGT AT ACCGAD V E E L T R G E G K S Y S V G G E Y T E

* . . . . SaIlA A C GA A C C GC T C CT T C C GC GGC C CT T T GGC GGA G C A GT TT AT C C GC C A GT T C A AT GT C GA CA A ACT CAT TT N R S F R G P L A E a F R 0 F N V D K L

C T C A A C G C G G C G T C GA T C GA T G T T GA T C G C G GA T T GA T CT G T A C G T C G T C G C C C G T GA A C G C GA G T G T C GL N A A S D V D R G L C T S S P V N A S V A

C T C G C G C C A T GA T C GA A G T T T C G A G C C G C G T G A T C G T C GT C G C G G A T C A T T C G A A A T T C A C A A A A T C G A GR A M E V S S R V V V A D H S K F T K S S

C CT CT C GGT GA C GGC C A GGA T C GA GGA T GT C GGC GT GA TC GT C A C C GA CT CT GGA A C C C GA A C CAT CAT TL S V T A R E D V G V V T 0 S G T R T

* . . . . Xms II IGA GA C A A TA C C GG A AA GC T GC GGA A GA A GT T C GT T GT TGC GA A C TA GGT T C GA C GOGC C C GC GC GA AT TE T P E K L R K K F V V A N a

oR . . . .A A AG C CT CAT T C A A C A GAG CT G OGAGO AA A A ACCAC.CT TAA GA C C A AT C G C AG GA AT T T TAT GA T G G G

cr:

/

TnG/GTGORF1 H-

322

ORF2

0

\1 /

(D (0

CMcoco co

tO o0 0)

co

CY)

-_ TAG

1096

ATG

1156

UD

C>iQ

70

140

210

20

350

420

450

580

700

770

840

910

980

100D

112D

1190

21

0

(.0

C't

ORF4

ATG _.ON. TAG......595

AGT GCTAORF3 -

336 960

1096

FIG. 3. Partial DNA sequenceand organization of ORFs withinEcoRI fragment 26 from pTiC58.(A) The sequence of the first 1190bp of EcoRI fragment 26 showsthe proposed TTG/GTG start siteof ORF1, preceded by two RBsequences. Upstream sequencessimilar to the virE -10 and the E.coli -35 promoter sequences areunderlined. ORF2 begins with anATG at position 595. The 5-bpdeletion in the DNA sequence ofthe fragment from pTiC58TraC isindicated by "D." The amino acidsequence translatable from ORF1and ORF2 is shown in one-lettercode. ORF3 begins at an ATG atposition 960. Two related invertedrepeats are indicated by divergentarrows. (B) The schematic map ofEcoRI fragment 26 shows the re-lationships of ORF1, ORF2, andORF3 and the location of ORF4.The cat cassette was inserted atthe Sal I site at position 826.

in the N-terminal domains of all five proteins has the pre-

dicted helix-turn-helix motifcommon to many DNA bindingproteins (10, 25, 26). The C-terminal domains feature a

second conserved sequence that may define an inducerbinding site (10).

Subclones containing ORF1 complement the AccC and TraCphenotypes associated with pTiC58TraC (Fig. 2). RepressionofTra by ORFi responds to agrocinopine induction (Table 1),which is consistent with ORF1 encoding the opine-responsive gene regulator. Ih contrast, repression of acc inthese merodiploids, as characterized by resistance to agrocin84, is not responsive to agrocinopine induction (Fig. 2B). Wehave no explanation for this observation. However, it ispossible that repressor-operator interactions regulating theexpression ofacc are more complex than those at the tra loci.The ORF1 gene product acts at the level of transcription

(Table 1), which is consistent with our proposal that accRencodes a repressor protein. The question remains as towhether ORF2 constitutes a separate functional gene. catexpresses in pOCi even though the deletion in ORFi shouldcause the translational termination of AccR and a concomi-tant polar effect on expression of the downstream antibioticresistance gene. This suggests that ORF2 may be separatelytranscribed.

Our results provide genetic and molecular data that con-firm the model of Ellis et al. (8) proposing that the regulationof tra and acc ofpTiC58 involves a common repressor. These

Table 1. .8-Galactosidase activities from acc and Tra II lacZgene fusions under repressed, constitutive, andagrocinopine-induced conditions

,B-Galactosidase activity,*units min- OD6j-o1

Without WithPlasmid content agrocinopine agrocinopinet

pTiC58Trac 2 NDpTiC58TraCtra749 1176 1522pTiC58TraCtra749/pOW1 3 560pTiC58Tractra749/pOC1 1314 1037pTiC58acc289 1 2pTiC58Tracacc289 584 192pTiC58TraCacc289/pOW1 2 2pTiC58TraCacc289/pOC1 482 422ND, not determined.

*Measured as described in ref. 19.tA mixture of agrocinopines A and B was added to a final concen-tration of 200 ,uM.

B

Proc. Natl. Acad. Sci. USA 89 (1992) 647

AccRLacRDeoRGuIRFucR

LV F NST DR AKIVELLRDE FAV AT A R R D SEH EA G L LRR T H GMKESLHMNKKRR[ IR | KIDGTITUKEIIDELDftSIDMTARRDLDLjEA[ LTR THG

METR R E EI LLQOELIKRSDK[[HLKDAAALLG VSEMTIRRDLNNHSAPVVL ---GMKPROR QAlLPYLOKQGKCS VEEAQY[UDTTGT|T|I|RNDLV E TV YGM K - A A DKLLNHT SgTTEAASgLKVgK ETRR DLN T GKIL G

AccR G AVASV T 0 V TFD- FIN A - AfA V WE A A G V V A G VLLDAGTTABEV KMALacR GAO LLSSKKPLE[SJTHI EKKSLNTK- EK I AKKACSLJ K DpT FI GPG TT L VL AL EWKDeaR GYI VLEPRSASHYLLSDQKSRLVE- EKRRAAKLAATLVEPDO LFFDGTTTPWI EAI DGutR G VvL - - - NKEESDnPPIDHKT L ET H KKE L IAEAAVSF H DSII LD AGST VQ M V P LL-FWCR RAKYIHRONO0NDSGDPFHIFL KSHYEHKDIAR E A L AW I EGMV AL ST C WY LE1R QLP

AccR DRR - FMSNG LD-EELIV- [ElYT N R LRQL[ENVDKLLacR G Y K R VITNS L P V F LI L NDSETI DLLLL- GGE RERI T G AFVGS T N L K A MR FAADOOR N E - -I PFFTAVCYSLNTFLAL KEKPHCRAFL- CGGEH A S[EA F KPI DAFQ@TLNNFGutR - S R F N I IT V M T|S L HFF ALSELDNEQTELMPGGRFRKKSA[ 3OKAE A F E HIF TFICGuIR-SRFNNITVMTNSLH~~~~~IVNALSELDNEOTE1LMPGGTFRKKSA ljHMOLANAFHTFID~FUCR [D I- - - NIaVFTNSHPICHEL GK[RERIa-LI[Sm GTLERKVGCVVN[SLIsaLKSLEI JLF

AccR 31 L N -AAlS| DVIDRflL| C T N7SSIP A RFAE1ARE3 E V S S R VVD H SK F TIK S SIL S T A R FE1DFVG VLacR FVR.ALtA-VT-.HNSW D K KEGVIaOLALNNAVEKFLLVIDIS 3KFIDRYDFFNFYNLDQLDTDeOR F Y SIA AG V H vJS K JA T F N -LEE L P V K H WA MS MA QK H V LV V DHS K F GEV R PAR MG DL KR F DIGMAR F MGT D G[ - - L N AGFVDJ T F N E V YT VIS KAM1C N A A R E[EjL MA D S S K F G R K[S]P NW]V C S L[IFSVVD KFuCR FF S C E G[-S SGALWD NA 7D YKSMLLKRAAasL L L K N RSG E A R G H L D EIVT H

ACCR 1 V T DSTRTI ETI PF KL[SK[EGFVVANLJCR TIDIN QI S P O H L E E F S VY T T L K A DDeOR V SDGCC P E D E Y V K Y AO TQRI 1RL M YGMAR LITITDA[1 DIPAFROAL[jEKGIDVIITGESNEFcAR [[JI S]EROVATSLVTA

FIG. 4. Amino acid sequence homologies between AccR and other repressor proteins. Amino acids showing exact matches between twoor more members of the family are boxed. Gaps were introduced to generate optimal alignments. The N-terminal region with a predictedhelix-turn-helix motif (10) is indicated by a bar-ine-bar. The potential inducer binding site (10) is indicated by a hatched bar. Sequences forDeoR, GutR, FucR, and LacR are taken from van Rooijen and de Vos (10).

workers described a second spontaneous TraC mutant thatwas, however, Acc-. We show here that this phenotyperesults from a 1.1-kb deletion that eliminates part of the accRcoding sequence and extends into ORF4, which encodes thefirst gene of the acc locus (unpublished results).The negative regulation ofagrocinopine catabolism by AccR

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