Vol. 175, No. 17JOURNAL OF BACrERIOLOGY, Sept. 1993, p. 5575-55840021-9193/93/175575-10$02.00/0Copyright © 1993, American Society for Microbiology

Genetic Analysis of the Agrocinopine Catabolic Region ofAgrobacterium tumefaciens Ti Plasmid pTiC58, Which

Encodes Genes Required for Opine and Agrocin 84 TransportG. THOMAS HAYMAN,1t SUSANNE BECK VON BODMAN,2 HEENAM KIM,3 PING JIANG,3 AND

STEPHEN K. FARRAND2,3*Department ofMicrobiology, Loyola University Medical Center, Maywood, Illinois 60153,1 and Departments

ofPlant Pathology2 and Microbiology,3 University of Illinois, Urbana, Illinois 61801

Received 26 April 1993/Accepted 1 July 1993

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacteriumtumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy.acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined bycomplementation analysis using subcloned fragments and transposon insertion mutations of acc carried ondifferent plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least fourare required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with thehypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first ofthese genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to belocated in the periplasm. The DNA sequence of the accA4 region revealed an open reading frame encoding apredicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar tothe periplasmic binding proteins OppA and DppA ofEscherichia coli and Salmonella typhimurium and OppAof BaciUus subtilis.

The diseases crown gall and hairy root are caused by thesoil bacteriaAgrobacterium tumefaciens andAgrobacteriumrhizogenes, respectively. In both cases, neoplasias in dicoty-ledonous plants result from the transfer and integration intothe plant genome of segments of large plasmids (Ti or Riplasmids) harbored by these strains. The transferred DNA,called T-DNA, contains genes which direct the formation ofthe neoplasia as well as the synthesis of novel compoundscalled opines. These compounds are produced and secretedby the tumors and can be utilized by the inducing agrobac-teria. The genes encoding catabolism of the opines are alsolocated on the Ti or Ri plasmid but are outside of the Tregion (5, 16, 25; see reference 47 for a review). Over 20different opines are known, and they comprise severalstructural groups (7-9, 11, 15, 16, 27, 28, 43; for a review, seereference 45). In addition to acting as nutritional sources, asubclass of opines functions as signal molecules, inducing Tiplasmid conjugal transfer (14, 36). Classical nopaline-typestrains of A. tumefaciens cause tumors that produce theopines nopaline, nopalinic acid, and agrocinopines A and B(5, 16). The agrocinopines are sugar phosphodiesters, andthey are the conjugal opines for this type of Ti plasmid (14,38).

Certain strains ofA. tumefaciens are susceptible to agro-cin 84, an antibiotic produced by avirulent Agrobacteriumradiobacter K84 (16, 35). Agrocin 84, an adenosine analog, isspecifically toxic to nopaline- and agropine-type A. tume-faciens strains and to someA. rhizogenes andA. radiobacterisolates (23, 29, 34). Susceptibility to this antibiotic is due touptake of agrocin 84 mediated by a transport system en-

* Corresponding author.t Present address: Phytoproducts Research, National Center for

Agricultural Utilization Research, USDA Agricultural ResearchService, Peoria, IL 61604.

coded on the Ti, Ar, or At plasmid (16, 23, 25, 35). Theillogic of a system specific for uptake of a toxic compoundled to the search for the true substrates of this transportsystem. This resulted in the discovery of agrocinopines Aand B (16). Several lines of evidence indicate that agrocin 84and the agrocinopines both are taken up by this Ti plasmid-encoded transport system. First, Ellis and Murphy (16)showed that agrocin 84 susceptibility and transport wereboth induced by agrocinopine A, suggesting a link betweenthe opine and the antibiotic. Second, spontaneous mutantsconstitutive for agrocinopine transport are also supersensi-tive to agrocin 84 and take up the antibiotic at an acceleratedrate (17, 22). Third, spontaneous and transposon-inducedmutants resistant to agrocin 84 no longer transport agrocin-opine A or the antibiotic (16, 22). Lastly, agrocinopine Ainhibits transport of agrocin 84, presumably by a competitivemechanism (16).The region of pTiC58 responsible for agrocinopine catab-

olism and susceptibility to agrocin 84, called acc, is locatedat kb 130 on the Ti plasmid map (22). It is approximately 6 kbin size, and is transcribed in a clockwise fashion at arelatively high basal level. This is consistent with the factthat strain C58 is susceptible to agrocin 84 in the absence ofthe inducer opines. Both agrocin 84 susceptibility and acctranscription are induced to higher levels by either of the twoopines. All mutations within the acc region abolish transportof both agrocin 84 and the agrocinopines and also suscepti-bility to the antibiotic (22, 25). Extensive analysis of mutantshas failed to separate these three phenotypes, stronglysuggesting that they all are associated with a single region ofDNA.

Here, we report a detailed genetic and structural analysisof the acc region, including studies of opine catabolism,genetic complementations, analyses of acc-specific proteinprofiles, and the DNA sequence of a part of this region.

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TABLE 1. Strains and plasmids

Strain or plasmid Relevant phenotype' Source or reference

StrainsE. coliLCD44 Rec- MetF L. C. DeVeaux1830(pJB4JI) Km Nm Cm Gm 20

A. tumefaciensC58b Agrs Acp+ 22NT1 Agrr Acp- 22C58C1RS Rf Sm 22UIA143 Agrr Acp- Rec- 19NT1(pAgK84-A1) Agr+ Agr' Km 20NTI(pTiC58Trac) Agr'S Acp+ Trac C. Kado

PlasmidspCP13 Km Tc 10pSal52 Cm Gm Km Sp 44pTHH206 Agrs Acp+ Cm Gm Km 22pTHB112 Agrs Acp+ Tc 22pTHSB5 Agrr Km Sp This studypTHSE16 Agrr Cm Km This studypTHSE26 Agrr Cm Km This studypTHSH11 Agrr Cm Km This studypTHSS9 Agrr Km Sp This studypTHVEl6 Agrr Tc Km This studypTHVHll Agrr Tc This studypTHB422C Agrr Acp- Tc Cb 22pTHB426C Agrr Acp- Tc Cb 22pTHB476C Agrr Acp- Tc Cb 22pTHB555C Agrr Acp- Tc Cb 22pTHB566C Agrr Acp+ Tc Cb 22pTHB578C Agrs Acp+ Tc Cb 22pTHB587- Agr' Acpm TcGb 22pTHH30ld AgrF Cm Km Nm This studypTHH306d Agrr Cm Km Nm This studypTHH312d Agrr Cm Km Nm This studypTHH313d Agrs Cm Km Nm This studypTHH317d AgF Cm Km Nm This studypTHH348d AgF Cm Km Nm This studypTHH349d Agrr Cm Km Nm This studypTHH352d Agr Cm Km Nm This study

a Symbols: Acp, agrocinopine transport; Agr', agrocin 84 immunity; Agr+, agrocin 84 production; Agrf, agrocin 84 resistance; Agr5, agrocin 84 susceptibility;AgryS, agrocin 84 supersensitivity; Cb, carbenicillin resistance; Cm, chloramphenicol resistance; Gm, gentamicin resistance; Km, kanamycin resistance; Met,methionine auxotrophy; Nm, neomycin resistance; Rec-, recombination deficiency; Rf, rifampin resistance; Sp, spectinomycin resistance; Sm, streptomycinresistance; Tc, tetracycline resistance; Trac, constitutive conjugal transfer.

b Contains pTiC58.Tn3-HoHol insertion derivative of pTHB112 (22).

d TnS insertion derivative of pTHH206.

(A preliminary report of some of these results has beenpublished previously [21].)

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains and plasmidsused in this work are listed in Table 1. Agrobacterium strainswere grown at 28°C, and Escherichia coli strains were grownat 37°C. pTHB112 is a large cosmid clone derived frompTiC58 and contains the entire acc region located centrallyon its insert (22).

Media. Nutrient agar, L broth, AB minimal medium, ATNminimal medium, and Stonier's medium have been describedelsewhere (22). M63 medium containing 0.4% glucose and 20,ug of methionine per ml (42) was used to grow E. colimaxicell strain LCD44. Antibiotic concentrations (in micro-grams per milliliter) were as follows: chloramphenicol, 25;

gentamicin, 50; kanamycin, 50; neomycin, 150; rifampin, 50;spectinomycin, 150; streptomycin, 200.

Chemicals. Agrocinopines A and B are both biologicallyactive (14, 16). Mixtures of the two opines isolated fromtumor extracts (generously provided by M. Ryder) were notpurified further except for catabolism experiments as notedbelow.

For growth experiments, crude extracts containing agro-cinopine B were biologically purified by incubating in ATNmedium (22) with A. tumefaciens NT1 at 28°C for 5 days.This strain lacks a Ti plasmid and cannot catabolize theopines. However, it can utilize other catabolites contaminat-ing the opine preparation. The cells were removed bycentrifugation, and the supernatants were further purifiedessentially by the method described by Ryder et al. (38).Chloroform sterilization of the spent culture medium con-taining agrocinopine B was followed by Dowex AG1-X2

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(acetate form) chromatography. The column was washedwith several volumes of distilled water and developed withan increasing pyridine acetate 10-step gradient (20 mMpyridine, 10 mM acetate to 200 mM pyridine, 100 mMacetate). Fractions (1 ml each) were collected, and thosegiving a positive reaction with the phloroglucinol reagent(12) were pooled. To convert the opine to a sodium salt, thepooled fractions were applied to a 1.5-ml bed of Dowex 50(Na+ form). The column was washed with distilled water.Again, the phloroglucinol-positive fractions were collected,pooled, evaporated to dryness, and redissolved to a concen-tration of 50 mM agrocinopine B.

Plasmid DNA isolation, manipulation, and transformation.Plasmid DNA was isolated by the method of Hayman andFarrand (23) and the alkaline lysis method of Maniatis et al.(32). Procedures used for restriction endonuclease digestionand agarose gel electrophoresis of plasmid DNA have beendescribed previously (22, 33). Recombinant plasmids weregenerated by ligation using T4 DNA ligase (GIBCO BRL).Procedures for transforming agrobacteria with plasmid DNAand for mobilizing plasmids into agrobacteria have beendescribed previously (22). Transformation of E. coli wascarried out in accordance with the method of Maniatis et al.(32).Transposon mutagenesis. Plasmid pTHH206 was muta-

genized with TnS by using pJB4JI, as described previously(20). Cells from filter matings between E. coli 1830(pJB4JI)and A. tumefaciens recipient strain C58C1RS(pTHH206)were plated on nutrient agar containing neomycin, chloram-phenicol, rifampin, and streptomycin. Plasmid DNA isolatedfrom pooled colonies was used to transform E. coli HB101,selecting for neomycin resistance, to isolate the target plas-mid containing TnS insertions.Growth experiments. To test growth with agrocinopine B

as the sole carbon and energy source, appropriate strainswere cultured at 28°C in ATN medium containing 0.2%glucose to mid-exponential phase, harvested by centrifuga-tion, and washed twice with ATN medium. Microcentrifugetubes containmng ATN medium (15 ,ul) supplemented withpurified agrocinopine B (final concentration, 5 mM) wereinoculated with 1 ,ul of washed cells and incubated at 28°C.Samples (2 p,l each) were removed every 24 h with sterilecalibrated capillary tubes, and appropriate dilutions wereplated on nutrient agar plates for viable counts. Additional2-p,l samples were removed at selected time points andsubjected to high voltage paper electrophoresis (HVPE) todetect disappearance of the opine from the medium aspreviously described (22). Agrocinopine B was quantitatedby the phloroglucinol colorimetric method (12), with arabi-nose (Sigma) as the standard.Opine and agrocin 84 assays. Susceptibility to agrocin 84

and uptake of agrocin 84 and of agrocinopines were deter-mined as described elsewhere (22). Separation and detectionof opines by HVPE was performed as reported previously(39).

Maxicel analysis. Plasmid-encoded proteins were radiola-belled by using the maxicell technique as described bySilhavy et al. (42). Proteins were separated by electrophore-sis in 12.5% polyacrylamide gels containing SDS (SDS-PAGE) as described by Laemmli (30). For visualization ofproteins, gels were subjected to fluorography (6) and ex-posed to X-ray film (XAR 5; Kodak) at -80°C.

Fractionation and analysis of periplasmic proteins.Periplasmic protein fractions were prepared from Agrobac-terium strains essentially as described by Schardl and Kado(40). Strains to be assayed were grown until late exponential

phase in 200 ml of L broth containing appropriate antibiotics.The cells were harvested by centrifugation, washed twicewith 0.9% NaCl, and resuspended in ice-cold Tris-HCl-EDTA-sucrose (pH 7.4). Proteins present in the osmoticshock fraction were separated by SDS-PAGE (12% poly-acrylamide) and visualized by staining with Coomassie blue.DNA sequencing. Sequencing of both strands of DNA

fragments cloned into vectors pUC18 and pUC19 was per-formed with the Sequenase kit, version 2.0 (U.S. Biochem-icals), in accordance with the instructions provided by themanufacturer. DNA sequence analysis was performed withDNA* (DNASTAR).

Nucleotide sequence accession number. The nucleotidesequence shown in Fig. 5 has been assigned GenBankaccession number L14678.

RESULTS

Agrocinopine B catabolism. The subcloned acc region ofpTiC58, contained on the 8.5-kb fragment insert inpTHH206, encodes agrocinopine transport and agrocin 84susceptibility (22). To determine whether this region issufficient to confer opine utilization as a sole carbon andenergy source, strains C58, NT1, and NT1 containing clonepTHB112 or pTHH206 were incubated at 28°C in ATNmedium containing 5 mM purified agrocinopine B. Biologicalpurification of the opine (see Materials and Methods) wasnecessary because Ti plasmid-less strain NT1 was able togrow in the presence of crude agrocinopine B preparations(Fig. 1A). All four strains were inoculated at the same initialcell concentration, but by 72 h strains C58, NTl(pTHB112),and NTl(pTHH206) increased to cell densities approxi-mately 2 orders of magnitude greater than that of strain NT1(Fig. 1A). Growth was accompanied by disappearance of theopine from culture supematants (Fig. 1B). Strain NT1,which did not show any increase in cell numbers, failed toremove the opine from the medium (Fig. 1).

Complementation of acc mutants with subcloned fragments.If, as previously postulated (22), the acc region consists of asingle transcriptional unit, it should not be possible tocomplement in trans Tn3-HoHol insertions within acc byusing subcloned fragments containing portions of the region.To test this, overlapping subclones carried in vector pSa152(IncW) (44) were constructed. None of these subclones bythemselves conferred susceptibility to agrocin 84 (data notshown). We generated a series of merodiploids by introduc-ing the subclones into Agrobacterium strains containingderivatives of pTHB112 with Tn3-HoHol insertions thatabolished acc expression (22). The recA Agrobacteriumstrain UIA143 (19) was used to prevent homologous recom-bination between the two plasmids. Each merodiploid strainwas tested for susceptibility to agrocin 84, as well as for thepresence of both plasmids. Results of the complementationanalyses are shown in Fig. 2. Restriction fragment EcoRI 26overlaps the insertions in both pTHB476 and pTHB426 butfailed to complement either mutation. However, BamHI-5,which extends further to the left and to the right, comple-mented both of these mutations. HindIII fragment 11 did notcomplement the insertion in pTHB426 but complementedthe insertions in pTHB566 and pTHB422. BamHI fragment 5and EcoRI fragment 16 also complemented mutations inpTHB566 and pTHB422. BamHI fragment 5 did not comple-ment the insertion in pTHB555, but HindIIl 11 and EcoRI16, which contain sequences to the right of BamHI 5, bothcomplemented this insertion mutation.We then tested pairs of subcloned fragments for the ability

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A

80

.0co

AcpB -

Ori

- OG

- xc

- F

0 24 48 720 66 0 66 0 66 0 66NT1 C58 NT1 NT1

Hours (pTHB112) (pTHH206)FIG. 1. Utilization of agrocinopine B as the sole carbon and energy source. (A) Growth assays were performed as outlined in Materials

and Methods. 0, C58; *, NT1; 0, NT1 cultured in minimal medium supplemented with an unpurified preparation of agrocinopines; A,NT1(pTHB112); V, NTl(pTHH206). (B) HVPE analysis of culture samples. Samples taken at the beginning (0 h) and the end (66 h) of thegrowth experiments were subjected to HVPE in formic acid-acetic acid buffer. AcpB, agrocinopine B; F, fructose; XC, xylene cyanole; OG,orange G; Ori, origin of electrophoresis; + and -, anode and cathode, respectively.

to reconstitute agrocin 84 susceptibility. A strain harboringEcoRI-16 [pTHVE16 (IncP1)] and BamHI-5 [pTHSB5 (IncW)]was not susceptible to agrocin 84. In contrast, a strainharboring pTHVH11 [HindIII-11 (IncP1)] and pTHSB5 wasfully sensitive to the antibiotic (Fig. 2A).

Strains containing either subcloned EcoRI-16 or sub-cloned BamHI-5 in trans to any of the Tn3-HoHol insertionsfor which complementation was observed showed a zone ofinhibition parallel to and completely surrounding the paperstrip impregnated with agrocinopines (Fig. 2B, plates 2 and4). This raised the possibility that these strains were sensi-tive to agrocinopines A and B as well as to agrocin 84. Totest this, Stonier's medium plates lacking agrocin 84 wereoverlaid with these merodiploid strains and paper discsimpregnated with agrocinopines A and B were placed on topof the overlays. Following incubation at 28°C for 24 h,growth of the strains was found to be inhibited by the opines.This inhibitory effect was most pronounced in merodiploidscontaining EcoRI-16 (Fig. 2B, plate 5). No such growth-inhibitory effect was observed when strains containing anyone of the five Tn3-HoHol insertion plasmids complementedwith subcloned fragment HindIII-11 or SmaI-9 were testedin a similar manner (Fig. 2B and data not shown).Complementation of acc mutants with TnS insertion muta-

tions. Results from these complementation analyses areinconsistent with our previous hypothesis that acc is tran-scribed as a single unit from left to right (22). To address this,the locus was further defined by complementing the Tn3-HoHol insertion mutants with a second set of insertionmutations generated in a compatible acc clone. This secondgroup of mutants was obtained by mutagenizing acc sub-clone pTHH206 with TnS as described in Materials andMethods. Over 60 insertions within pTlHH206 were mapped,and eight plasmids were selected for further study. Seven ofthe eight, when tested alone in strain UIA143, failed toconfer susceptibility to agrocin 84 (Fig. 3). These results areconsistent with the results of our previous analyses (22),indicating that acc is approximately 6 kb in size (Fig. 2 and3). The insertion in pTH313, which maps at the far left endof the cloned fragment, had an atypical effect on agrocin 84sensitivity. The zone of growth inhibition was somewhatobscured, and induction of supersensitivity to the antibioticby agrocinopines was abolished.We then tested the seven TnS acc insertion mutations for

their abilities to complement in trans the five Tn3-HoHolacc insertion mutations in pTHB112 (Fig. 3). Three TnSinsertions mapped to the right of the insertion in pTHH313,in the same area as the Tn3-HoHol insertions in pTHB476

FIG. 2. Complementation analysis of the acc region using pTHB112::Tn3-HoHol derivatives paired with subcloned fragments. (A)Restriction map of acc-containing cosmid clone pTHB112, showing the sites of Tn3-HoHol insertions (revised from that published previously[22]) and the results of the complementation analyses. Transcriptional direction and agrocin 84 phenotype for each Tn3-HoHol insertionconstruct (plasmid name prefix pTHB) in strain NT1 are indicated above the map. Restriction fragments shown below the map, cloned intopSal52 (44), were introduced into strain UIA143 containing pTHB112::Tn3-HoHol derivatives. Agrocin 84 susceptibility (+) or resistance(-) of merodiploids, determined by plate bioassay, is indicated above the complementing fragment for each of the five subcloned fragmentstested. Brackets indicate two subcloned fragments tested in the same strain. S, susceptibility; R, resistance. (B) Agrocin 84 bioassay platesof complementations depicted in panel A. Plates were inoculated in their centers with agrocin 84 producer strain NT1(pAgK84-Al) andoverlaid with different strains. Chloroform-sterilized paper strips containing 10 nmol of agrocinopines A and B were placed on the overlaysprior to incubation. Each of plates 1 through 4 is representative of all positive complementation assays using the indicated subclone. Overlaidstrains are UIA143(pTHB476, pTHSS9) (plate 1), UIA143(pTHB476, pTHSB5) (plate 2), UIA143(pTHB566, pTHSH11) (plate 3), andUIA143(pTHB566, pTHSE16) (plates 4 and 5). Plate 5 contains no agrocin 84. Instead, a chloroform-sterilized paper disc containing 20 nmolof agrocinopines A and B was placed on the overlay prior to incubation.

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AAGROCIN

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1KAE

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R A B C DI-F-- 4I--H-----S R R RRAGROCIN

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FIG. 3. Complementation analysis using strains containing paired pTHB112::Tn3-HoHol and pTHH206::TnS derivatives. Agrocin 84susceptibility (+) or resistance (-) of merodiploids, determined by plate bioassay, is indicated beneath the corresponding Tn3-HoHolinsertion and above the line representing the TnS derivative present in trans. TnS insertion positions are marked (V). Agrocin 84 reactionsfor TnS derivatives alone in strain NT1 are indicated on the right. S, susceptibility; R, resistance. The bar above the map indicates themaximum (open) and the minimum (hatched) limits of the acc locus. R indicates the location of the accR gene (2). A through E denote thefive complementation groups described in the text.

and pTHB426. These three TnS insertions, in pTH352,pTHH301, and pTHH349, complemented all of the Tn3-HoHol insertions except those in pTHB476 and pTHB426.The TnS insertion in pTHH312, within EcoRI fragment 16,complemented four of the five Tn3-HoHol insertions, in-cluding the one in pTHB422, but failed to rescue the nearbymutation in pTHB566. Similarly, the TnS insertion inpTHH306 complemented all Tn3-HoHol insertions exceptthe insertion in pTHB422. The TnS insertion in pTHH348,mapping within SmaI fragment 22, complemented all five ofthe Tn3-HoHol insertions. Lastly, the TnS insertion inpTHH317 and the Tn3-HoHol insertion in pTHB555 mapclosely together at the right end of the region. The latter isthe only Tn3-HoHol mutation that could not be comple-mented by the TnS insertion in pTHH317 (Fig. 3). Theseresults together indicate that acc consists of at least five

complementation groups which we designate accA throughaccE (Fig. 3).

Proteins encoded by the acc region. Recombinant plasmidscontaining all or a portion of the acc region were analyzedfor expression of encoded protein products in E. coli maxi-cell strain LCD44. Cells harboring plasmids pTHH206 andpTHSB5 expressed a protein of 60 kDa that was not seen inextracts of strains containing only the vectors (data notshown). A protein of 48 kDa was expressed in cells contain-ing pTHSE26. No acc-specific proteins were detected instrains containing pTHSE16 or pTHSH11 (data not shown).Maxicell analysis of Tn3-HoHol insertion derivatives ofpTHB112 showed that the 60-kDa protein was synthesizedby all mutants except those harboring pTHB476 andpTHB426 (Fig. 4, lanes 2 and 3). The 48-kDa protein was notsynthesized at detectable levels by any of these strains.

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60-

1 2 3 4 5 6 7 8 9

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FIG. 4. Maxicell analysis of strains haboring constructs contain-ing Tn3-HoHol insertions into the acc region. 3S-labeled cellextracts were subjected to electrophoresis in SDS-12.5% polyacryl-amide gels. Bands were visualized by fluorography. Insert-encodedproteins are indicated (-) on the left. Lanes 1 and 9 containedprotein size markers, and molecular masses in kilodaltons are

indicated on the left. Lanes 2 through 8 contained extracts from cellsharboring plasmids pTHB476 (lane 2), pTHB426 (lane 3), pTHB566(lane 4), pTHB422 (lane 5), pTHB555 (lane 6), pTHB587 (lane 7),and pCP13 (lane 8).

However, a polypeptide of 43 kDa was synthesized by thestrain harboring pTHB426, which contains a Tn3-HoHolinsertion at the right end of the region corresponding toEcoRI fragment 26 (Fig. 2A and Fig. 4, lane 3).DNA sequence analysis of accA. The entire double-stranded

sequence of the region encoding complementation group Awas determined (Fig. SA). This region contains a single,1,566-bp open reading frame (ORF) having a transcriptionalorganization consistent with the direction defined by thelacZ fusions in plasmids pTHB476 and pTHB426. This ORFcan encode a 522-residue protein of 59,147 Da. The N-ter-minal portion of this protein contains a putative signalpeptide motif and a consensus signal peptide cleavage site(Fig. 5A). A FASTA-based computer search of the GenBankprotein data bases uncovered significant homologies be-tween this protein and the periplasmic binding proteins ofthe oligopeptide and dipeptide transport systems of E. coliand Salmonella typhimurium and the oligopeptide transportsystem of Bacillus subtilis. Amino acid identities betweenAccA and these periplasmic binding proteins were between20 and 23%. Relatedness increased to about 65% whenconservative and neutral changes were considered (data notshown). The alignment between AccA and OppA of E. coli ispresented in Fig. 5B.

Periplasmic location of AccA. Periplasmic protein fractionswere prepared from strains NT1, NT1(pTiC58Trac),NTl(pTHB112), and NT1 harboring derivatives of pTHB112with Tn3-HoHol insertions in accA. This fraction from NT1harboring an intact Ti plasmid that expresses acc at the fullyderepressed level (22) contained a protein with an Mr ofapproximately 60 kDa (Fig. 6, lane 3). A protein of this sizewas absent in samples prepared from the Ti plasmid-lessstrain (Fig. 6, lane 2). A protein of the same mobility was

detected in the periplasmic fraction from a strain harboringthe acc+ clone pTHB112 (Fig. 6, lane 4) but was missing insamples from strain NT1 harboring the pTHB112 derivativescontaining transposon insertions in accA (Fig. 6, lanes 5 and

6). No proteins of altered mobility were detected in fractionsfrom either of the two mutant strains.

DISCUSSION

Growth studies with agrocinopine B as the sole carbon andenergy source show that acc, contained on pTHH206, en-codes functions sufficient for utilization of this opine. Agro-cinopines are, therefore, not only conjugal opines but alsocan serve as a sole source of carbon and energy. We usedagrocinopine B because agrocinopine A can be converted toagrocinopine B and glucose by several Agrobacteniumstrains, including NT1 (16). Ti plasmid-less strains can growon glucose or other contaminating nutrients in crude agroci-nopine preparations (Fig. 1A), and this would obscure opine-specific growth. Strain NT1 lacking the clone showed nogrowth and failed to take up the opine over the culture period(Fig. 1). Strain NT1 containing pTHB112 reached a highercell density than did NT1 containing pTHH206. This may bedue to plasmid copy number differences between pTHB112and pTHH206 (13, 18) or, possibly, to additional growthadvantages conferred by the larger cosmid clone.Our complementation analyses show that the acc region is

composed of at least five expression groups (Fig. 2A). Eachis less than 2 kb in size and may correspond to a single gene.The TnS insertion in pTHH313 lies not within the accstructural genes but, rather, in accR, the gene that encodesa negative regulator associated with the acc region (2). Thisinsertion interferes with the regulation of expression of theagrocin 84 susceptibility phenotype in a manner consistentwith its abolishing repressor activity. The TnS insertions inpTHH352, pTHH301, and pTHH349 define complementa-tion group A of acc. These three mutations complementedall tested Tn3-HoHol insertions except those in pTHB476and pTHB426 (Fig. 3). Furthermore, these two Tn3-HoHolinsertions were complemented by BamHI fragment 5 andSmaI-9 but not by EcoRI-26 (Fig. 2A). This indicates thatpart of accA lies to the right of EcoRI-26. This is confirmedby the accA sequence data, which indicate that this ORFextends into EcoRI fragment 16 (Fig. SA). HindIlI-11 did notcomplement the Tn3-HoHo1 insertion in pTHB426 but com-plemented the insertions in pTHB566 and pTHB422. Both ofthese two mutations also were complemented by fragmentsBamHI-5 and EcoRI-16. The fact that SmaI fragment 9complemented the mutations in pTHB476 and pTHB426 butnot the one in pTHB566 is additional evidence that theinsertions in pTHB476 and pTHB426 constitute a comple-mentation group different from that of the insertions inpTHB566 and pTHB422 (Fig. 2A). The latter two Tn3-HoHol mutations could be placed into separate geneticgroups on the basis of their different complementation reac-tions with the TnS insertions in pTHH312 and pTHH306.Complementation analyses indicated that the TnS mutationin pTHH312 and the Tn3-HoHol mutation in pTHB566 liewithin a second complementation group, accB. The thirdcomplementation group, accC, is delineated by the Tn3-HoHol insertion in pTHB422 and the TnS insertion inpTHH306. The TnS insertion in pTHH348 abolished suscep-tibility to agrocin 84 conferred by parent plasmid pTHH206and complemented all five Tn3-HoHol mutations inpTHB112. Thus, it defines a fourth complementation group,accD. Lastly, our data indicate that the TnS insertion inpTHH317 and the Tn3-HoHol mutation in pTHB555 definea fifth complementation group, accE (Fig. 3).

Restriction fragments BamHI-5 and HindIll-11 reconsti-tute a functional acc region when placed in trans to each

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A-35 -10 S.D. 1160

GTTGTlTrFXkCTAGGTTCGACGGCCGCGCGCGCGlmGCCTCATTCcAACAGAGCTGGGEGIAAAACCACATGCTT

1200AAGACCAATCGCAGGAATTTTATGATGGGQACCGCGGCGATAGCGGTCGCTTCAACTGCCGGTGCGAAGTTCACCTTC

WT N SK R N F MM G TA A IA VAST A ASK F T F1250 1300

GCGCAAGAACGCCGGGCGCTTCGCCTGGGTGTGAACGGATTGCCGAACTCGCTGGAGCCGGTCAATGCAATCAGCAATA Q E R R A L R L G V N G L P N S L E P V N A I S N

1350GTCGGCCCGCGTATCGTCAACCAGATATTCGACACGCTGATAGCGCGCGATTTTTTCGCGAAGGGAGCTCCCGGCAATV G P R I V N Q I F D T L I A R D F F A G A P G N1400 1450

GCCATCGACCTCGTTCCGGCACTTGCCGAGAGCTGGGAGCGCATCGATGAGAAATCGGTTCGCTTCAAGCTGCGGCAGA I D L V P A L A E S W E R I D E S V R F K L R Q

1500 1550AAGGTCATGTTCCACGATGGTGTTGAACTGACCGCGGACGATGTCGCATACACCTTTTCTTCCGAACGCCTTTGGGGTV M F H D G V E L T A D D V A Y T F S S E R L W G

1600

CCCGAAGCGATCAAGAAAATCCCGCTTGGGAAATCCTATTCGCTTGATTTCGATGAGCCCGTGGTCGAGGACAAATACP E A I R R I P L G S Y S L D F D E P V V E D K Y

1650 1700ACAGTCACACTTCGCACAAAGACCCCGAGCTATCTCATCGAGACCTTCGTCGCATCCTGGATGAGCCGCATTGTTCCCT V T L R T R T P S Y L I E T F V A S W H S R I V P

1750AAAGAATACTACAAGAAACTGGGAGCAGTAGATTTCGGTAACAAGCCCGTTGGAACAGGCCCGTACAAGTTCGTGGAA

E Y Y R R L G A V D F G N K P V G T G P Y K F V E1800 1850

TTTGTTGCCGGCGACCGTGTGGTGCTGGAAGCCAACGACGCCTACTGGGGTCCAAAGCCGACTGCATCGAAGATCACCF V A G D R V V L E A N D A Y W G P K P T A S K I T

1900TACCAGATCGTCGCAGAGCCAGCCACACGCGTCGCGGGTCTGATCAGCGGTGAATACGATATCATCACTACCCTGACGY Q I V A E P A T R V A G L I S G E Y D I I T T L T

1950 2000CCGGACGACATTCAGCTGATAAATAGCTATCCAGACCTCGAAACGCGCGGGACGCTCATCGAAAACTTCCACATGTTCP D D I Q L I N S Y P D L E T R G T L I E N F H M F

2050 RindlIIACCTTCAATATGAACQAGAAGTTTTCAAGGATAAG CGGCGCGCTCTTGCTCTGGCTGTCAACCGGCCAATCT F N M N Q E V F D K L R R A L A L A V N R P I

2100 2150ATGGTTGAAGCGCTCTGGAAGAAACAAGCATCTATCCCGGCCGGCTTCAATTTCCCGAACTATGGGGAAACCTTCGATM V E A L W Q A S I P A G F N F P N Y G E T F D

2200 2250CCAAAGCGTAAGGCCATGGAATATAATGTCGAGGAGGCCAAGCGTCTCGTCAAGGAGAGCGGCTACGATGGAACGCCGP R A M E Y N V E E A K R L V K E S G Y D G T P

2300ATCACCTATCACACGATGGGCAACTATTATGCCAACGCCATGCCTGCGTTGATGATGATGATCGAAATGTGGAAGCAGI T Y H T M G N Y Y A N A M P A L M M M I E M W Q

2350 2400ATCGGTGTGAACGTGGTCATGAAAACCTACGCACCGGGTAGCTTCCCCCCGGACAACCAGACCTGGATGAGAAACTGGI G V N V V M K T Y A P G S F P P D N Q T W M R N W

2450 S.ORITCCAACGGCCAATGGATGACCGATGCCTACGCCACGATCGTCCCCGAATFGGGCCGAATGGTCAGGTTCAAAAGCGTS N G Q W M T D A Y A T I V P E F G PR G Q V Q R

2500 2550TGGGGTTGGAAGGCGCCGGCCGAGTTCAACGAATTATGTCAGAAGGTCACGGTTCTGCCGAATGGCAAGGAGCGCTTCW G W A P A E F N E L C Q V T V L P N G K E R F

2600GACGCCTACAATCGCATGCGGGACATTTTCGAAGAGGAGGCGCCAGCCGTCATTCTCTATCAGCCGTATGACGTTTATD A Y N R M R D I F E E E A P A V I L Y Q P Y D V Y

2650 EgQSR12700GCCGCGCGCAAGGATGTCCACTGGAAGCCCGTGAGCTTCGAGATGATGGAATTCCCGCAACAATCTCAGCTTCGGCTGAA A R K D V H W K P V S F E H M E F R N N L S F G

2750ACTCGTTAATCGAGAAGCCGGCGCGCGCGAGATGTGCGCGTCGCCCTGGAGGAGC

other (Fig. 2A). In contrast, restriction fragments EcoRI-16and BamHI-5 in trans did not produce an agrocin 84-susceptible phenotype, presumably because sequences tothe right of EcoRI fragment 16 are required for agrocin 84susceptibility (Fig. 2A). However, fragment EcoRI-16 over-laps and complements the Tn3-HoHol insertion inpTHB555, which lies in accE. Although sequence dataindicate that the accE ORF extends to the right of EcoRI-16into EcoRI fragment 33 (29a), our genetic data indicate thatEcoRI-16 may encode a truncated, yet functional, AccEprotein. The fact that EcoRI-16 and BamHI-5 together didnot produce an agrocin 84-susceptible phenotype (Fig. 2A)suggests that there is a sixth expression unit located to theright of accE.

,-Galactosidase activities of the Tn3-HoHol insertionsindicate that at least accA, accC, and accE are transcribedfrom left to right (22) (Fig. 2A). If acc is expressed as a singletranscript, transposon insertions should exert polar effectson downstream genes and none of the TnS insertion muta-tions should have complemented any of our Tn3-HoHolmutations. Our positive complementation data suggest twopossibilities. Either the region consists of a single transcriptand genes downstream of each insertion are being expressedfrom transposon-specific promoters (4) or there are promot-ers associated with each of the separate genes of this region.The fact that all TnS and Tn3-HoHol insertions within theacc region are complementable in trans, when tested inappropriate pairwise combinations or with subcloned frag-ments of acc (Fig. 2 and 3), supports the hypothesis thateach complementation group can be expressed indepen-

B10 20 30 40 50

AccA MLKTNRRMNFMGTAAIAVASTAGAKFTFAQERRALRLGVNGLPNSLEPVNAIS:. . .. ::.. . .: . ...

OppA MTNITKRSLVAAGVAVVALAADVPAGVTLAEKQTLVMNNGSEV-QSLDPHKIEG10 20 30 40 50

60 70 80 90 100 110AccA NVGPRIVNQIFDTLIARDFFAKGAPGNAIDLVPALAESWEMIDEKSVRFKLRQKVMFHDG

OppA VPESNISRDLFEGLLVSDL--DGHPA------PGVAESWDNKDAKVWTFHLRKDAKWSDG60 70 80 90 100 110

120 130 140 150AccA VELTADDVAYT------------FSSERLWGP-EAIKKIPLGKSYSLDFDEPVVED-KYT

. .:: .: . :X ..: .: . .: .: ::. : .. ...:

OppA TPVTAQDFVYSWQRSVDPNTASPYASYLQYGHIAGIDEILEGKKPITDLGVKAIDDHTLE120 130 140 150 160 170

160 170 180 190 200 210AccA VTLRTKTPSYLIETFVASWMSRIVPKEYYKKLGAV-DFGNKPVGTGPYKFVEFVAGDRVV

OppA VTLSEPVP-YFYKLLVHPSTSP-VPKAAIEKFGEKWTQPGNIVTNGAYTLKDWVVNERIV180 190 200 210 220

220 230 240 250 260 270AccA LEANDAYW-GPKPTASKITYQIVAEPATRVAGLISGEYDIITTLTPDDIQLINSYPDLET

OppA LERSPTYWNNAKTVINQVTYLPIASEVTDVNRYRSGEIDM--TYNSMPIELFQKLKKEIP230 240 250 260 270 280

280 290 300 310 320 330AccA RGTLIENFH-MFTFNMNQEVFKDKKLRRALALAVNRPIMVEALWKKQASIPAGFNF-PNY

OppA DEVHVDPYLCTYYYEINNQKPPFNDVLWRTALKLGMDRDIIVNKVKAQGNMPAYGYTPPY290 300 310 320 330 340

340 350 360 370 380 390AccA GETFDPKRKAMEYNVEEAKRLVKESGYDGTPIAYHTMGNYYANAMPALMMMIEMWKQIGV

OppA TDGAKLTQPEWF GWSQEKRNEEAKKLLAEAGYTADKPLTINLLYNTSDLHKKLAIAASSL350 360 370 380 390 400

FIG. 5. (A) DNA sequence and predicted protein sequence ofaccA. The predicted signal sequence is underlined. >, potentialcleavage site. Potential -35, -10, and Shine-Dalgarno (S.D.) re-gions and relevant restriction sites are overlined. Base numberingbegins at the left EcoRI site of EcoRI fragment 26. (B) Comparisonof the predicted AccA amino acid sequence with that of E. coliOppA. Exact matches (:), conserved changes (.), and the boundariesof the region of high similarity (X) are indicated.

dently. Alternatively, it is possible that acc is transcribed asa single unit but contains gene-specific internal promotersthat function as well. This is similar to the pattern ofexpression of btuCED, the vitamin B12 transport systemoperon of E. coli (37).

Surprisingly, certain merodiploid strains were sensitivenot only to agrocin 84 but also to agrocinopines A and B(Fig. 2B). It may be that the opines are taken up butincompletely catabolized by these strains, such that somemetabolic intermediate accumulates to toxic levels. Similareffects have been seen with certain glycolysis mutants of E.coli (26). Alternatively, these merodiploids resemble themaltose-sensitive E. coli mutants containing lamB::lacZfusions constructed by Silhavy et al. (41). These workersproposed that when certain LamB-LacZ fusion proteins aresynthesized in large amounts, as when induced by maltose,they block the cellular export machinery, resulting in celldeath. It may be that, in certain of the merodiploid Agro-bacterium strains containing acc::lacZ fusions and acc sub-clones, agrocinopine-induced transport proteins which havebeen truncated or fused to LacZ are also jamming proteinexport machinery and killing the cells.

Maxicell analysis showed that the acc region encodes a60-kDa polypeptide (Fig. 4 and 6). Proteins of similar massesencoded by this region have been detected by two othergroups (27a, 40a). Although the 60-kDa protein was presentin extracts from cells containing insertion plasmidspTHB566, pTHB422, and pTHB555, it was not detected incellular extracts from strains containing insertion plasmidspTHB476 and pTHB426 (Fig. 4). These insertions are within

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106

45.5-

32

275

FIG. 6. Periplasmic protein profiles of plasmid-containingAgro-bacterium strains. Protein fractions prepared from Agrobacterium

strains by the sucrose-EDTA osmotic shock method were separated

by SDS-PAGE in 12% polyacrylamide gels and stained with

Coomassie blue. Lanes 1 and 7 contained protein markers whose

sizes are indicated on the left. Lanes 2 to 7 contained periplasmic

proteins from strain NT1 containing no plasmid (lane 2), pTiC58Trac(lane 3), pTHBll2 (lane 4), pTHB426 (lane 5), or pTHB476 (lane 6).The accA gene product (approximately 58 kDa) is marked (0-).

accA, indicating that the 60-kDa protein is encoded byaccAi.

A second protein of 48 kDa, synthesized in maxicells con-

taining subcloned EcoRI fragment 26 (data not shown), is

most likely a truncated form of the 60-kDa accA product.This is supported first by the observation that it is not

detectably synthesized by cells containing either the sub-

cloned BamHI fragment 5, which entirely contains EcoRI

fragmnent 26, or any of the Tn3-HoHo1 insertion plasmids.Second, the DNA sequence of accA4 shows that the portionof this ORF contained on EcoRI fragmnent 26 could encode a

truncated protein of the size observed. A novel polypeptideof 43 kDa was synthesized by cells containing pTHB426,whose Tn3-HoHol insertion lies at the right end of EcoRI

fragment 26 (Fig. 4, lane 3). This protein was not detected in

cellular extracts of strains containing any other construct

and most probably represents a version of the 60-kDa

protein truncated by the transposon insertion in pTHB426.Three lines of evidence indicate that accA encodes a

periplasmic binding protein, a common component of ABC-

type bacterial transport systems (3, 24, 31; reviewed in

reference 1). First, our analysis of periplasmic proteins from

strains containing wild-type and mutant forms of accA (Fig.6) clearly shows a periplasmically located 60-kDa proteinexpressed from this locus. Second, the predicted amino acid

sequence of AccA has marked similarity to OppA (Fig. SB)and DppA of E. ccli and S. typhimurium and to OppA of B.

subtilis (data not shown). Each of these is a periplasmicbinding protein associated with the dipeptide or oligopeptidetransport systems of these bacteria. Lastly, these results are

consistent with those of Murphy and Roberts (35) that

showed the existence of an agrocin 84-binding activity

present in the periplasmic protein fraction of nopaline-typestrains. It is interesting that uptake of two other opines,octopine and nopaline, is mediated by cognate ABC-typetransport systems (46, 48).We conclude from these studies that acc consists of at

least five genes required for transport of agrocinopines and

agrocin 84. Furthermore, the region containing these genes

is sufficient to allow growth on agrocinopines as the sole

carbon and energy source. However, these results do not

prove that functions required for catabolism of the sugarphosphate opines are encoded in this region. It is conceiv-able that genes encoding such degradative activities arelocated on some other replicon in the cell. The available datasupport the idea that the acc regulon of A. tumefaciensencodes a multicomponent periplasmic binding protein-as-sociated transport system for agrocinopines A and B. Thefact that this system also transports the antibiotic agrocin 84has been exploited by A. radiobacter K84, presumably toenhance its competitive ability in the tumor rhizosphere, andby humans for the biological control of crown gall.

ACKNOWLEDGMENTS

This work was supported by grant R01 CA44051 from the Na-tional Cancer Institute to S.K.F. G.T.H. was the recipient of adissertation fellowship from the Graduate School of Loyola Univer-sity of Chicago.We thank L. C. DeVeaux for providing strain LCD44 and for

assistance with maxicell analysis, M. Ryder for supplying agrocino-pine and agrocin 84 samples, and Y. Dessaux for many helpfuldiscussions and suggestions.

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