JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 841-849 Vol. 174, No. 30021-9193/92/030841-09$02.00/0Copyright C 1992, American Society for Microbiology

Opine Transport Genes in the Octopine (occ) and Nopaline (noc)Catabolic Regions in Ti Plasmids of Agrobacterium tumefaciens

HANS ZANKER, JOHANNES VON LINTIG, AND JOACHIM SCHRODER*Institut fur Biologie II, Universitat Freiburg, D-7800 Freiburg, Germany

Received 26 July 1991/Accepted 18 November 1991

The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsiblefor the catabolic utilization of octopine and nopaline, respectively. Opine-inducible promoters, genes forregulatory proteins and for catabolic enzymes, had been identified in previous work. However, both regionscontained additional DNA stretches which were under the control of opine-inducible promoters, but thefunctions were unknown. We investigated these stretches by DNA sequence and functional analyses. Thesequences showed that both of the catabolic regions contain a set of four genes which are transcribed in thesame direction. The occ and noc region genes are related, but the arrangement of the genes is different. Thededuced polypeptides are related to those of binding protein-dependent transport systems of basic amino acidsin other bacteria. The comparison suggested that three of the polypeptides are located in the membrane andthat one is a periplasmic protein. We constructed cassettes which contained either the putative transport genesonly or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters). Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins.The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake ofnopaline requires the putative transport genes and additional functions from the left part of the noc region.

Plant tumors induced by Agrobacterium tumefaciens syn-thesize a group of substances (opines) which are metabolizedby the bacteria. This is an important part of the interactionwith plants and presumably was the driving force in theevolution of the systems (10). The genes that are essential foropine catabolism are located on the Ti plasmids. In the caseof nopaline and octopine plasmids, the regions are called theocc and noc regions, respectively.

Ti plasmids with the occ region confer octopine [N2-(1-D-carboxyethyl)-L-arginine] but not nopaline [N2-(1,3-D-dicar-boxypropyl)-L-arginine] utilization to A. tumefaciens. Intypical octopine Ti plasmids, the region has been located toa stretch of 12 kbp (4, 21, 38). Figure 1A summarizes theorganization. The genes so far identified code for octopineoxidase (EC 1.5.1.19; oox, two proteins) (46), ornithinecyclodeaminase (EC 4.3.1.12; ocd) (5, 7, 33), and a positiveregulator (occR) which is necessary and sufficient for theactivation of an octopine-inducible promoter (Pil[occ]) (36,42). Figure 1A also shows a gap of approximately 3.3 kbpbetween Pil[occ] and the identified catabolic genes. Thefunctions encoded in this stretch were not known. Theavailable data indicated that the inducible promoter con-trolled the expression of the complete occ region (42);therefore, it seemed likely that the unknown sequences wereexpressed and that they contained genes which contributedin some way to the efficiency of opine utilization.The noc region allows A. tumefaciens to utilize nopaline

and also octopine; the catabolism of octopine, however,requires induction by nopaline (20). The noc region waslocated directly to the right of the T region, in a stretch ofabout 17 kbp. An analysis of mutants also indicated that theregion is split into two parts, which are separated by at least5 kbp of sequences with unknown functions (17, 19, 32). Thefunctional organization is summarized in Fig. 1B. The left

part codes for five proteins and, except for that of a 40-kDapolypeptide, their functions have been identified (30, 31, 35).In contrast to the occ region, the noc region contains anarginase gene (EC 3.5.3.1; arc). The expression of theproteins is controlled by inducible promoter Pil[noc]. Theright part of the region harbors nocR, the gene for a positiveregulator which is closely related to occR (42). NocR con-trols not only Pil[noc] but also a second inducible promoter,which is in the right part of the noc region (Pi2[noc]) andwhich reads leftward into a DNA sequence of unknownfunction (Fig. 1B). The fact that this promoter was inducedin the presence of nopaline (42) suggested that the sequencescode for functions which contribute to the utilization of theopine. The precise left border of this part of the noc region isnot known. Studies of Ti plasmid mutants suggested astretch of several kilobase pairs (19) and the possibility thata protein involved in nopaline uptake might be encoded inthis region (32).The available data suggested that, for both the occ region

and the noc region, the enzymes encoded by the identifiedgenes are sufficient for the catabolism of opines in A.tumefaciens (36). Figure 1, however, shows that both re-gions contain additional sequences which are under thecontrol of opine-inducible promoters. These sequences andtheir functions are the topic of the investigations describedhere. The results indicate that both of the catabolic regionscontain a set of genes for four polypeptides which are relatedto each other and to proteins involved in transport functionsin other bacteria. Functional studies support a conclusionthat the polypeptides are involved in the induced uptake ofopines.

MATERIALS AND METHODS

Agrobacterial strains and plasmids. A. tumefaciens APF2is a plasmid-free derivative of LBA275 (=C58C1) (18).Plasmid pGV3850 (45) is a derivative of nopaline plasmid* Corresponding author.

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842 ZANKER ET AL.

A. Q region35 40 (kbp)

I I aA

eI 1

m<c --------- --- --- --- --- --- --- _,

E 211E N E N

iIi occi

B. noc region5 10

------ left part ---------H N E K E E l

gEd 9 am~u& am£ tC

*i I DMno)

IS (kbp)I4--- risht pwt ---a

N E N X

*i2CnocJ

FIG. 1. Functional organization of the occ region in octopineplasmid pTiAch5 and the noc region in nopaline plasmid pTiC58.The boxes indicate the regions analyzed in this study. The mapcoordinates refer to the SmaI site in the T region, which isconserved in octopine and nopaline Ti plasmids (6). (A) ocd,ornithine cyclodeaminase (33); ooxA and ooxB, two proteins neces-sary for octopine oxidase activity (46); occR, positive regulator ofinducible promoter Pil[occ] (42). (B) ocd, ornithine cyclodeaminase(30); 40k, 40-kDa polypeptide of unknown function; arc, arginase(35); noxA and noxB, two proteins necessary for nopaline oxidaseactivity (31); nocR, positive regulator of inducible promotersPil[noc] and Pi2[noc] (42). B, BamiHI; E, EcoRI; H, HindlIl; K,KpnI; X, XbaI.

pTiC58. It contains the complete noc region, but a part of theT region is replaced by pBR322. Plasmid pGV2260 (3) isderived from octopine-type plasmid pTiB6S3. It harbors thecomplete occ region, but the T region and some adjoiningsequences are replaced by pBR322.

Bacteria were routinely grown in liquid cultures (1% Bactotryptone, 0.5% Bacto yeast extract, 0.5% NaCl, 1 mMMgSO4, 0.2% glycerol [pH 7.5]) with the appropriate antibi-otics (rifampin, 50 ,ug/ml; carbenicillin, 50 ,ug/ml; kanamy-cin, 25 ,ug/ml; tetracycline, 2 p.g/ml). For long-term storage,cultures from the stationary phase were mixed with dimethylsulfoxide (final concentration, 3%) and stored at -70°C.

Escherichia coli strains and plasmids. Strains JM109 (43)and S17-1 (37) were used routinely. E. coli S17-1 contains thetrans-acting mobilization factors of plasmid RP4 integratedin the chromosome. Subcloning of the Ti plasmid fragmentswas performed in the pTZ18R-pTZ19R system, which pro-vides multiple cloning sites in the polylinker (44). Themolecular techniques have been described previously (28).

Constructions with the broad-host-range vectors pCB303and pUCD2001. Plasmid pCB303 has been described previ-ously (34). It contains a small polylinker (SmaI, XbaI, PstI,Sall, BamHI, and EcoRI) which is flanked to the right byreporter gene lacZ and to the left by phoA. The Ti plasmidfragments were inserted into the unique cloning sites (XbaI,PstI, and BamHI). Suitable restriction sites were obtainedvia intermediate clones with the fragments inserted into thepTZ18R-pTZ19R polylinker. When this was not feasible, thefragments were excised and inserted blunt ended after fillingin of the protruding ends. The orientation of the inserts wasdetermined with asymmetric sites in the cloned fragmentsand with the help of the flanking restriction sites in thevector. Plasmid pUCD2001 (8) has been described previ-ously. Fragments were cloned into the Sall site of thetetracycline resistance gene. The constructions were carriedout in E. coli, and the plasmids were conjugated from E. coli

S17-1 into A. tumefaciens APF2 (42). The presence of theplasmids was routinely confirmed by miniscale plasmidisolation, retransformation into E. coli, and restriction anal-ysis.DNA sequences. The sequences were determined on both

strands by the dideoxy nucleotide chain termination tech-nique (29). The pTZ18R-pTZ19R system (44), helper phageM13K07 (41), and E. coli JM109 (43) (all from PharmaciaLKB Corp.) and a reverse sequencing primer (BoehringerGmbH, Mannheim, Germany) were used routinely. Suitableplasmids were obtained by subcloning of DNA fragments;standard molecular techniques were used (28). DNA poly-merization reactions were performed with [35 ]dATP (>37TBq/mmol; Amersham Corp.) and modified T7 DNA poly-merase (Sequenase; United States Biochemical Corp.). Thecomplete sequences were obtained with suitable subclones,with overlapping subclones from exonuclease III digests (11)(double-stranded nested deletion kit from Pharmacia LBKCorp.), or with the help of synthetic oligonucleotides.Computer analysis. Standard sequence compilation and

evaluation were performed with published programs (24).Analysis of the predicted polypeptides was carried out withthe ALIGN2 program of M. Trippel (Infomed GmbH,Freiburg, Germany), a PCGENE software package (Intelli-Genetics Inc., Mountain View, Calif.), and the PROSITEdictionary compiled by A. Bairoch (1).

Synthesis of [3H]octopine. [3H]octopine was synthesizedessentially as described previously (2). The incubation mix-ture contained, in 0.1 ml, the following: 0.75 MBq ofL-[2,3,4,5-3H]arginine (1.5 TBq/mmol; Amersham Corp.)mixed with unlabelled L-arginine to a final concentration of0.73 mM; 2.5 mM sodium pyruvate; 8 mM P-NADH; 0.1 Uof octopine dehydrogenase (Sigma Biochemicals); and 0.1 Msodium phosphate-EDTA buffer (pH 6.6). After 4 h at 35°Cunder anaerobic conditions, the labelled octopine was puri-fied by preparative paper electrophoresis (pH 1.8; formicacid-acetic acid-water, 3.4:6:90.6, vol/vol/vol). It was elutedwith distilled water, lyophilized, and redissolved to 0.83kBq/ul.Measurements of opine uptake. (i) With preinduction with

opines. After growth in LB medium at 28°C to an A6. of 0.8to 1.2, the cells were harvested by centrifugation and resus-pended to an Awo of 0.5 in AB-glucose medium (0.5%glucose in 13 mM K2HPO4-7.2 mM NaH2PO4-18.7 mMNH4Cl-1.2 mM MgSO4 7H20-2 mM KCI-68 ,uM CaCl2-9nM FeSO4 7H20 [pH 7.1]; bacteria grew slowly in thismedium [50% increase in 3 h]) containing either 0.4 mMunlabelled octopine or 0.4 mM unlabelled nopaline. After 3 hof induction, the cells were collected and resuspended to anA6. of 0.5 in AB-glucose medium containing 150,000 cpmof either [3H]octopine or [3H]nopaline (a gift from Dr. U.Langridge, Adelaide, Australia) per ml.

(ii) Without preinduction with opines. Cultures grown inLB medium were harvested by centrifugation and resus-pended to an A6. of 0.5 in AB-glucose medium containingeither 0.2 mM octopine and 150,000 cpm of [3H]octopine perml or 0.2 mM nopaline and 150,000 cpm of [3H]nopaline perml. The cultures were incubated on a rotary shaker at 28°C.In both procedures, bacteria in samples of 0.2 ml werecollected on nitrocellulose filters and washed with 10 ml ofAB-glucose medium, and the radioactivity was determinedby scintillation counting.

Nucleotide sequence accession numbers. The nucleotidesequence data reported in this paper will appear in theEMBL, GenBank, and DDBJ nucleotide sequence data

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A. TUMEFACIENS OPINE TRANSPORT GENES 843

A. o region38 39 40 41 (kbp)I II

Rs 2S Rs SSS PI I I III

H P

s--->--->*- - -> p- -- - ->

Rs---> *---> *--> S----> H----><--* <---* <--S <-----H

<---S ---Rs<---S <----P <----P

occP OCCQ

(. (

OCCT ,occ

B. no region14 15 16 17 (kbp)I I

--IE5SSm S Sl HH N EE5 P HI II I II II

E5--> o-->o--> H->o->o-> 0--> P---->a-->o-->S--->H--> E--> 0-->

<---0<--O<-0 <--O <--E <---P<--o<--o<-o < -N < -* C----H

,ocQ nocP

nocN nocT

FIG. 2. Sequencing strategies. Symbols: *, sequences obtainedwith synthesized oligonucleotides; 0, sequences obtained withexonuclease deletion clones. E, EcoRI; E5, EcoRV; H, HindIll; N,NaeI; P, PstI; Rs, RsaI; S, Sall (2S, two closely spaced SalI sites);Sm, SmaI. The bottom lines show the extent of the ORFs for thepolypeptides related to bacterial transport proteins.

1 TAAACGCACCATAACAT101 GGCCAACCGGGATCAGA201 TATGCTCAGGGCGACCG301 TCTGGAGCGCTTCAGGC401 TCATTTCCAATGTCGCT501 CCAAACGCAGGTCCTGC601 ATCGTCCTGCCTCAGGC701 TCGAACTGATGCGCCAG801 CTCGGGGCAGGTCTTCC901 TCGCTTTGCTTTCCGGC1001 TCGCCTCTGGTGGCTTG1101 CAATTTCCCGATGTTCG1201 CAGAGATCATGCGTGGA1301 TATTCCGCAGGCGATAC1401 ATTACCGGGATCGCAGC1501 CGCGTCTTTTCACGTTG1601 ACCCAGTTCGCCCAGCT1701 GATCTCCATTCTCGGCT1801 GAAGAGATCGCTCTCGF1901 GCTTTAATCTCTGGTCA2001 GGCGCTGCTTGAACGCG2101 GCGATGCGGCCGGACGT2201 AGGGGCGCACCATGCTG2301 CCCCTCCTCCGAAATGT2401 TCGCGTGTCCAGAAAAC2501 GAAAAGTCGATTACGAT2601 CTCTCTGCGAAAAGATG2701 CGCGATGAGCGTCACGC2801 GCCGAAATGCCAGGCTT2901 CTGTTGGCGTGCAGGGC3001 CCTCGACCTGACGAGTG3101 GGGCCGCTGTTCTCCGG3201 CAAGCGAAGACGGCACG3301 GTATCCC

bases under the accession numbers M77784 (occ) andM77785 (noc).

RESULTS AND DISCUSSION

Nucleotide sequences and protein coding regions. The se-quencing strategies are summarized in Fig. 2, and the resultsare presented in Fig. 3 (occ region, 3,307 bp) and Fig. 4 (nocregion, 3,373 bp). Both of the analyzed regions contain fourlarge open reading frames (ORFs), with the orientationsfrom right to left in the presentations used in Fig. 1 and 2;note that the sequence data are printed in the oppositeorientation to facilitate discussion. Table 1 summarizes thepositions of the deduced polypeptides in the DNA sequencesand their sizes. The proteins can be demonstrated in E. coliminicells, but only when all four ORFs are expressed to-gether under the control of a weak promoter. Experimentsinvolving stronger promoters or the expression of singleproteins were unsuccessful, indicating that these conditionswere detrimental to E. coli. The precise protein starts weretherefore not determined experimentally. The bases for theproposals in Table 1 are (i) the presence of methioninecodons which are preceded by a sequence reasonably resem-bling a Shine-Dalgarno sequence and (ii) the similarity of theproteins in the occ and noc regions to each other and toproteins in other bacteria. Table 1 also contains proposalsfor the names of the genes. These are based on the pro-nounced similarities of the deduced polypeptides to polypep-tides in other bacteria.occQ and nocQ. The amino acid sequences (Fig. 5A)

deduced from the first ORF in the occ region and the thirdORF in the noc region are related. The presence of aribosome binding site suggests that NocQ starts with the first

;CGATGACGATGGCTGTGGCTTTCAGCGGCTTCACAATCGGCCTTGTCTTCGGCTGTCTTGGAGCCGCCGCAAGCTTGTCAAGCCAGCGGCATCTGGATACACGACCGCGCTTCGTGGCATCCCCGACCTACTTGTCATCTATCTATTCTACTTCGGGTCGAGTTCTGrTCACTCTTCGGAAGTAGTGGGTTCGTTGGCGCTTCGACGTTTCTGATAGGTGCCCTCGCCATCGGCGTGGTTTCGGGTGCCTACGCGGTGCGGTTCTGGCTCTCAACAAGGGCGAGATCGAGGCTGGTCGAGCGTATGGCATGGGCGCCI

-

TTGCTGTTGTTCCGGCGG:GGCGCGGTACGCCCTGCCCGGTGTCGGCAATGTCTGGCAACTGGTCCTGAAAGAATCCGCCCTGATCTCGGTGATTG 3GCCTCG

CGTCTGGCAGAGACGCGCTCTATGCGCGGACTGCAGAGGGGCGTGTGATGCCGTTCGATCCCGCATTTCTCTGGCAGACCTTCGCATTCCGCTTGCACTGCAACTCGCTGTGTTCTCGGTGGCGCTCGGAACGGTTCTCGCTTTCGGCCTGGCGCTGATGCGCGTGTCLATCTGCCGGCACGCTTCTACATTTTCGCGTTTCGTGGCACGCCGCTTCTGGTTCAGATCTACATCATTTACTACGGGCTGAGC

ATAGCTTCATCTGGCCCATTCCTGCGCGATGCTTACTGGTGCGCGATGGCGGCACTGGCATTGAATACCGCTGCTTACACAG

CCAAACTGATCTCGGAAAGCTACCGGACAGTCGAAGTCTTCTCCTGTGCCGGGGCTATCTACCTTATCCTCAACTTCATCGTCGGCTTGAATGGGCTCTCTGGCCCGMCAATAGACTTACAACAGATCCTGTCGACCGAAAGGGCGAACTCCATGCCTA'GTTCAGCTGAAGGACATCAGG TTTCGGTAACCTGGAGGTTCTCCACGGCGTATCGCTGAGCGCCAATGAGGGAGAAGTrCGTCCGGCTCCGGCAAGTCGACGCT'TCTGCGATGTGTCAACATGCTCGAGGTCCCAAATGCCGGAAGCGTTGCGATCATGGGC

___-. __ .____--.-__.-____. .-_. -_-.__ -__ __-. -______ _- . ._-_-_ -____-.-.-GCATCGGGCCGGCCGGCTCGCTCGCCCGAAGGATTTGAAGCAGGTCAACAGACTCCGCGAGCGGGCGGCAATGGTCTTCCAAGCATCAGACGATCCTGCAAAATGTCATGGAGGCGCCGGTCCACGTCCA CG TCGTAAGGCGTGCCGCGACGAAGCTGAGTAGGCATTGCCTCCAAACGCGATGCGTATCCGTCCGAGCTATCTGGCGGCCAGCAACAACGCGCTGCGATTGCCCGTGCCCTG

GATAGTCACGCACGAAATGGATTTTGCCCGAGATGTCTCATCGCGAACGGTCTTCTTGCACCAGGGCGTGATTGCCGAAGAAGGTTCGCCCACCCCCGCACGGATCGCTTCCGGCAATTCCTGCGGCGCGACGGCGGTACATCCCACTGACGACGAACTCGTCACGGCCACTGAACAGAGGTGAACTTATGAAACTCAAAACTATTCTGTGCGCAGCGCTTCTCCTTGTTGCCGGGCAGGCGGCAGCTCAGTCGCGACGGAGGGCGGCTATGCGCCCTGGAACTTCTCCGGGCCGGGCGCACTCGACGGTTTCGAGATCGATCTGGCCAACG

CCAAAACGGCAAGAGGTGATCGGCTTCTCTATCCCCTACGCTGCTGGCATCAATGGGTTTGCCGTGATGGGTGACAGCAAGTTGTGGGTGAAACCTATTCTCTCGATAGCCAGGCGGACGCTGCCAAGAAGGCTATTGCCGATATTTCGAGCTTCCTCAACGGGACAA

GGTCGGCTGGATGCTGTTCTGGCGAACGCGACCGTGCTTGCCGCAGCAATCGAGAAGCCGGAAATGAAGGGCGCGAAGCTCGTCGTGGCGAGTTCGGCGTAGTCGCCGTTGGTCTTCGCAAGGAGATACCGCCCTCAAGGCTGATTTCGACGCGGCCATCAAGGCGGGATCAAAACACTGTCGCTGAAGTGGTTCAAGGTGGATGTCACCCCCCAATGATCGATCGCTTTTAGGGATACGACGTTCGCGCC

FIG. 3. DNA sequence of the occ transport protein region. The sequence is printed in the orientation in which the ORFs read from leftto right. The sequence starts with the stop codon defining the beginning of the ORF for occQ and ends with the putative stem-loop sequenceafter occT.

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1ITAGAACTGGGTCCTACTCTCGAAGAATGCACTGCGGTCACGTCAACACGTGCTGCACCGTTGAGAATGAATGCTGGGCAGATT TC101 ATTTTCGGCTGTCCCGTCCTCACGGTTTTGCGCTGCATCGAGGTTGGGAATGATAACAGACGCG AATGGACGCTACTCAACCCACCCTGGT201 GGCAGAAGACGTGCATAACTTTGGGACGCTCGAAATTCTGAAGGGGATTTCACTTACGGCCAATAAAGGCGACGTGGTCTCGATTATCGGCAGCTCC301 GGCTC GUCGATTsCCTGCGTTGTATGAACTTCCTCGAACGCUCrA-AC---TtATCGCGGTTGGGCAGGAAGAAGTTGTCGTCAAGACCG401 ATGCGGCTGGCGTTGATCGGCGTCGAT- GACCTGCGCATTGCCGGCATGGTGTTCCAGGTTTCAACCTGTGOGACATAT501 GACCGTCCTGCAGAACGTCATG CTCTCCACGTTCTG ACAAGGGCGAAGTGCACCGCGCGATGATTTTCTGGACAAGGTCGGC601 AT ;CA AUCOCCCG= CATCCGTCTCAACTG -GCA-CUXGCTCAGCATCCACGCGCCTGGCCAT AeTTC701 TCTTCGACGAACCGACCTCTGCTCTCGATCCGGAATTGGTGGGCGAAGTCCTGAAGGTGATCAGGAAGCT C CGGC GTGGT801 GACGCACGAA TGGGTTTTGCTCGCGATTACGGCAA TTCTCTTCCT TGAGG CCA GGTTCCAG901 AATCCGACGTCACCAAGGTGTCGGGCCTTCCTCCACTTTT-YC--=--GCCGCAGTCAACAGCTTCGAAAT CACGCACGA1001 GGTGAACGTTATGAAATTCTTCAACTTGAATGCGCTTGC6GUCGTCGTGACTGGTGTGCTTCT ;;-GCGCCGCIG^TCUG1 101 ATTACGATC nA TCCTATGCGCCGTACAACTT %CGA ACGCGTAACGATCGGCTTTGATATCGATCTCGGATGACCTTTGCA1201 AGCGCATGAATCGAATGTAATTCGTCGAGCAGGCTTGGGTAGATCATTCCTTCGCTCACGGCCGGTCGTTATGATGCCATCATGGGGTGGG1301 AATTCAAG-C-- GGCG-UUGTATTGCGTTCTCCC GGCCGTATCTCCTCACGCCGATGACGTTCTTG^GAC GCG-GAC-AGCCCGTTCTGAAGACC1401 CAGGTGGCAAT GTCTTCCGCTCGACAATATCG=CCGAC TGGAC AATTCACCAAGATTTTCGAAGGCGTTAAGTTCGGCG1501 TTCAGGCCGGCACATCTCATGAAGCATTCATGAACAGTGATGCGGGTGCAGATCTCGACTTACGACACCATAGATAACGTCGTGATGGATCTGAA1601 GGGGCGTCGA CGAGCTGGCTTCGGTCAGCTTCCTGAACCGCT AC AG ACTCAAGATGTCGGCCArATG1701 ACGGGCCGTCGAGGGCTGT GTCGGTGTTGGCATtCU GTlCGGACCTCAAAGCCCTGTTCG ^GGATTGATGCCGCCATCGCGG1801 TTCAGAAGCTGTCCCAACAGTGGTTCGGCTACGACGCCTCtttAACtCG CtGCAttC6C

1901 GCTCTTTGGTTGCAAAtGCTGAGCGTG^GAGGCCATGTACCTATCTGGGGtTt^CACGGCTGtGCGATGACTGC Tt2001 GATGACCGTTGTCGTTGCAGCCTTsTCGTACTTTTTCGGAATCATTTCGGTTGCGCTCTTYGCT C GCTTTCCCGTTTTTGGTCACAGCTTG2101 CTCGGCGACGTCT tCGAC-AGTTGTTCGCGGTTCCssCCGACTGCTCACATCATTTCTGGTGTTCTTCGGCGGTGGGACACTGTGCGAATtCGCCA2201 ACCt;GCTTTTCGGATACAGGTACATCGAGCCTCCGATCTTCGTGATCGGTGTGCTGCtGATCAGGGGGCGTGAGTCGTCATt2301 TCGCACCGTTCCTtCGCTTTC ttCGAAGCCGAATG tCCATGGATGTTGGC TGCGCCTGCGCCGGGTGTTGTCCCCA2401 GCtTCAATCGCGCTGCCGGGCTGGGATGTCGGCAGTTCA = T TCGCttTtGACCTGTCGCGGTCTAGTCGA^GACGAT2501 CAGCGGCCATGGGTGCGGGTTGACGAACAGCCATTTACCTTCTACACttACCGUCCTCGATSCCtCGTTCCTtGCTTTCCCCGTCTCAtCMGGC;TT2601 TCTCAAAGCTGAAATnCC U GTGGGAGtCTAGTATGGATATTCAACTCACATCCGAGTCTCCGAAACTCTC T=C2701 CACGACGCTCA CCTCGCGTTCATATCGCTCCTCATCGGGTGTTGTTTCGTGCCTGTTGCCtGCTGATGGCGGCTTT TCGATCGTTsTCTCA2801 TTGGCAT TGCTACtACACATACAGT ----TCTGtGTCAGATTTCCTGATTTACTACGGCTCCGCTATTCAGGGTTCTCTtCCG2901 MGTTGGCTTGTGGTCGTCATTCCCACCATGGTTCTGCGCGATCCT TGGCCCTGCAAT AAACCGCAGCCTCATCCGGGCG3001 CATCCAGTCGGTTTCGCTCGGCAGACT CAGCMTGCAGTCGGAATGTCGACCTTTCTTCAATTCCGAGTCGTGTTCCCGATCGCCATTCGG3101 CAAGCATTGCCGGCATTGCACGAAGTGATGCTCATCATCAAATCGACGTCGCTTCTTCGACAATACCATGCAAGTGACCGGTCGGCGAAGC3201 AGTCATCTCGGCCAUtCC TC^GAAGTttGTTCATCGTGCGTGCGATCTACCTGTTTATCACCGTTCGTTGCAGCCGTCTGGTCATGCTGGC3301 GGAGTGGTGGCTATCGCAtCAT GCGCGCCCGGGTGGGCGGCGT A t0CMGMACATTAG

FIG. 4. DNA sequence of the noc transport protein region. The sequence is printed in the orientation in which the ORFs read from leftto right. The sequence starts with the stop codon defining the beginning of the ORF for nocP and ends with the stop codon for nocM.

AUG a few base pairs after a stop codon. It seems likely thatOccQ utilizes the second AUG in its ORF (positions 151 to153 in the DNA), since a sequence reasonably resembling aribosome binding site sequence is present here but not infront of the first AUG. The proteins share 50.4% identity.Both OccQ and NocQ are related to other bacterial proteins.The highest values (41.7 and 40.4% identities) were foundwith HisQ, a membrane protein involved in the transport ofhistidine in Salmonella typhimurium (13). A computer anal-ysis predicted several transmembrane helices for the poly-peptides encoded by the Ti plasmids (underlined in Fig. SA).occM and nocM. The second ORF in the occ region

encoded a polypeptide which is related to that encoded bythe fourth ORF in the noc region (Fig. SB). OccM and NocMare related to each other (51% identity) and are similar to

TABLE 1. Proteins predicted from the sequences ofthe occ and noc regions

Proposed Putative Coding ProteinRegion name of Shine-Dalgarno region size

gene sequence' (bp)b (Da)

occ oocQ GCAA&SGAACGCT ATG 151-861 24,801occM CAGAGGGGCGTGT ATG 864-1598 27,486occP AAGGGCGAACTCC ATG 1594-2379 28,954occT CAGAGGTGAACTT ATG 2438-3265 28,986

noc nocP CAADGGAACGCGA ATG 175-945 28,188nocT AGAGGTGAACGTT ATG 1011-1859 30,721nocQ TGAGGCGTAGGCC ATG 1936-2643 25,524nocM AGGAGCCAATAGT ATG 2648-3370 26,424

a Underlining indicates identity with Shine-Dalgarno sequence.b Position in the DNA sequence.

other bacterial proteins involved in the transport of basicamino acids (13, 26). The examples showing the highestdegrees of similarity were HisM from S. typhimurium(OccM, 40.9%; NocM, 43.4%) and GlnP from E. coli (OccM,40.2%; NocM, 37.4%). Like these, the Ti plasmid-encodedpolypeptides contain several stretches which probably rep-resent transmembrane helices (underlined in Fig. SB).occP and nocP. The polypeptides deduced from the third

ORF in the occ region and the first ORF in the noc region aresimilar (57.5% identity) (Fig. 5C). Both OccP and NocPshare extensive homology with several other bacterial pro-teins involved in transport functions (9, 13, 22, 26). Thehighest values were found with HisP from S. typhimurium(57.4 and 62%), HisP from E. coli (56 and 61.9%), GlnQ fromE. coli (54.2% with both OccP and NocP), and ProV from E.coli (44.3 and 42.5%). All of these polypeptides contain thecharacteristic ATP or GTP binding site motif (A) and theATP binding protein "active transport" family signature (1,14, 25), and these motifs are also present in OccP and NocP.occT and nocT. The polypeptides deduced from the fourth

ORF in the occ region and the second ORF in the noc regionare homologous (48% identity) (Fig. 5D). They contain at theN-terminal end a hydrophobic stretch with similarity tosignal sequences for transport through membranes. Thepotential cleavage sites (1) are indicated in Fig. SD. Bothpolypeptides are related to proteins of transport systems inother bacteria (12, 13, 16, 27). The highest degrees ofsimilarity were found with ArgT from E. coli (47.9 and45.9%), ArgT from S. typhimurium (43.5 and 41.5%), andHisJ from S. typhimurium (37.7 and 43.6%). All of these arelocated in the periplasm and contain a protein transportsignal at the N-terminal end.

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A. TUMEFACIENS OPINE TRANSPORT GENES 845

A. B.

OccO NDYSOLW.FGPDG.YDILRATATMAVAFSGFTIGLVFGCLGASLSS 50

NcO" WLTLLQ-GD -ELARGAiMITffW FGI IFGLFALSR 49

OccO SGAL SGYTAL RGIP2LLFYFGSSSVISNVAS-LFGSSGFVG 99

NcO" FWSLRLLGDVYTTGVPELLI IFLVFFGGGTLLRTI_NGLFGYEGYIE 99

OccO ASTFLIGALIGWVSYOT@VLRG.AVLALKGEIEAAYQIGALLLFF 149

Noce PIESIG LRLR 149

OccO RIVLPQAYALPGVGNVUQLVLKESALISVIGLVELNRQAQVGSGSTRQ 199:...c: ::---::::-::::-::: 199

NOC RVLIPQ FALPGLLVWFTLKDTSLI LAISLK KO 199

OccO PFSFLUTA VIITFV FRUAETR1GLGEV* 237

NocO PFTFYITAFVIFLLLSSMRGFLKAEKIMNRGV-RSO 236

C.NATPN

OccP MMPVRPALKDIRKNFGMLEVLHGVSLSAE EGEVISILW =STL 50

NOcP NDATQPTLVAEDVNKNFGTLEILKGISLTMKGDVVSIIGSSSGKTF 49

OccP

NOcP

LRCVILEVPNAGSVAINGEEIALEHRAIGLLMCDLKAV LRERAA 100

LRCNNFLETPNKGRIAVGEEWWITDAAGRLIGVDRKKIERNRLGNV 99

OccP FaGFNLWSNTILUR EA LERVGIASKRDA 150

NocP FQSFNLWGNTVLQNVNEGPLHVLKQPCGEWMRFLDKVGIMKNAA 149

m ctive trarotH.OccP tPSELSGOMILDAWNLFDEPTSAPELVDEKVNL 200

MacP LFDEPTSALDPELVGEVLKVIRKL 199

OccP MEGRTNLItVTHENDFA VSSRTVFLNVIAEEGPS FAMPRTDRF 250

NocP AEEGRTMYVVTMENGFARDVSSKVLFLEKGQIEEQGTPQEVFQNPTSPRC 249

OcCP RQFLRRDGGTSN 262

MacP RAFLSSVL* 257

OcCN NPFDPAFLUOTFVALLSGIPLALQLAVFSVALGTVLAFGLALNRVSRL%W 50* *E*FP LL -*-...AVTLLGV.PL... .. 4... 48

NOC NDIQLI IESFPKLLAAVPTTLTLAFISLLIGFWVPVAfLSKNRI 48

OccN LDLPARFYIFAFRGTPLLVQII IYYGLSQFPDVRHSFI-WPFLRDAYK 99

NacN VSSLAYGYVYI IRSTPLLVWIFLIYYGSAQFRGVLSEVGLWSSFREPWFC 98

OccN AALALNTAAYTAEINRGGLLSVPAGQIEAAKACGNGRVKLFRRIVIPO 149

MacN AILALALNTAAYTSEIIRGGIQSVSLGQIEAARAVGNSTFLQFRRIVFPI 148

Occl AIRQNLPGYSNEVILNVSTSLASTITINEITGIAAKLISESYRT1YY[ 199

NOCN AIRQALPAYGN ,LI IKSTSLSTITGKQI ISATYSPY 198

OccN CAGAIYLILNFIVARLFTLLEWALLPERRNNRLTTDPVDRKGELHA* 245

NoCN VAGAIYLFITFVSRLVNLAEWJLNPHNRARVGGTAPKAAETH* 241

D.OccT NKLKTU-C;ALLLVA - ----KSITI ATEGGY FSGPGGK 4

NocT NKFMI MAlMATVLL TQDYKSITIATEGSYAPYNFKDAGGK 50

OccT LDGFEIDLAMALCEKNKCQIVAMDGINPSLTGKKYDAINAASVTP 94

NocT LIGFDIDLGNDLCIKRNIECKFVEQAWGIIPSLTAGRYDAINAAGIQP 100

OccT RQEVIGFSIPYAGINGFAVNGSIAEMGLGETTSLDS AAKKAI 14"

NocT AREKVIAFSRPYLLTPNTFLTTADSPLLKTQVAIENLPLDNIAPEQKAEL 150

OccT ADISSFLNGTTVVGSTTASTFLDKYFKGSIDIKEYKSVEEHNLDLTSG 194

MocT DKFTKIFEGVKFGVQAGTSNEAFN-KQNNPSVQISTYTIDDMWNDLKAG 199

OcCT RLDAVLANATVLAAAIEKEGAKLVGPLFSGGEFGV-VAVGLRKEDTA 243

NocT RIDASLASVSFLKPLTDKPD0MDLKNFGPRNTGGLFGKGVGVGIRKEDAD 249

OccT LKADFDAAIAMS TIKiLSLKWFKVDVTPO* 276

NocT LKALFDKAIDAAIADGTVQLSIWFG 283FIG. 5. Alignment of the polypeptides predicted from the ORFs in the occ and noc regions. Underlining indicates putative transmembrane

helices or signal peptides (OccT and NocT). Symbol: ><, potential cleavage sites of signal sequences. ATP and active transport indicate theATP or GTP binding site motif (A) and the ATP binding protein active transport family signature (1), respectively.

Relationship to amino acid transport in enteric bacteria.The occ and noc regions code for a set of polypeptides whichare closely related to the components of binding protein-dependent transport systems in other bacteria. Amongthese, the proteins for the transport of histidine and argininein S. typhimurium (12-14) showed the closest relationship;therefore, we propose a similar nomenclature. These trans-port systems consist of four proteins, and each appears tohave a related counterpart in the occ and noc regions: aperiplasmic binding protein for arginine, lysine, and orni-thine or for histidine (ArgT and HisJ, related to OccT andNocT, respectively); two integral membrane proteins (HisMand HisQ, related to OccM and NocM and to OccQ andNocQ, respectively); and HisP, which is equivalent to OccPand NocP. The relationship not only is an overall similarityin the amino acid sequences and in the sizes of the proteinsbut also extends to the presence of similar domains andsignatures: (i) the amino acid sequences of OccT and NocT

encode a signal peptide as the only hydrophobic domain, asin ArgT and HisJ; (ii) like HisM and HisQ, the M- andQ-type proteins from the Ti plasmids are basic proteinscontaining several putative transmembrane segments; and(iii) like HisP, OccP and NocP contain the ATP or GTPbinding site motif (A) and the active transport family signa-ture.

Intergenic regions. Intergenic regions are in some casesvery short or nonexistent (nocQ-*nocM and occQ-occM--occP; Table 1). NocM starts 2 bp after NocQ stops (TAGTATG), the stop codon of OccQ overlaps with the start codonof OccM (TGATG), and the AUG of OccP is part of the lasttwo codons of OccM (CTCCATGCCTAA). Such a tightcoupling was also found with hisQ-*hisM--+hisP and appearsto be typical for proteins which are expressed to approxi-mately the same extent (see reference 13 for a review).The region between NocT and NocQ contains a sequence

which potentially could form a stem-loop structure (AG =

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region

T CT GG:CC:G

A CG:CC:GA:TT:AA:TG:CG:C

3282-G:C-330753 33

FIG. 6. Potential stem-loop structures after the coding regionsfor NocT and OccT. The numbers indicate the positions in the DNAsequences.

-15.9 kcal/mol [ca. -66.5 kJ/mol]), and a sequence with thesame potential is present downstream of the OccT codingregion (AG = -17.5 kcal/mol [ca. -73.2 kJ/mol]) (Fig. 6).Both showed some relationship with the repetitive extra-genic palindromic sequences identified in E. coli. Thesestem-loop structures are probably involved in differentialgene expression in polycistronic operons (15), and such afunction has been discussed for extragenic palindromicsequences after the coding region for periplasmic proteinHisJ in the histidine transport operon (13). It seems possiblethat the Ti plasmid sequences downstream of the relatednocT and occT have a similar role in A. tumefaciens.

Analysis of function. We constructed plasmids which al-lowed in A. tumefaciens the investigation of the transportfunctions encoded by the DNA sequences. All of the con-structs were made with the broad-host-range vector pCB303or pUCD2001; the Ti plasmid portions are shown in Fig. 7.The strategy for these experiments may be summarized asfollows: (i) construction of cassettes which contain thecomplete catabolic regions (pOCC300 and pNOC166) and (ii)construction of a second set in which at least the initialenzymes in catabolism, the opine oxidases, are inactivated

(pOCC290) or completely absent (pNOC158). All of theconstructs included the regulatory genes (occR and nocR)and the inducible promoters, which are necessary and suffi-cient for the activation of the genes (Fig. 1) (42). A. tume-faciens plasmids pGV2260 (octopine type) and pGV3850(nopaline type) served as positive controls, and the negativecontrols were the vectors used in the construction of thecassettes.The occ and noc region genes in Ti plasmids are inactive in

the absence of opines (42). The cassettes retained the naturalpromoters and the regulatory genes; therefore, the opineuptake studies required the induction of protein expression.We used two different protocols: (i) induction with unla-belled opines for 3 h, removal of the opines, and uptakestudies with radioactive opines; and (ii) simultaneous addi-tion of unlabelled and labelled opines (see Materials andMethods for details). The results with the first protocolindicated linear uptake rates for at least 30 min, and theincorporation in cells containing the transport functions wasmuch higher than that in the controls (data not shown). Theonly exception was pNOC158; the cells with this cassetteincorporated very little radioactive nopaline, with valuesonly slightly higher than those of the controls. The plasmidcontained the transport functions but not the catabolicgenes; therefore, it seemed possible that loading of the cellswith unlabelled nopaline during the 3-h induction led to aninhibition of further opine uptake. The second protocolavoided this problem; therefore, the experiments were re-peated with all plasmids under these conditions. It should benoted that this approach measured not only Ti plasmidgene-dependent opine uptake but also the lag period ofinduction. These conditions appeared acceptable becausethe major purpose of these experiments was not a detailedcharacterization of the uptake kinetics but the identificationof the gene functions. In this context, the simultaneousdetermination of the lag period was an advantage, because italso tested whether the functions were induced with a lagphase comparable to that of the induction of the promotersresponsible for the expression of the genes. The experimentsdiscussed below were performed with the second protocol.

occ region. No significant octopine uptake was observedwith vector pCB303 or pUCD2001 (only the data for pCB303

A. o regionzosd- <o0xM-<ooxM-V cTE<MoccRSt E StI pOCC300

S-J pOCC290

B. noc region

CPt¶I DWIB K deleted B

1~, , |~~~~~~~~~~~

q4 VI rocRx(WO 4WZ4Pf21noc3

x

x

FIG. 7. occ and noc region cassettes. All plasmids contain the regulatory gene (occR or nocR) and the inducible promoters (Pi) which arenecessary for the activation of the functions (42). All cassettes were cloned in vector pCB303, except for pOCC300, which was inserted invector pUCD2001. (A) pOCC300 harbors all genes of the occ region. pOCC290 lacks part of ooxA and all of ocd, eliminating the catabolicactivities (octopine oxidase and ornithine cyclodeaminase). (B) pNOC166 contains all genes of the noc region; the deleted KpnI-BamHIfragment contains no genes necessary for nopaline catabolism. pNOC158 harbors only the putative transport functions, and all of the genesfor the catabolic enzymes are absent. B, BamHI; E, EcoRI; K, Kpnl; S, Sall; St, StuI; X, Xbal.

no regionTTT

G TC:GG:CT:AT:AT:AC:G

T CC TG-TC:G

G AC:GC:G

1896-G:C-19285' 3'

E

pNOC166pNOC158

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A. TUMEFACIENS OPINE TRANSPORT GENES 847

251

20 -

15-

10 -

6

A.

uptake (%)

I II I

0 15 30 46 60 76 90 105 120 135 160 65 180

time (min)

B.

0 16 30 46 60 76 90 105 120 135 150 165 180

time (min)FIG. 8. Time course of induced opine uptake into A. tume-

faciens. (A) occ region (induction with octopine). (B) noc region(induction with nopaline). The plasmid constructs are described inFig. 7; pCB303 was the vector for the cloned fragments. Thecultures were pregrown to an A6. of 1 in the absence of opines.After concentration by centrifugation, they were resuspended to an

A6. of 0.5 in 0.2 ml of AB-glucose medium with a 0.2 mMconcentration of the appropriate opine (including 30,000 cpm ofradioactive opine) and incubated on a rotary shaker at 28°C. Thecells were collected after the times indicated, and the amount ofradioactivity in the cells was determined by liquid scintillationcounting. 100% = total radioactivity offered to the cells at 0 h.

are shown in Fig. 8A). Bacteria with plasmids pGV2260 andpOCC300 (complete cassette; vector pUCD2001) showed acomparable level of octopine uptake, with a lag period ofabout 30 min. The highest rates and a lag phase of only 15min were obtained with pOCC290 (based on vector pCB303),

which expressed only the putative uptake genes. The copynumber of pCB303-based plasmids is about 10, whereaspGV2260 and pUCD2001 are present in only 1 or 2 copies (3,8, 34). Therefore, it seems likely that the difference betweenpOCC290 and pOCC300 or pGV2260 reflected the copynumber of the plasmids. The length of the lag period corre-sponded to that observed for the induction of the promoter(Pil[occ] in Fig. 1A) (42; unpublished data), indicating thatthe lag in octopine uptake represented the time required forthe expression of the proteins. The results indicate that theopine uptake observed in the experiments was due to in-duced expression of the transport proteins.

noc region. Cells with vector pCB303 showed no signifi-cant uptake of nopaline (Fig. 8B). Ti plasmid pGV3850 andpNOC166 (complete cassette; vector pCB303) yielded com-

parable values, with a slow increase in the first 120 min andhigh uptake rates afterward. Similar lag periods were ob-served in experiments measuring the induction of the pro-moter which controls the expression of this part of the noc

region (Pi2[noc] in Fig. 1B) (42; unpublished data), suggest-ing that the lag period for uptake reflected the induction ofthe proteins.

Plasmid pNOC158 yielded no significantly increased no-

paline uptake compared with the vector control (Fig. 8B).The same result had been obtained with the preinductiontechnique, and the negative results with the second protocolappear to exclude the possibility that preloading of the cellsinhibited further opine uptake. Plasmid pNOC158 differedfrom plasmid pNOC166 only by the absence of the genes fornopaline degradation (Fig. 7B). A. tumefaciens shows low-level Ti plasmid-independent opine uptake (Fig. 8) (23), andone could argue that the effects observed with pNOC166were the result of such uptake coupled with efficient degra-dation of nopaline by the catabolic enzymes. In this case,however, one would predict the same result if only thecatabolic functions were present, and we established a

binary system in A. tumefaciens to test this hypothesis. Thefirst plasmid contained the left part of the noc region(inducible promoter plus all catabolic genes), and the secondplasmid harbored the regulatory gene (nocR) from the rightpart of the noc region which is necessary for the activation ofthe promoter (Fig. 1). The results with various concentra-tions of nopaline showed that the catabolic enzymes were

induced but that the incorporation of radioactivity was nothigher than that observed with control plasmid pCB303.These results indicated that the high uptake rate observedwith pNOC166 was the result of the activated transportfunctions and not of the catabolic enzymes.The reasons for the negative result with pNOC158 remain

unclear. Cloning artifacts appear to be excluded, becausepNOC158 was the basis for the construction of pNOC166and because the data were reproducible with different plas-mid isolates and with different A. tumefaciens colonies. Onepossible explanation could be that gene products from theleft part of the noc region influence the expression or thefunction of the transport protein. This explanation deservesfurther attention, in particular since the expression of thecatabolic functions (left part) and the expression of thetransport genes (right part) are controlled by different pro-moters (Fig. 1B).A. tumefaciens with nopaline-type Ti plasmids can also

utilize octopine, provided the catabolic functions are in-duced with nopaline (20). This result suggests that noc regionfunctions are also capable of transporting octopine. Therequirement for nopaline and the interest in maintainingcomparable induction conditions led to the following proto-

/ ~~pGV2260

pOCC300

PCB303U - vv

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col for these experiments: after pregrowth of the culture inthe absence of opines, the cells were resuspended in 0.2 mMunlabelled nopaline plus 1 ,uM 3H-labelled octopine, and thetime course of radioactivity incorporation was determinedover 3 h. The data indicated the following: (i) no significantuptake of radioactive octopine in the presence of only thevectors; (ii) after a lag phase of about 60 min, high rates ofuptake with pNOC166 and pGV3850 (in terms of percentradioactivity in the same range as that shown in Fig. 8B); and(iii) very little incorporation with pNOC158. The resultsparalleled those obtained with radioactive nopaline, includ-ing the low activity with pNOC158. Additional investigationswith the preinduction technique led to the same conclusions.It should be noted, however, that the results for octopineuptake by noc region functions required precultures from theearly logarithmic growth phase. Cells from later phasesshowed Ti plasmid-independent uptake of octopine whichpartly obscured the activity of the noc region genes. Thiseffect was detected only at micromolar concentrations of theradioactive opine. It has been proposed that such low-levelopine uptake represents a side activity of a pathway forL-arginine transport (23).

It seems likely that the Ti plasmid-encoded transportsystems for octopine and nopaline function in the same wayas the arginine-lysine-ornithine and histidine transport sys-tems in S. typhimurium (13), although this hypothesis mustbe tested in detail. Both octopine and nopaline are L-argininederivatives, a likely explanation for the similarity withtransport systems for basic amino acids in other bacteria. Inthis context, it is of interest that plants transformed withoctopine Ti plasmids synthesize not only octopine but alsoopines containing other basic amino acids, e.g., lysopine[N2-(1-D-carboxyethyl)-L-lysine], octopinic acid [N2-1-D-carboxyethyl)-L-ornithine], and histopine [N2-(1-D-carboxy-ethyl)-L-histidine]. Similarly, nopaline-type tumors producenot only nopaline but also nopalinic acid [N2-(1,3-D-dicar-boxypropyl)-L-ornithine] (39). Previous studies have shownthat many of these substances are also inducers of opineuptake in A. tumefaciens (40), and presumably they aretransported via the same permease function (20). On thebasis of these data, it appears likely that the transportfunctions identified in the catabolic regions possess a broadsubstrate specificity.

ACKNOWLEDGMENTS

We thank U. Langridge for radioactively labelled nopaline and G.Lurz for competent help.

This work was supported by Deutsche Forschungsgemeinschaft(SFB206) and Fonds der Chemischen Industrie.

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