Journal of Bacteriology, February 2002, p . 1121-1131, Vol . 184, No . 4

Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR

Philippe Oger^1, and Stephen K . Farrand^1,2*

Departments of Crop Sciences,¹ Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801²

Received 31 July 2001/ Accepted 13 September 2001

While TraR directly activates the tra regulon, conjugation is dependent on a second environmental signal, the conjugal opine . Opines are highly specific imine or phosphodiester conjugates of amino acids or sugars produced by crown gall tumors, neoplasias induced by A . tumefaciens on its host plants (reviewed in references 7 and 10) . The opines are used by the bacterium as a source of carbon and energy via catabolic systems also encoded by the Ti plasmid . A subset of these compounds, the conjugal opines, also serve as signals, inducing conjugal transfer of the Ti plasmid resident in the agrobacteria that caused the tumor (reviewed in reference 18) . The conjugal opines regulate transfer by controlling the expression of an operon of which traR, the gene coding for the quorum-sensing activator, is a member . For example, the sugar phosphodiester opines agrocinopines A and B (12), which are produced by tumors induced by A . tumefaciens strain C58, are used as a nutritional source by the bacterium . The acc operon codes for the transport and catabolism of these two opines (25, 27, 31), and expression of this set of Ti plasmid genes is controlled by the opine-responsive repressor AccR (2, 31) . Agrocinopines A and B also serve as the conjugal opines of pTiC58 (16); in addition to controlling expression of the acc operon, AccR negatively controls expression of the adjacent, divergently oriented arc operon, a set of five genes of which traR is a member (43) . Thus, in the absence of the conjugal opine, AccR represses expression of arc, and therefore neither traR nor the tra regulon is expressed . This arrangement of regulatory genes results in a hierarchical control process in which the opine signal is required to induce the quorum-sensing system (43) .

The mechanism by which opines regulate transfer has been determined for three types of Ti plasmids (23, 39, 43) . Although the overall strategy by which opines control quorum sensing is conserved among the three plasmids, the conjugal opines are specific to each plasmid type . Thus, agrocinopines A and B induce the transfer of the nopaline-type Ti plasmids (43), while octopine (23) and agrocinopines C and D (39) induce transfer of the octopine- and chrysopine-type Ti plasmids, respectively . Furthermore, the gene sets of which traR is a member differ among the Ti plasmids; while traR is a member of the five-gene arc operon on pTiC58 (43), the activator gene is a member of the 14-gene occ operon on the octopine-type Ti plasmids such as pTi15955, pTiR10, and pTiA6 (23) . Aside from traR, the genes of the occ operon share no relatedness to those of arc or acc . Similarly, the opine-responsive transcriptional regulators are unrelated; while agrocinopines regulate gene expression via AccR, a FucR-type repressor (2), octopine serves as a coinducer for OccR, a LysR-type activator (24) . This conservation of function through variable gene arrangements prompted us to propose that opine responsiveness plays an important role in the ecology of Ti plasmids by promoting transfer only in habitats where acquisition of the Ti plasmid is of benefit to the recipient bacterium (43) . We also predicted that the gene arrangements resulting in such responsiveness arose by fortuitous fusions of opine-controlled gene sets to the resident allele of traR that controls the conjugal transfer component of the particular Ti plasmid core replicon (reviewed in reference 19) .

Opine-induced conjugation is not limited to the Ti plasmids . Of particular interest is a class of Agrobacterium elements called the opine-catabolic plasmids, which generally are found in nontumorigenic members of the genus . These plasmids may share extensive DNA homology with Ti plasmids, but they do not confer upon their host the ability to induce tumors (6, 37) . On the other hand, these extrachromosomal elements often code for the ability to utilize opines produced by tumors induced by tumorigenic agrobacteria (6, 15, 37) . Some of these plasmids are transmissible by mating (11, 16), although it remains to be demonstrated that they are self-conjugal . Moreover, transfer is dependent upon opines . One such plasmid, pAtK84b, codes for the catabolism of two opine families, agrocinopines A and B and nopaline (15, 26), thus resembling nopaline-type Ti plasmids such as pTiC58 . However, pAtK84b lacks the virulence regulon and T-region characteristic of Ti plasmids (6) and does not confer tumorigenicity on its host bacterium, Agrobacterium radiobacter strain K84 . Both nopaline and agrocinopines A and B induce transfer of this plasmid from wild-type A . radiobacter strain K84 to an Agrobacterium recipient (11, 16) . This observation is of interest for several reasons . First, nopaline is not known to induce the transfer of any Ti plasmid . Second, induction of conjugal transfer by more than one opine class has not been described for any Ti plasmid . Third, the region of pTiC58 responsible for catabolism of the agrocinopine opines exhibits little if any homology to pAtK84b (6, 26) . Finally, two regions from pAtK84b hybridize strongly with the traR gene from pTiC58 (40), suggesting that transfer is directly regulated by quorum sensing and that the process is controlled by more than one copy of traR . We therefore sought to determine whether pAtK84b is self-conjugal, and if so, to characterize the molecular mechanism and gene arrangements responsible for the control of transfer of this plasmid by two opines of radically different chemical classes .

 
Media, substrates, and growth conditions. Cultures of Agrobacterium spp . and E . coli were grown at 28 and 37°C, respectively . L broth (low salt) (GIBCO-BRL, Rockville, Md.) was used as the rich liquid medium, and nutrient agar (Difco, Detroit, Mich.) or L agar (GIBCO) was used as the rich solid medium . AT minimal medium supplemented with 0.15% (NH₄)₂SO₄ and 0.2% mannitol as the sole source of carbon (ATNM) (41) was used for the growth of Agrobacterium strains . Medium was solidified with 15 g of agar per liter . Octopine, nopaline, and mannopine were obtained from Sigma Chemical Company, while mannopinic acid and cucumopine were the generous gifts of Yves Dessaux (ISV, CNRS, Gif sur Yvette, France) . Preparations of agrocinopines A and B and of agrocinopines C and D were obtained from tumors induced on tomato plants by the nopaline-type strain C58 and the agropine-type strain Bo542, respectively (12) . The agrocinopines were purified by affinity chromatography as described previously (39, 44) . Purified agrocinopines from each tumor type were redissolved in 50 µl of water per g (fresh weight) of tumor tissue extracted . For Agrobacterium, antibiotics were used as follows (with concentrations in micrograms per milliliter): kanamycin, 100; streptomycin, 250; tetracycline, 1; carbenicillin, 100; and gentamicin, 30 . For E . coli, antibiotics were as follows: kanamycin, 50; tetracycline, 10; and ampicillin, 100 (48) . Biotin (2 µg per liter) was added to minimal medium to allow growth of A . radiobacter strain K84 .

DNA manipulations. DNA manipulations were performed essentially as described by Sambrook et al . (46) . pAtK84b was isolated and purified from NT1(pAtK84b) as described by Hayman and Farrand (26) .

Southern analysis. pAtK84b and cosmid clones of this plasmid (Table 1) (6) were digested with HindIII, EcoRI, or BamHI, as well as with combinations of these enzymes . The DNA fragments were separated by electrophoresis in 0.8% agarose gels and transferred to a charged nylon membrane (Boehringer GmbH, Mannheim, Germany) . Southern blots were hybridized at medium to low stringency using two probes as described by Dessaux et al . (8) . Alleles of the traR gene were detected using the trlR open reading frame (ORF) from pTi15955 (40), while accR was probed for using EcoRI fragment 26 of pTiC58, which contains the complete accR gene and most of accA (2) Probes were labeled using the digoxigenin kit from Boehringer GmbH as instructed by the manufacturer .

Bacterial matings. Agrobacterium donors harboring pAtK84b or its derivatives were mated with A . tumefaciens C58C1RS (Table 1) essentially as described by Oger et al . (40) . Donor strains were incubated with shaking for 36 h in conjugal transfer induction medium, which consisted of AT medium supplemented with 0.2% glucose, 0.15% (NH₄)₂SO₄, and the opine being tested for induction of transfer . Octopine, nopaline, mannopine, mannopinic acid, and cucumopine were included in induction medium at a final concentration of 5 mM . Five-microliter volumes of purified solutions of agrocinopines A and B or C and D were added per milliliter of induction medium .

Nucleotide sequencing and DNA sequence analysis. Double-stranded sequencing was performed using dye terminator chemistries by the Keck Genomics Center of the University of Illinois and by Bio S&T (Lachine, Canada) . DNA sequences were assembled using the Sequencher program (Gene Codes Inc., Ann Arbor, Mich.), and sequence analyses were performed using the Genetics Computer Group (Madison, Wis.) package (version 10) and DNA Strider (36) . Sequences were compared with those in the databases by using the Blast protocols accessible at www.ncbi.nlm.nih.gov/BLAST (1) . When necessary, local alignments were done with the ClustalW program (50) .

Construction of traR::lacZ reporter fusions. The traR_noc::lacZ fusion was created by ligating a 1,896-bp BstYI fragment containing part of the traR_noc ORF into the compatible BamHI site of pLKC482 (51) to form pPOtraR_noc (Table 1) . The cloning resulted in a translational fusion between amino acid (aa) 77 of TraR_noc and LacZ . The ocd::lacZ fusion was created by ligating a 711-bp EcoRI-MscI fragment between the EcoRI and SmaI sites of pVIK107 (29) to form pPO ocd (Table 1) . This cloning resulted in a translational fusion between aa 298 of ornithine cyclodeaminase (OCDase) and LacZ . The traR_acc::lacZ fusion was created by ligating a 2.7-kb HindIII fragment containing the first 600 bp of the activator gene into the HindIII site of pLKC482 to form pPOtraR_acc (Table 1) . This cloning resulted in a translational fusion between aa 200 of TraR_acc and LacZ . All constructs were electroporated into NT1(pAtK84b) to create single-crossover Campbell-type insertions as well as insertion disruption mutations in their respective loci (Table 1), as described previously (29, 39) . The correct structures of all such insertions were confirmed by Southern analysis .

Construction of a traR_noc insertion mutant. A gentamicin resistance gene from pMGm (38) was inserted into the unique SmaI site in traR_noc cloned in pPONH16a to form pPOtraR_nocGm (Table 1) . The wild-type allele of traR_noc on pAtK84b in strain NT1 was replaced with the traR_noc::Gm gene construct by homogenotization to create pPONtraR_nocGm, using the pPOtraR_nocGm as a suicide vector as previously described (20, 40) . The correct double-crossover event was confirmed by Southern analysis .

Construction of the traR_noc traR_acc double mutant. Plasmid pPOtraR_acc was cointegrated into pPONtraR_nocGm to produce the double mutant pPONtraR_noctraR_acc (Table 1) .

Selection of regulatory mutants. Regulatory mutants with mutations in the noc locus were obtained essentially as described by Petit and Tempé (41) . Briefly, approximately 10⁹ cells of NT1(pAtK84b) were spread onto plates of AB minimal medium (4) containing 0.2% arginine as the sole carbon source and incubated for up to 14 days at 28°C . Strain NT1 lacks a functional OCDase gene (9) but can utilize arginine when the nopaline-regulated OCDase coded for by the noc regulon of a Ti or At plasmid is expressed (14, 41) . Among mutants growing on arginine plates, many are constitutive for expression of the noc operon because of mutations yielding nopaline-independent variants of NocR, the activator regulating expression of noc (35, 52) . Candidate noc-constitutive mutants were purified on the same medium and tested for their ability to catabolize arginine . Regulatory mutations in the arc locus were screened for as described by Ellis et al . (13) .

Assays for gene induction. Cells harboring plasmids with traR::lacZ or ocd::lacZ reporter fusions were grown to late exponential phase in ATNM medium containing the appropriate opine being tested as an inducer . The cells were collected and washed with distilled water by centrifugation and were assayed in duplicate for levels of ß-galactosidase as described previously (40) . Activity is expressed as units per 10⁹ CFU . Each experiment was repeated a minimum of two times, and values from a representative experiment are shown .

Nucleotide sequence accession numbers. The sequences of the arc_K84 and nox_K84 operons, including their respective traR genes, have been deposited in GenBank under accession numbers AF065245 and AF065244, respectively .

 
Mapping the traR genes on pAtK84b. By Southern analysis two fragments of pAtK84b, HindIII 16a and HindIII 21b, hybridized with a traR probe (40), suggesting that the plasmid codes for two copies of this regulatory gene . The fragments map to the 9 and 12 o'clock positions on the standard map of pAtK84b (6), with fragment 21b being located in the vicinity of the region believed to be responsible for transport of agrocinopines A and B and fragment 16a mapping to a region at the end of the noc locus, which codes for the transport and catabolism of nopaline (6) (Fig . 1) .

 
Cloning and sequence analysis of the conjugation-regulatory loci from pAtK84b. The locations of the hybridizing segments suggest that each of the two copies of traR is linked to a locus required for catabolism of one of the two conjugal opines . To assess this hypothesis, we sequenced the two traR alleles and the regions surrounding these genes .

Analysis of the sequence of a 6,080-bp segment including fragment 21b identified four complete ORFs, all oriented in the same direction (Fig . 1) . The last ORF, which we named traR_acc, could encode a 234-residue protein closely related to the products of other traR genes from Agrobacterium Ti plasmids (Table 3) . traR_acc is preceded by a 1,464 bp-ORF, spl (Fig . 1), that could encode a 488-residue protein that is 87% similar in sequence to the product of splA from the arc operon of pTiC58 (Table 3 and Fig . 2) . spl is preceded by an 1,182-bp ORF, orf1 (Fig . 1), which is not present in the arc operons of pTiC58 and pTiChry5 (Fig . 2 and data not shown) . The 394-residue translation product of this ORF is 52% identical and 64% similar in sequence to the product of an ORF, AGR-L-1564, located on the linear chromosome of strain C58 (Table 3) . The products of both ORFs also are related to YihR, a gene product of E . coli K-12 that is itself related to aldose-epimerases (Table 3) . orf1 is preceded by a 1,641-bp ORF, orfB (Fig . 1), that could code for a protein of 547 residues, the sequence of which is 85% similar to the product of orfB from the arc operon of pTiC58 (Table 3 and Fig . 2) . Directly downstream of traR_acc is a 90-bp nucleotide sequence that is identical to a portion of mcpA, a gene located immediately downstream of traR_C58 in the arc operon of pTiC58 (Fig . 2) . Finally, the 3" end of an incomplete ORF oriented in the same transcriptional direction is located just upstream of orfB (Fig . 1) . The deduced amino acid sequence of this partial ORF is not related to any protein in the databases . Based on sequence and organizational similarities with the arc operon of pTiC58 (Fig . 2), we named this gene set of pAtK84b arc_K84 .

 
Nucleotide sequence analysis of a 4,860-bp region including HindIII fragment 16a identified four complete and two incomplete ORFs (Fig . 1) . One of the incomplete ORFs, located on the far right-hand (clockwise) side could code for a protein that is closely related to the product of the ocd gene, which is the last ORF in the nox operon of pTiC58 (Table 3) . The four genes of this operon from pTiC58 code for the enzymes responsible for degrading nopaline to proline (47, 55), with ocd specifying OCDase (47), an enzyme required for the cyclization of ornithine to proline (55) . The middle ORF, which we named traR_noc, is transcribed in the same direction as ocd (Fig . 1) . Like for traR_acc, the 234-residue translation product of this gene is closely related to the products of other traR genes (Table 3) . traR_noc and ocd are separated by two slightly overlapping ORFs, orf1 and orf2, of 1,128 and 399 bp, respectively . The 376- and 133-residue translation products of these two ORFs are not related to any sequences in the protein databases . traR_noc, orf1, orf2, and ocd are oriented in the same direction and could be transcribed as an operon (Fig . 1) . The remaining two ORFs, sml and msl, located on the left-hand side of the sequenced region, are oriented convergently to the first four ORFs (Fig . 1) . sml (for smoC-like) could encode a 340-residue protein weakly related to SmoC, a sorbitol-responsive repressor from Xanthomonas species, while msl (for mannopine synthase-like) could code for a protein related to the N-terminal portion of Mas1", an oxidoreductase required for the synthesis of mannopine, another opine coded for by the T regions of several Ti and Ri plasmids (Table 3) . A segment of the product of the msl ORF also is related to a domain strongly conserved among the phosphogluconate mutase family .

The conjugal opines induce expression of their cognate traR genes. In the three examples described to date, opines regulate conjugation of Ti plasmids by controlling expression of traR (23, 39, 43) . Given that pAtK84b contains two full-sized copies of the traR gene and is induced for transfer by two opines we reasoned that expression of each gene would respond to one or the other of the conjugal signals .

traR_noc is located directly downstream from an ocd homolog within the nopaline-catabolic region of pAtK84b, suggesting that the expression of this allele is controlled by nopaline . We tested this hypothesis by determining the influence of the arginyl opine on the expression of the ocd and traR_noc genes of pAtK84b . As assessed by analysis of ß-galactosidase activity from lacZ reporter fusions integrated into pAtK84b, both genes are expressed at very low basal levels in cells grown with mannitol as the sole carbon source (Table 4) . However, addition of nopaline to the growth medium resulted in 15- to 20-fold increases in the levels of expression of these two genes . No other opine tested, including agrocinopines A and B, induced the expression of either gene (Table 4) .

 
On pTiC58 the single copy of traR is located within arc, an operon of five genes the expression of which is induced by agrocinopines A and B (43) . Given the relatedness between arc_C58 and arc_K84 (Fig . 2), we reasoned that expression of traR_acc is controlled by the agrocinopines in a similar manner . As assessed by analysis of ß-galactosidase activity, traR_acc was expressed at very low levels in the absence of the agrocinopines . However, addition of these sugar phosphodiester opines to the growth medium resulted in an eightfold increase in the expression level of the lacZ fusion (Table 4) . No other opines, including nopaline, mannopine, and the closely related sugar phosphodiester opines agrocinopines C and D, induced expression of the traR_acc::lacZ reporter (Table 4 and data not shown) .

Mutations in the traR genes abolish induction of transfer but only by the cognate controlling opine. In addition to creating reporters, the ocd::lacZ and traR_noc::lacZ fusions produced ocd- and traR_noc-null mutants of pAtK84b . Strains harboring the ocd mutant plasmid failed to catabolize nopaline, while the strain harboring the traR_noc mutation grew as well as the wild type with the opine as the sole carbon source (Table 5) . Both mutant plasmids failed to transfer from donors pregrown in medium containing nopaline (Table 6) . Providing a constitutively expressing copy of traR in trans restored conjugal transfer to both mutant plasmids (data not shown) . Moreover, the complemented mutant plasmids transferred even in the absence of induction by nopaline .

 
In a manner analogous to that for the traR_noc fusion, the traR_acc::lacZ construct created a traR_acc-null mutation . This mutant plasmid failed to transfer from donors pregrown in medium containing agrocinopines A and B (Table 6) . However, providing traR_acc or traR_noc in trans to the mutant plasmid restored conjugal transfer (data not shown) . Moreover, as with the traR_noc mutant, transfer of the complemented traR_acc::lacZ construct is independent of induction by agrocinopines A and B .

traR_noc and traR_acc are regulated independently. The presence of two copies of traR, coupled with our observation that these two genes code for functional proteins, suggested that the two alleles on pAtK84b are regulated independently by the two conjugal opines . We tested this hypothesis by assessing the influence of the alternate conjugal opine on the transfer of the traR-null mutant plasmids . The mutant plasmids transferred at frequencies similar to those of pAtK84b when the donors were pregrown in medium containing the alternate conjugal opine, the cognate opine for the wild-type copy of traR (Table 6) . Thus, pPONtraR_acc (pAtK84btraR_acc::lacZ) transferred from donors induced with nopaline but not with agrocinopines, while pPONtraR_noc (pAtK84btraR_noc::lacZ) transferred from donors induced with agrocinopines A and B but not with nopaline . Growth in medium containing both opines did not increase the frequency of transfer of pAtK84b from strains K84 or NT1(pAtK84b) (data not shown) .

While derivatives of pAtK84b disrupted in one of the two traR genes transferred when induced with the second opine, pPONtraR_noctraR_acc (pAtK84btraR_acc::lacZ-traR_noc::Gm), in which both traR genes are mutant (Table 1), failed to transfer from donors grown in medium containing either opine alone or in medium containing both opines (Table 6) . However, transfer of this mutant plasmid was restored by providing either traR gene in trans (data not shown) .

Isolation of opine-regulatory mutants constitutive for conjugal transfer. Agrocinopines A and B control transfer of pTiC58 by regulating expression of the arc operon, and therefore traR, via the opine-responsive repressor AccR (43) . Transfer-constitutive (Tra^c) mutants of pTiC58 are easily isolated by selecting for rare transconjugants following matings conducted in the absence of the inducer opines (13) . Such mutants invariably contain null alleles of accR (reference 2 and unpublished results) . However, despite repeated attempts, we were unable to isolate such spontaneous Tra^c mutants of pAtK84b .

Expression of the noc and nox operons of pTiC58 is controlled by NocR, a LysR-type transcriptional activator (35, 52) . Mutants of pTiC58 exhibiting constitutive expression of the two operons can be obtained by selecting for growth on medium containing arginine as the sole carbon source (14, 41) . Among such variants are those that presumably code for a constitutive form of NocR (NocR^c) that no longer requires the opine ligand to activate expression of the noc and nox operons . Such Noc^c mutants also utilize octopine, a chemically related but noninducing arginyl opine (41) . Given that nopaline regulates the expression of ocd and traR_noc on pAtK84b, we predicted that this plasmid codes for a NocR-like activator and that at least some Noc^c mutants of this plasmid should be constitutive for conjugal transfer .

NT1(pAtK84b) did not grow on minimal medium containing arginine or octopine as the sole carbon source (Table 5) . However, after incubation for 1 to 2 weeks, about 100 colonies appeared on arginine plates inoculated with ca . 10¹⁰ cells of NT1(pAtK84b) . Ten such colonies from each of four selection plates were isolated and purified . All 40 clones utilized arginine and octopine in the absence of nopaline (Table 5 and data not shown) . Of the Noc^c mutants tested, all transferred pAtK84b even when pregrown in the absence of nopaline (Table 7 and data not shown) . Moreover, while NT1(pAtK84b) produces barely detectable amounts of AAI when grown without a conjugal opine (3), the Noc^c mutants produced large amounts of the quormone under these noninducing conditions (data not shown) . However, transfer frequencies, as well as levels of AAI produced by these Noc^c Tra^c mutants under noninducing conditions, were consistently lower than those of NT1(pAtK84b) grown with nopaline (Table 7 and data not shown) . Transfer frequencies and signal production exhibited by these Noc^c Tra^c mutants could be fully induced by providing nopaline or agrocinopines A and B in the growth medium (Table 7) .

 
In this study we show that regulation of transfer of pAtK84b by two opines, first reported by Ellis et al . (16), results from the independent control of two copies of traR, each by one of the two tumor-produced opine signals . In this regard, pAtK84b is the first of the Ti-like plasmids from the genus Agrobacterium to be shown to contain two functional copies of the quorum-sensing activator . The octopine-type Ti plasmids pTi15955 (40) and pTiR10 (57) also contain two independently regulated traR genes . However one such gene, called trlR, codes for a mutant protein that lacks the C-terminal DNA binding domain and consequently fails to activate transcription (40, 57) . The product of this gene is induced by mannopine, a member of the mannityl opine family, and strongly inhibits activity of the functional quorum-sensing activator coded for by the traR gene located within the octopine operon . Although it is the first example, it is likely that pAtK84b is not unique; pAtK112, an opine catabolic plasmid in a second isolate of A . radiobacter, is induced for transfer by both nopaline and agrocinopines A and B (16), suggesting that this element also contains two functional copies of traR . Moreover, A . radiobacter K299, which catabolizes both nopaline and agrocinopines A and B, contains at least two copies of the traR gene (40) . Like K84, both K112 and K299 were isolated from orchards in South Australia . However, it is unlikely that the plasmid in either isolate is identical to pAtK84b; both pAtK112 and pAtK299 confer susceptibility to agrocin 84 (26; J . G . Ellis, personal communication) .

Among the Ti plasmids examined to date, opines control conjugation because traR is located within an operon regulated in response to the tumor signal (Fig . 3) (23, 39, 43) . Such also is the case for pAtK84b; clearly, expression of the two traR genes is induced by one or the other of the two conjugal opines (Table 4), and each is a member of a gene set regulated by one or the other opine inducer (Fig . 1) . The agrocinopine-responsive copy, traR_acc, is located within a group of four genes all oriented in the same direction . Within this group, the two-gene set of traR_acc and spl is found in the arc operons of pTiC58 and pTiChry5 (Fig . 2 and 3) (39, 43) . Moreover, arc_K84 contains a homolog of orfB and remnants of mcpA, the terminal gene of the arc operon of pTiC58 (Fig . 3) . In pTiC58 (43), and probably also in an otherwise unrelated Ti plasmid, pTiChry5 (39), the arc operon, and therefore traR, is negatively regulated by AccR, the transcriptional repressor for which agrocinopines serve as the inducers (2) . Thus, in the presence of the sugar phosphodiester opines, AccR releases from the arc promoter, the operon is expressed, and TraR is produced at levels sufficient to activate expression of the tra regulon . On pTiC58, accR, the gene coding for the opine-responsive repressor, is the first member of the acc operon located just upstream of and in the opposite orientation to arc (31) . The acc operon of this Ti plasmid, in turn, is responsible for the uptake and catabolism of agrocinopines A and B (25, 27, 31) . Thus, arc and acc with its attendant accR form a tightly linked but separately transcribed pair of operons on pTiC58 that couple opine catabolism to conjugal transfer . While traR_acc and portions of arc are conserved on pAtK84b, it is not at all clear how the agrocinopines regulate expression of this operon on the opine-catabolic plasmid . pAtK84b, although conferring uptake of agrocinopines A and B (26), does not hybridize significantly with elements of the acc operon from pTiC58 (6, 26) . Consistent with this observation, acc_K84, the to-date-uncharacterized agrocinopine transport locus of pAtK84b, does not confer uptake of or sensitivity to the toxic opine analog agrocin 84 (26) . Susceptibility to this antibiotic is a hallmark associated with all other known agrocinopine catabolism systems, including those of pTiC58, pTiBo542, and pTiChry5 (21, 26, 39) . Furthermore, the region immediately upstream of the arc operon on pAtK84b does not contain a homolog of accR (data not shown) . By Southern analysis we did detect two regions of pAtK84b that hybridized weakly with an accR probe (data not shown) . However, neither region is linked closely to arc_K84, suggesting that if either segment does encode the agrocinopine-responsive regulatory gene, it, and perhaps the rest of acc_K84 has diverged from the acc operon of pTiC58 and, unlike the Ti plasmid, is spatially separated from arc . Finally, we were unable to isolate agrocinopine-independent transfer-constitutive mutants of pAtK84b . Such mutants, invariably with lesions in accR, are obtained readily from pTiC58 (13; our unpublished results) . Despite these differences, the structural similarities between arc_K84, arc_C58, and arc_Chry5 (Fig . 3) clearly indicate that the three gene sets have evolved from a common ancestral operon .

 
Expression of the second activator gene, traR_noc, is induced by nopaline but not by agrocinopines A and B (Table 3) . The gene is located downstream from and in the same transcriptional orientation as ocd, the gene coding for OCDase, an enzyme essential for catabolism of nopaline (47) . On pTiC58, the genes required for nopaline catabolism are organized as two operons separated by about 5 kb of sequence of unrelated function . The first operon, noc, codes for the four genes of the transport system (54), while the second, nox, codes for the three enzymes, including OCDase, required for catabolism of the opine (55) . Hybridization analysis suggests that the nopaline catabolism region of pAtK84b is organized in a similar manner (6) . Two lines of evidence indicate that traR_noc is part of the nox operon . First, an insertion mutation in ocd_K84 abolishes nopaline-inducible transfer but has no effect on transfer induced by agrocinopines A and B (Table 6) . We take this to mean that the mutation in ocd is strongly polar on nopaline-induced expression of the downstream traR_noc gene . Consistent with this conclusion, the ocd mutant can be complemented for transfer by traR_noc expressed in trans . That the lacZ fusion to ocd is induced by nopaline (Table 4) and that the mutation arising from this fusion abolishes nopaline utilization (Table 5) confirm that this gene is indeed part of the noc system . Second, mutants of pAtK84b constitutive for nopaline catabolism also transfer in the absence of induction by either conjugal opine (Table 7), However, as first noted by Ellis et al . (15), such Noc^c variants are not fully constitutive for transfer; growing donors harboring such mutant At plasmids with nopaline increases the transfer frequency by almost two orders of magnitude (Table 7) . We suspect that the noc regulon of pAtK84b is controlled by a transcription factor similar, if not identical, to NocR, the LysR-like activator responsible for controlling noc of pTiC58 (35, 52) . NocR is closely related to OccR, the LysR-like activator of the octopine operons of Ti plasmids such as pTiR10 and pTi15955 (24, 52) . A subset of such octopine-independent mutants of OccR are only partially constitutive and respond positively to addition of octopine (5) .

While pAtK84b contains two copies of traR, the plasmid contains only one copy of the genes comprising the conjugation system . Southern analyses indicate that the traAFB and traCDG operons are arranged in a manner similar to that in pTiC58 and are located in the region just anticlockwise from traR_acc (Fig . 1) (6) . The trb operon, which also codes for TraI (28), the acyl-homoserine lactone quormone synthase, is located at about two o'clock on the map of pAtK84b (Fig . 1) (6) and is linked closely to the replication region, a feature that is conserved in all Ti plasmids analyzed to date (32) . Thus, it is likely that the two TraR paralogs, the expression of each of which is induced by its own opine, activate the single tra regulon .

In the four Ti plasmids for which nucleotide sequences are available, traR always is oriented opposite to and located within a few kilobases downstream of the traAFB operon 43, 49, 59; our unpublished results) . On pAtK84b, traR_acc associated with the arc_K84 operon represents this classical arrangement (Fig . 1), and thus the system of this plasmid derives from a lineage that includes the conjugal control regions of pTiC58 (43), pTiSAKURA (49), pTiChry5 (39), and pTiBo542 (our unpublished results) . Significantly, conjugal transfer of each of these plasmids is induced by one or the other of the two families of agrocinopine-type opines (16, 39) . Interestingly, the arc_K84 operon most closely resembles the sucP-traR gene set found on several Ti plasmids from Agrobacterium vitis (Fig . 3) (21) . Moreover, TraR_acc from pAtK84b is most closely related to the TraR-like gene product of these Ti plasmids (Table 3) . However, the TraR products from the A . vitis Ti plasmids apparently are not functional (43), and the sucP-traR gene set on these elements may be vestigial .

While arc_K84 represents another iteration of the arc-type conjugal control region, the association of traR_noc with the nopaline catabolism system of pAtK84b is novel in its arrangement . Nopaline is not known to induce transfer of any Ti plasmid, although this opine does activate conjugation of the opine-catabolic plasmid pAtK112 (16) . The association of traR_noc with the nopaline catabolism regulon is conceptually reminiscent of the association of traR_occ with the octopine system of octopine-type Ti plasmids (23) . However, although the organization of the downstream elements of occ resembles that of the downstream elements of nox, it is unlikely that the associations of traR_noc and of traR_occ with their respective noc and occ systems derive from a common lineage . While traR_noc is separated from ocd_noc by two ORFs, traR_occ is located almost 8 kb downstream of ocd_occ, with the intervening region coding for six genes on pTiR10 (Fig . 3) (23) . Moreover, the two ORFs between ocd_noc and traR_noc on pAtK84b are not related to any of the six genes located between ocd_occ and traR_occ (Fig . 3) . Thus, the association of traR with the noc and occ systems most likely arose following the divergence of these two opine catabolism systems from some common ancestor . The nox-traR_noc gene set also is novel with respect to placement; this traR control operon is not closely linked to the traAFB operon (Fig . 1) . Instead, traR_noc abuts two ORFs in the opposite orientation located at the edge of a region of pAtK84b showing no detectable relatedness with pTiC58 or the octopine-type Ti plasmid pTiAch5 (6) .

Clare et al . (6) suggested that pAtK84b arose as a deletion derivative of a nopaline-type plasmid such as pTiC58 . We think that such an origin is unlikely for several reasons . First, while the tra regulon, replication region, and nopaline catabolism system of pAtK84b are closely related to those of pTiC58, the agrocinopine utilization system differs significantly in function and sequence from that of the nopaline-type Ti plasmids (6, 26) . Second, although the arc conjugal control regions of pTiC58 and pAtK84b clearly share a common phylogeny (Fig . 3), they have diverged significantly . Third, pTiC58 lacks any association of traR with the nopaline catabolism system, suggesting that the nox-traR arrangement on pAtK84b is novel and arose independently . Finally the noc-nox-traR interval of pAtK84b is contiguous to a ca . 10-kb region of the plasmid sharing no detectable relatedness to the nopaline-type Ti plasmids (6) . On pTiC58 nox is contiguous with the T region, a segment crucial for pathogenicity that is not present on pAtK84b . This last observation suggests that this portion of pAtK84b could have derived from some other type of nopaline-catabolic element of Agrobacterium . We think it likely that pAtK84b is chimeric and, as such, has arisen as an assemblage of segments of other Ti and At plasmids .

pAtK84b is central to the biology of strain K84 . This bacterium, although itself unable to induce tumors, can, by virtue of its ability to catabolize nopaline and agrocinopines A and B, co-opt those galls produced by nopaline-type pathogens . Moreover, agrocin 84 produced by strain K84 is lethal to and targets specifically pathogenic nopaline-agrocinopine-type strains of A . tumefaciens (17) . The two traits combined allow strain K84 to occupy the niche provided by the tumor and to eliminate competition by the pathogenic agrobacteria that induced the neoplasia (18) . In this regard, the bacterium by virtue of its resident plasmids is a highly evolved fratricidal parasite . Finally, pAtK84b, the plasmid responsible for these catabolic traits, utilizes both opines as signals to induce conjugal transfer and can be induced for transfer within nopaline-agrocinopine type tumors or within tumors induced by other agrobacteria that produce only one or the other of the two opine types .

Present address: Center of Advanced Studies and Research, 38111 Bonn, Germany .

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