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

Opine catabolism and conjugal transfer Qf the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor (Agrobcterium tumefaciens/gene regujation/AccR)

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

Communicated by Luis Sequeira, September 16, 1991

ABSTRACT The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gail tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for' opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accRencoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

negative regulatory proteins that control sugar catabolic operons in several unrelated bacteria (10). Our findings provide a molecular framework for the coregulation model and illustrate the diversity of regulatory mechanisms that govern the interaction between Agrobacterium and its plant hosts. 11

MATERIALS AND METHODS Strains and Plasmids. A. tumefaciens strains used were C58 (11), NT1(pTiC58Trac) (12), C58ClCE(pWI1003) (8), NT1(pAgK84-A1) (13), and C58C1RS (rifampin resistant, streptomycin resistant) (8); Escherichia coli strains were DH5a, RR1, and S17-1 (14). Broad host range vectors pRK415 (15) and pLAFR6 (16) were used for subcloning and complementation analysis; pCM1 was the source of the cat cartridge (17); and pUC18 served as cloning vector for DNA sequencing. Reagents. Antibiotics, 5-bromo-4-chloro-3-indolyl 8-Dgalactoside, o-nitrophenyl 13-galactoside, and nopaline were purchased from Sigma. Mutant Construction. Plasmids pTHB58 (18) and pSVB20 (12) were mutagenized with Tn3HoHo1 (19), and the mutations were homogenotized into Ti plasmids by standard methods (20). Agrocin 84 Sensitivity Assays. Because amounts of purified agrocinopine A and B sufficient for catabolic studies are not available, sensitivity to agrocin $4, assayed on Stonier medium plates as described by Hayman and Farrand (18), was used to monitor expression of acc. Uptake of agrocin 84 depends upon the acc-encoded opine transport system and therefore serves as a measure of acc expression (18). Strains to be tested for sensitivity were grown in LB medium and washed in 20 mM phosphate buffer (pH 7.0) before addition to the soft agar overlays. Induction by agrocinopine was assessed by placing paper strips impregnated with -20 nmol of agrocinopine onto the overlay agar (18). Conjugal Transfer Assays. The quantitative spot mating technique used in these studies has been described (12). The Ti plasmidless recipient strain C58C1RS was spread over the surface of an AT minimal medium (6) selection plate containing streptomycin, rifampin, and a mixture of nopaline (1 mM) and arginine (9 mM). Ten-microliter volumes of serial dilutions of donors were spotted onto the recipient plate. For opine induction, donors were grown overnight on small agar blocks containing -100 nmol of agrocinopine A and B. The cells were resuspended in 0.5 ml of AT minimal medium and diluted, and 10-.lI sam,3les were spotted onto recipient plates. Plates were incubated at 280C for 2-3 days and colonies

During interactions between Agrobacterium tumefaciens and its plant hosts, the bacterium senses and responds to several plant-produced signals. For example, virulent agrobacteria use a two-component signal transduction system to sense small phenolic compounds released from wounded plant tissue. In response, the bacteria express Ti plasmid-encoded Vir functions, which, in turn, facilitate T-strand excision and transfer to susceptible plant cells (1). The resulting plant neoplasias synthesize unique low molecular weight carbon compounds, called opines, which are thought to provide a source of carbon for tumor-colonizing agrobacteria (2). The opines also act as signals. For example, the Ti plasmidencoded functions required for opine catabolism are specifically induced by their cognate substrates (3, 4). In addition, conjugal transfer of the Ti plasmid, which is normally repressed, is induced by a subclass of opines, the conjugal opines (5, 6). A model proposing that conjugation and catabolism of the conjugal opines are coregulated emerged from the observation that mutants constitutive for conjugation are often derepressed for conjugal opine catabolism. Similarly, mutants selected for constitutive expression of these opine catabolic functions are generally transfer-constitutive (5, 7-9). For nopaline-type Ti plasmids such as pTiC58, conjugation is induced by the sugar phosphate opines, agrocinopines A and B (6). We report here that the coregulation of agrocinopine catabolism (Acc) and conjugal transfer (Tra) of pTiC58 is mediated by a repressor, AccR. This repressor is related to

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

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 643

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Biochemistry: Beck von Bodman et al.

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

DNA Sequence Analysis of the EcoRI 26 Region. Fig. 3A shows a 1190-base-pair (bp) DNA sequence of the wild-type EcoRI fragment 26 starting from the left EcoRI site. Computer analysis identified four open reading frames (ORFs). ORFI, which could encode a 28-kDa protein, begins with a TTG (bp 322) or a GTG (bp 325) and terminates with a TAG at position 1096. The two potential initiation codons are preceded by sequences that match the E. coli ribosomal binding (RB) consensus sequence. Upstream from the potential initiation codons are sequences similar to the virE -10 region (22) and with nearly perfect identity to the E. coli -35 consensus sequence (23). In addition, the potential -10 region is part of a 17-bp palindrome (CGCTCAIAGTATGiAGC~G). A similar repeat (GCCTCATTCAACAiA C) is present in the 3' noncoding region that separates ORFi from ORF4. ORF2 begins with an ATG at bp 595 and is in frame with ORFi. It is preceded by a potential RB site and - 10 and -35 sequences that resemble those of the virC promoter region (22). ORF2 could encode an 18-kDa protein. ORF3 proceeds in the opposite direction from an ATG at bp 960 and largely overlaps ORFs 1 and 2 (Fig. 3B). There are no identifiable 5' regulatory features associated with this ORF. ORF4 reads in the same direction as ORFi and ORF2, beginning with an ATG at bp 1156 (Fig. 3B) and extends beyond the right EcoRI site (data not shown). The sequence from pTiC58Trac is identical to that of the wild-type Ti plasmid except for a 5-bp deletion (bp 336-340) in the ORFi (ORF3) coding region (Fig. 3). This deletion creates a frame shift in ORFi that results in a new stop codon 22 bp downstream from the site of the mutation (Fig. 3A). The deletion is outside of and 5' to ORF2. ORF1 Encodes a Protein Homologous to Regulators of Other Sugar Catabolic Operon Systems. In a search of the National Biomedical Research Foundation Protein Information Resources data base, the regulatory proteins GutR (10), FucR (10), and GlpR (24) from E. coli showed between 26% and 34% amino acid identity with the ORF1-encoded protein. In addition, the ORFi amino acid sequence aligns well with those of the LacR proteins from Lactococcus lactis and Staphylococcus aureus and with DeoR, FucR, and GutR from E. coli (10) (Fig. 4). Although there are stringently conserved amino acids throughout the sequences, regions of

within the spots were counted. Conjugation frequencies are expressed as number of transconjugants per input donor. ,f-Galactosidase Assays. Strains were grown in AT minimal medium to an OD6w of 0.2. The cultures were divided into two tubes, a solution of agrocinopines (final concentration, 200 ttM) was added to one set, and incubation was continued for 3 hr. Levels of 8-galactosidase were measured as described by Stachel et al. (19). DNA Sequencing. EcoRI fragments 26 from the wild-type and mutant Ti plasmids were cloned into pUC18 and both strands were sequenced using the Sequenase kit, version 2.0 (United States Biochemical). The DNA sequences were analyzed using the DNA* computer program (DNASTAR, Madison, WI). Chloramphenicol Acetyltransferase (CAT) Assay. Cells were grown in LB broth to late exponential phase, and cell-free extracts were obtained by sonication. CAT protein was detected using the CAT ELISA kit marketed by 5 Prime 3 Prime, Inc., following the recommended procedure.

RESULTS Mutations and Genetic Complementations Identify a pTiC58-Encoded Function that Regulates tra and acc. Two cosmid clones, pTHB112 and pTHB55, repressed conjugal transfer and acc-encoded supersensitivity to agrocin 84 when in trans to the spontaneous Trac/Accc mutant Ti plasmid pTiC58Trac. These two cosmid clones contain DNA inserts from pTiC58 that overlap the agrocinopine catabolic locus (acc) and Tra region I (Fig. 1). pSVB20, a similar cosmid clone from pTiC58Trac, showed no effect on either phenotype (data not shown). We then subcloned regions of overlap from the wild-type and mutant cosmid clones into pRK415. pRKW26, containing EcoRI fragment 26 from wild-type pTiC58, repressed conjugal transfer to levels below 10-8 per input donor. This repression was largely overcome by addition of agrocinopines (Fig. 2A). Similarly, pRKW26 repressed the Accc phenotype; merodiploid strains containing this clone were nearly resistant to agrocin 84 (no zone of growth inhibition) (Fig. 2B). pRKC26, containing EcoRI fragment 26 from pTiC58TraC, had no effect on the Trac or Accc phenotypes (Fig. 2). T-REGION

1

749 1 Traill EcoRl

['oIs I

1

20

acc

2 2i _33_l

I

1-120

15

289

Tral 4

I----

'25

FIG. 1. Organization of the pTiC58 tra/acc region. The positions of the Tn3HoHol lacZ fusions in acc (289) and TraIl (749) are shown as flagstaffs with

-I

i

I

I

1-66 I

-

+

.t

pTHB58 * pWI 1003 A

pTHB55 pSVB20 pTHB1 12

pTHH26--

the transcription polarity indicated by the direction of the flag. "R" denotes the coding region for the tra/acc repressor. The lines below the map represent cloned segments of the Ti plasmid: pTHB55, pTHB58, and pTHB112 are cosmid clones derived from wild-type pTiC58 (18), whereas pSVB20 contains an insert from pTiC58Trac (12). Plasmid pTHH206 is the smallest subclone encoding a functional acc locus (18). Arrowheads indicate that the clones extend beyond the map. The deletion in pWI1003 is indicated by a hatched box. Coordinates (kilobases) are from Depicker et al. (21).

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

Biochemistry: Beck von Bodman et al. A

Agrocinopine

NT1 (pTiC5BTra )

C58

NT1 (pTiC58Tra )

+

pRKC26

-
iQ

0 (.0

C't

TAG 1096

ATG

ORF4

1156

_.ON.

TAG 1096

GCTA 960

in the N-terminal domains of all five proteins has the predicted helix-turn-helix motif common to many DNA binding proteins (10, 25, 26). The C-terminal domains feature a second conserved sequence that may define an inducer binding site (10). Subclones containing ORF1 complement the AccC and TraC phenotypes associated with pTiC58TraC (Fig. 2). Repression ofTra by ORFi responds to agrocinopine induction (Table 1), which is consistent with ORF1 encoding the opineresponsive gene regulator. Ih contrast, repression of acc in these merodiploids, as characterized by resistance to agrocin 84, is not responsive to agrocinopine induction (Fig. 2B). We have no explanation for this observation. However, it is possible that repressor-operator interactions regulating the expression of acc are more complex than those at the tra loci. The ORF1 gene product acts at the level of transcription (Table 1), which is consistent with our proposal that accR encodes a repressor protein. The question remains as to whether ORF2 constitutes a separate functional gene. cat expresses in pOCi even though the deletion in ORFi should cause the translational termination of AccR and a concomitant polar effect on expression of the downstream antibiotic resistance gene. This suggests that ORF2 may be separately transcribed.

100D

112D

.

/

595 AGT

.

A A ACCAC.CT TAA

-_

ATG

ORF2

.

0

\1

/

GT C GGC GT GA TC GT C A C C GA CT CT GGA A C C C GA A C CAT CAT T 0 G T G T R T V V S V . . . Xms II I A GT T C GT T GT TGC GA A C TA GGT T C GA C GOGC C C GC GC GA AT T A N F a V V

1190

FIG. 3. Partial DNA sequence and organization of ORFs within EcoRI fragment 26 from pTiC58. (A) The sequence of the first 1190 bp of EcoRI fragment 26 shows the proposed TTG/GTG start site of ORF1, preceded by two RB sequences. Upstream sequences similar to the virE -10 and the E. coli -35 promoter sequences are underlined. ORF2 begins with an ATG at position 595. The 5-bp deletion in the DNA sequence of the fragment from pTiC58TraC is indicated by "D." The amino acid sequence translatable from ORF1 and ORF2 is shown in one-letter code. ORF3 begins at an ATG at position 960. Two related inverted repeats are indicated by divergent arrows. (B) The schematic map of EcoRI fragment 26 shows the relationships of ORF1, ORF2, and ORF3 and the location of ORF4. The cat cassette was inserted at the Sal I site at position 826.

Our results provide genetic and molecular data that confirm the model of Ellis et al. (8) proposing that the regulation of tra and acc of pTiC58 involves a common repressor. These Table 1. .8-Galactosidase activities from acc and Tra II lacZ gene fusions under repressed, constitutive, and agrocinopine-induced conditions ,B-Galactosidase activity,* units min- OD6j-o1

Plasmid content

pTiC58Trac pTiC58TraCtra749 pTiC58TraCtra749/pOW1 pTiC58Tractra749/pOC1 pTiC58acc289 pTiC58Tracacc289 pTiC58TraCacc289/pOW1 pTiC58TraCacc289/pOC1 ND, not determined.

Without agrocinopine 2 1176 3 1314 1 584 2 482

With

agrocinopinet ND 1522 560 1037 2 192 2 422

*Measured as described in ref. 19.

tA mixture of agrocinopines A and B was added to a final concentration of 200 ,uM.

Biochemistry: Beck von Bodman et al. AccR

LacR DeoR GuIR FucR

AccR LacR DeaR GutR

FWCR

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

647

AKIVELLRDE FAV AT A R R D SEH EA G L LRR T H G IR | KIDGTITUKEIIDELDftSIDMTARRDLDLjEA[ LTR THG METR R E EI LLQOELIKRSDK[[HLKDAAALLG VSEMTIRRDLNNHSAPVVL ---G

LV F NST DR MKESLHMNKKRR[

VEEAQY[UDTTGT|T|I|RNDLV E TV YG QAlLPYLOKQGKCS A DKLLNHT SgTTEAASgLKVgK ETRR DLN T GKIL G G A VAS V T 0 V TFD- FIN A - AfA V WE GVVAG A A FI GPG TT L VL AL KMA GAO EKKSLNTK- EK I AKKACSLJ K DpT VLLDAGTTABEV EWK

MKPROR MK- A

LLSSKKPLE[SJTHI

LFFDGTTTPWI EAI D GYI VLEPRSASHYLLSDQKSRLVE- EKRRAAKLAATLVEPDO G VvL - - - NKEESDnPPIDHKT L ET H KKE L IAEAAVSF H DSII LD A GST VQ M V P L LL KSHYEH KDIAR E A L AW I EGMV LA ST C WY LE1R QLP

RAKYIHRONO0NDSGDPFHIF

L QL[ENVDKL AccR DRR YT R FMSNG L D-EELIV [El N R LacR G Y K R VITNS L P V F LI L NDSETI DLLLL- GGE RERI T G AF VGS T N L K A MR FAA F DOOR N E - -I PFFTAVCYSLNTFLAL KEKPHCRAFL- CGGEH A S[EA F KPI DAFQ@TLNN GutRGuIR - -SRFNNITVMTNSLH~~~~~IVNALSELDNEOTE1LMPGGTFRKKSA 3OKAE S R F N I IT V M T |S L HFF ALSELDNEQTELMPGGRFRKKSA[ljHMOLANAFHTFID~ AFE m GTLERKVGCVVN[SLIsaLKSLEI JLF FUCR [D I- - - NIaVFTNSHPICHEL GK[RERIa-LI[S

HIF TFIC

AccR LacR

DeOR GMAR

FuCR

L N -AAlS| VDIDRflL| C T SIP S N7 FAE1A A RRE3 E V S S R V VD H SK F TIK S SIL S T A R FE1DFVG V 31FVR.ALtA-VT-.HNSW D K KEGVIaOLALNNAVEKFLLVIDIS 3KFIDRYDFFNFYNLDQLDT

F Y SIA AG V H vJS K JA T F N -LEE L P V K H WA MS MA QK H V LV V DHS K F GEV R PAR MG DL KR F DI F MGT D G[ - - L N A GFVDJ T F N E V YT VIS KAM1C N A A R E[EjL MA D S S K F G R K[S]P NW]V C S L[IFSVVD K FF S C E G[-S SGALWD NA 7D YKSMLLKRAAasL L L K N RSG E A R G H L D EIVT H

ACCR 1 V T DSTRTI ETI PF KL[SK[EGFVVAN LJCR TIDIN Q I S P O H L E E F S VY T T L K A D DeOR V SDGC C P E D E Y V K Y A O T QRI R 1 L MY GMAR LITITDA[1 DIPAFROAL[jEKGIDVIITGESNE FcAR [[JI S]EROVATSLVTA FIG. 4. Amino acid sequence homologies between AccR and other repressor proteins. Amino acids showing exact matches between two or more members of the family are boxed. Gaps were introduced to generate optimal alignments. The N-terminal region with a predicted helix-turn-helix motif (10) is indicated by a bar-ine-bar. The potential inducer binding site (10) is indicated by a hatched bar. Sequences for DeoR, GutR, FucR, and LacR are taken from van Rooijen and de Vos (10).

workers described a second spontaneous TraC mutant that was, however, Acc-. We show here that this phenotype results from a 1.1-kb deletion that eliminates part of the accR coding sequence and extends into ORF4, which encodes the first gene of the acc locus (unpublished results). The negative regulation of agrocinopine catabolism by AccR differs from that seen for catabolism of octopine, the conjugal opine for octopine-type Ti plasmids (5, 7). Two recent reports show that expression of occ is regulated by OccR, an activator related to the LysR family of positive regulatory proteins (27, 28). However, the role played by OccR in octopine-induced conjugal transfer of this Ti plasmid class has yet to be determined. Interestingly, noc, the nopaline catabolic operon of pTiC58, is regulated by an activator closely related to OccR (28). However, nopaline does not induce conjugal transfer of pTiC58. Although the nopaline- and octopine-type Ti plasmids are closely related (29), it is clear that they have evolved separate mechanisms to coregulate their opine-responsive catabolic and conjugal transfer functions. We thank S. Allen and S. Qin for constructing the tra749::lacZ fusion in pTHB58 and Drs. J. F. Gardner, C. G. Miller, and W. L. Ogren for critically reviewing the manuscript. The work was supported in part by Grants CA44051 from the National Cancer Institute and 89-AG-F-1932 from the U.S. Department of Agriculture to S.K.F. 1. Zambryski, P., Tempe, J. & Schell, J. (1989) Cell 56, 193-201. 2. Tempe, J. & Petit, A. (1983) in Molecular Genetics of the BacteriaPlant Interaction, ed. Puhler, A. (Springer, Berlin), pp. 14-32. 3. Bomhoff, G., Klapwijk, P. M., Kester, H. C. M., Schilperoort, R. A., Hernalsteens, J. P. & Schell, J. (1976) Mol. Gen. Genet. 145, 177-181. 4. Ellis, J. G., Kerr, A., Tempe, J. & Petit, A. (1979) Mol. Gen. Genet. 173, 263-269. 5. Klapwijk, P. M. & Schilperoort, R. A. (1979) J. Bacteriol. 139, 424-431. 6. Ellis, J. G., Kerr, A., Petit, A. & Tempe, J. (1982) Mol. Gen. Genet. 186, 269-274. 7. Petit, A., Tempe, J., Kerr, A., Holsters, M., van Montagu, M. & Schell, J. (1978) Nature (London) New Biol. 271, 570-572.

8. Ellis, J. G., Murphy, P. J. & Kerr, A. (1982) Mol. Gen. Genet. 186, 275-281. 9. Kerr, A. & Ellis, J. G. (1982) in Molecular Biology of Plant Tumors, eds. Kahl, G. & Schell, J. (Academic, New York), pp. 321-344. 10. van Rooijen, R. J. & de Vos, W. M. (1990) J. Biol. Chem. 265, 18499-18503. 11. Zimmerer, R. P., Hamilton, R. H. & Pootjes, C. (1966) J. Bacteriol. 92, 746-750. 12. Beck von Bodman, S., McCutchan, J. E. & Farrand, S. K. (1989) J. Bacteriol. 171, 5281-5289. 13. Farrand, S. K., Slota, J. E., Shim, J.-S. & Kerr, A. (1985) Plasmid 13, 106-117. 14. Simon, R., Priefer, U. & Puhler, A. (1983) in Molecular Genetics of the Bacteria-Plant Interaction, ed. POhler, A. (Springer, New York), pp. 98-106. 15. Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. (1988) Gene 70, 191-197. 16. Rahme, L. G., Mindrinos, M. N. & Panopoulos, N. J. (1991) J. Bacteriol. 173, 575-586. 17. Close, T. J. & Rodriguez, R. L. (1982) Gene 20, 305-316. 18. Hayman, G. T. & Farrand, S. K. (1988) J. Bacteriol. 170, 17591767. 19. Stachel, S. E., An, G., Flores, C. & Nester, E. W. (1985) EMBO J. 4, 891-898. 20. Ruvkun, G. B. & Ausubel, F. M. (1980) Nature (London) 289, 85-88. 21. Depicker, A., de Wilde, M., de Vos, G., de Vos, R., van Montagu, M. & Schell, J. (1980) Plasmid 3, 193-211. 22. Das, A., Stachel, S. E., Ebert, P., Montoya, A. & Nester, E. W. (1986) Nucleic Acids. Res. 14, 1355-1364. 23. von Hippel, P. H., Bear, D. G., Morgan, W. D. & McSwiggen, J. A. (1984) Annu. Rev. Biochem. 53, 389-446. 24. Larson, T. J., Ye, S., Weissenborn, D. L., Hoffmann, H. J. & Schweizer, H. (1987) J. Biol. Chem. 262, 15869-15874. 25. Harrison, S. C. & Aggarwal, A. K. (1990) Annu. Rev. Biochem. 59, 933-969. 26. Brennan, R. G. & Matthews, B. W. (1989) J. Biol. Chem. 264, 1903-1906. 27. Habeeb, L., Wang, L. & Winans, S. C. (1991) Mol. Plant-Microb. Interact. 4, 379-385. 28. von Lintig, J., Zanker, H. & Schroder, J. (1991) Mol. Plant-Microb. Interact. 4, 370-378. 29. Engler, G., Depicker, A., Maenhaut, R., Villarroel, R., van Montagu, M. & Schell, J. (1981) J. Mol. Biol. 152, 183-208.

Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a...
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