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 tume- negative regulatory proteins that control sugar catabolic faciens are conjugal elements whose transfer is strongly re- operons in several unrelated bacteria (10). Our findings pressed. Transfer is induced by the conjugal opines, a group of provide a molecular framework for the coregulation model unique carbon compounds synthesized in crown gail tumors. and illustrate the diversity of regulatory mechanisms that The opines also induce Ti plasmid-encoded genes required by govern the interaction between Agrobacterium and its plant the bacteria for' opine catabolism. We have cloned and se- hosts. 11 quenced 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. MATERIALS AND METHODS The gene, accR, is closely linked to the agrocinopine catabolic Strains and Plasmids. A. tumefaciens strains used were locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which C58 (11), NT1(pTiC58Trac) (12), C58ClCE(pWI1003) (8), constitutively expresses conjugal transfer and opine catabo- NT1(pAgK84-A1) (13), and C58C1RS (rifampin resistant, lism, was complemented in trans by a clone of wild-type accR. streptomycin resistant) (8); Escherichia coli strains were Comparative sequence analysis identified a 5-base-pair dele- DH5a, RR1, and S17-1 (14). Broad host range vectors tion close to the 5' end of the mutant accR allele from pRK415 (15) and pLAFR6 (16) were used for subcloning and pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and complementation analysis; pCM1 was the source of the cat opine catabolic structural genes demonstrated that the accR- cartridge (17); and pUC18 served as cloning vector for DNA encoded function is a transcriptional repressor. accR can sequencing. encode a 28-kDa protein. This protein is related to a class of Reagents. Antibiotics, 5-bromo-4-chloro-3-indolyl 8-D- repressor proteins that includes LacR, GutR, DeoR, FucR, and galactoside, o-nitrophenyl 13-galactoside, and nopaline were GlpR that regulate sugar catabolic systems in several bacterial purchased from Sigma. genera. Mutant Construction. Plasmids pTHB58 (18) and pSVB20 (12) were mutagenized with Tn3HoHo1 (19), and the muta- During interactions between Agrobacterium tumefaciens and tions were homogenotized into Ti plasmids by standard its plant hosts, the bacterium senses and responds to several methods (20). plant-produced signals. For example, virulent agrobacteria Agrocin 84 Sensitivity Assays. Because amounts of purified use a two-component signal transduction system to sense agrocinopine A and B sufficient for catabolic studies are not small phenolic compounds released from wounded plant available, sensitivity to agrocin $4, assayed on Stonier me- tissue. In response, the bacteria express Ti plasmid-encoded dium plates as described by Hayman and Farrand (18), was Vir functions, which, in turn, facilitate T-strand excision and used to monitor expression of acc. Uptake of agrocin 84 transfer to susceptible plant cells (1). The resulting plant depends upon the acc-encoded opine transport system and neoplasias synthesize unique low molecular weight carbon therefore serves as a measure of acc expression (18). Strains compounds, called opines, which are thought to provide a to be tested for sensitivity were grown in LB medium and source of carbon for tumor-colonizing agrobacteria (2). washed in 20 mM phosphate buffer (pH 7.0) before addition The opines also act as signals. For example, the Ti plasmid- to the soft agar overlays. Induction by agrocinopine was encoded functions required for opine catabolism are specif- assessed by placing paper strips impregnated with -20 nmol ically induced by their cognate substrates (3, 4). In addition, of agrocinopine onto the overlay agar (18). conjugal transfer of the Ti plasmid, which is normally re- Conjugal Transfer Assays. The quantitative spot mating pressed, is induced by a subclass of opines, the conjugal technique used in these studies has been described (12). The opines (5, 6). A model proposing that conjugation and catab- Ti plasmidless recipient strain C58C1RS was spread over the olism of the conjugal opines are coregulated emerged from surface of an AT minimal medium (6) selection plate con- the observation that mutants constitutive for conjugation are taining streptomycin, rifampin, and a mixture of nopaline (1 often derepressed for conjugal opine catabolism. Similarly, mM) and arginine (9 mM). Ten-microliter volumes of serial mutants selected for constitutive expression of these opine dilutions of donors were spotted onto the recipient plate. For catabolic functions are generally transfer-constitutive (5, opine induction, donors were grown overnight on small agar 7-9). blocks containing -100 nmol of agrocinopine A and B. The For nopaline-type Ti plasmids such as pTiC58, conjugation cells were resuspended in 0.5 ml of AT minimal medium and is induced by the sugar phosphate opines, agrocinopines A diluted, and 10-.lI sam,3les were spotted onto recipient plates. and B (6). We report here that the coregulation of agrocino- Plates were incubated at 280C for 2-3 days and colonies pine 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 ofAgriculture, Peoria, IL 61604. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" "The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession np. M81646). 643 Downloaded by guest on September 25, 2021 644 Biochemistry: Beck von Bodman et al. Proc. Natl. Acad. Sci. USA 89 (1992) within the spots were counted. Conjugation frequencies are DNA Sequence Analysis of the EcoRI 26 Region. Fig. 3A expressed as number of transconjugants per input donor. shows a 1190-base-pair (bp) DNA sequence of the wild-type ,f-Galactosidase Assays. Strains were grown in AT minimal EcoRI fragment 26 starting from the left EcoRI site. Com- medium to an OD6w of 0.2. The cultures were divided into puter analysis identified four open reading frames (ORFs). two tubes, a solution of agrocinopines (final concentration, ORFI, which could encode a 28-kDa protein, begins with a 200 ttM) was added to one set, and incubation was continued TTG (bp 322) or a GTG (bp 325) and terminates with a TAG for 3 hr. Levels of 8-galactosidase were measured as de- at position 1096. The two potential initiation codons are scribed by Stachel et al. (19). preceded by sequences that match the E. coli ribosomal DNA Sequencing. EcoRI fragments 26 from the wild-type binding (RB) consensus sequence. Upstream from the po- and mutant Ti plasmids were cloned into pUC18 and both tential initiation codons are sequences similar to the virE -10 strands were sequenced using the Sequenase kit, version 2.0 region (22) and with nearly perfect identity to the E. coli -35 (United States Biochemical). The DNA sequences were consensus sequence (23). In addition, the potential -10 analyzed using the DNA* computer program (DNASTAR, region is part of a 17-bp palindrome (CGCTCAIAGTAT- Madison, WI). GiAGC~G). A similar repeat (GCCTCATTCAACAiA C) is Chloramphenicol Acetyltransferase (CAT) Assay. Cells present in the 3' noncoding region that separates ORFi from were grown in LB broth to late exponential phase, and ORF4. ORF2 begins with an ATG at bp 595 and is in frame cell-free extracts were obtained by sonication. CAT protein with ORFi. It is preceded by a potential RB site and - 10 and was detected using the CAT ELISA kit marketed by 5 Prime -35 sequences that resemble those of the virC promoter 3 Prime, Inc., following the recommended procedure. 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 RESULTS identifiable 5' regulatory features associated with this ORF. Mutations and Genetic Complementations Identify a ORF4 reads in the same direction as ORFi and ORF2, pTiC58-Encoded Function that Regulates tra and acc. Two beginning with an ATG at bp 1156 (Fig. 3B) and extends cosmid clones, pTHB112 and pTHB55, repressed conjugal beyond the right EcoRI site (data not shown). transfer and acc-encoded supersensitivity to agrocin 84 when The sequence from pTiC58Trac is identical to that of the in trans to the spontaneous Trac/Accc mutant Ti plasmid wild-type Ti plasmid except for a 5-bp deletion (bp 336-340) pTiC58Trac. These two cosmid clones contain DNA inserts in the ORFi (ORF3) coding region (Fig. 3). This deletion from pTiC58 that overlap the agrocinopine catabolic locus creates a frame shift in ORFi that results in a new stop codon (acc) and Tra region I (Fig. 1). pSVB20, a similar cosmid 22 bp downstream from the site ofthe mutation (Fig. 3A). The clone from pTiC58Trac, showed no effect on either pheno- deletion is outside of and 5' to ORF2. type (data not shown). We then subcloned regions ofoverlap ORF1 Encodes a Protein Homologous to Regulators of Other from the wild-type and mutant cosmid clones into pRK415. Sugar Catabolic Operon Systems. In a search of the National pRKW26, containing EcoRI fragment 26 from wild-type Biomedical Research Foundation Protein Information Re- pTiC58, repressed conjugal transfer to levels below 10-8 per sources data base, the regulatory proteins GutR (10), FucR input donor.