agrocin-84 and agrocinopine

Periplasmic binding proteins (PBPs) in association with ABC transporters select and import a wide variety of ligands into bacterial cytoplasm. They can also take up toxic molecules, as observed in the case of the phytopathogen Agrobacterium tumefaciens strain C58. This organism contains a PBP called AccA that mediates the import of the antibiotic agrocin 84, as well as the opine agrocinopine A that acts as both a nutrient and a signalling molecule for the dissemination of virulence genes through quorum-sensing. Here, we characterized the binding mode of AccA using purified agrocin 84 and synthetic agrocinopine A by X-ray crystallography at very high resolution and performed affinity measurements. Structural and affinity analyses revealed that AccA recognizes an uncommon and specific motif, a pyranose-2-phosphate moiety which is present in both imported molecules via the L-arabinopyranose moiety in agrocinopine A and the D-glucopyranose moiety in agrocin 84. We hypothesized that AccA is a gateway allowing the import of any compound possessing a pyranose-2-phosphate motif at one end. This was structurally and functionally confirmed by experiments using four synthetic compounds: agrocinopine 3'-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate. By combining affinity measurements and in vivo assays, we demonstrated that both L-arabinose-2-phosphate and D-glucose-2-phosphate, which are the AccF mediated degradation products of agrocinopine A and agrocin 84 respectively, interact with the master transcriptional regulator AccR and activate the quorum-sensing signal synthesis and Ti plasmid transfer in A. tumefaciens C58. Our findings shed light on the role of agrocinopine and antibiotic agrocin 84 on quorum-sensing regulation in A. tumefaciens and reveal how the PBP AccA acts as vehicle for the importation of both molecules by means of a key-recognition motif. It also opens future possibilities for the rational design of antibiotic and anti-virulence compounds against A. tumefaciens or other pathogens possessing similar PBPs.

Topics: Adenine Nucleotides; Agrobacterium tumefaciens; Anti-Bacterial Agents; ATP-Binding Cassette Transporters; Bacterial Proteins; Crystallography, X-Ray; Molecular Sequence Data; Protein Conformation; Quorum Sensing; Sugar Phosphates

The acc locus from the Ti plasmid pTiC58 confers utilization of and chemotaxis toward agrocinopines A and B (A+B), as well as susceptibility to a highly specific antiagrobacterial antibiotic, agrocin 84. DNA sequence analyses revealed that acc is composed of eight open reading frames, accR and accA through accG. Previous work showed that accR encodes the repressor which regulates this locus, and accA codes for the periplasmic binding protein of the agrocinopine transport system (S. Beck Von Bodman, G. T. Hayman, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 89:643-647, 1992; G. T. Hayman, S. Beck Von Bodman, H. Kim, P. Jiang, and S. K. Farrand, J. Bacteriol. 175:5575-5584, 1993). The predicted proteins from accA through accE, as a group, have homology to proteins that belong to the ABC-type transport system superfamily. The predicted product of accF is related to UgpQ of Escherichia coli, which is a glycerophosphoryl diester phosphodiesterase, and also to agrocinopine synthase coded for by acs located on the T-DNA. The translated product of accG is related to myoinositol 1 (or 4) monophosphatases from various eucaryotes. Analyses of insertion mutations showed that accA through accE are required for transport of both agrocin 84 and agrocinopines A+B, while accF and accG are required for utilization of the opines as the sole source of carbon. Mutations in accF or accG did not abolish transport of agrocin 84, although we observed slower removal of the antibiotic from the medium by the accF mutant compared to the wild type. However, the insertion mutation in accF abolished detectable uptake of agrocinopines A+B. A mutation in accG had no effect on transport of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This finding suggests that agrocin 84 is activated by AccF after being transported into the bacterial cell.

Topics: Adenine Nucleotides; Agrobacterium tumefaciens; Amino Acid Sequence; Anti-Bacterial Agents; Arginine; ATP-Binding Cassette Transporters; Bacterial Proteins; Base Sequence; Biological Transport; DNA, Bacterial; Drug Resistance, Microbial; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genes, Reporter; Genetic Complementation Test; Molecular Sequence Data; Operon; Plasmids; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sugar Phosphates

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

Topics: Adenine Nucleotides; Agrobacterium tumefaciens; Amino Acid Sequence; ATP-Binding Cassette Transporters; Bacterial Proteins; Base Sequence; Biological Transport; Cloning, Molecular; DNA, Bacterial; Genetic Complementation Test; Kinetics; Molecular Sequence Data; Mutagenesis, Insertional; Plasmids; Restriction Mapping; Sequence Homology, Amino Acid; Sugar Phosphates

Agrobacterium tumefaciens strains C58, T37, K827 and J73, A. rhizogenes strains A4 and 15834, and A. radiobacter strain K299 were all susceptible to agrocin 84 and this sensitivity was enhanced in each case by addition of agrocinopines A and B. Analysis of transconjugants showed that sensitivity of strain A4 to agrocin 84 was encoded by pArA4a and not by the rhizogenic plasmid, pRiA4. The acc region of the A. tumefaciens nopaline-type Ti plasmid pTiC58, contained on the recombinant plasmid pTHH206, hybridized strongly to restriction fragments of plasmids from strains T37, K827, J73 and K299. Hybridizing fragment patterns generated with BamHI and EcoRI were identical among the four Ti plasmids while pAtK299 showed restriction fragment length polymorphisms at acc with the two enzymes. At moderate stringency, the pTiC58 acc region hybridized weakly to a single restriction fragment from the Ar plasmid of A. rhizogenes strain A4, but not to pTiBo542, which encodes catabolism of the closely related opines agrocinopines C and D. Plasmid pAtK84b of A. radiobacter strain K84 is induced for conjugal transfer by agrocinopines A and B. However, no hybridization was detected between this plasmid and acc from pTiC58 under conditions of moderate stringency. Like pTiC58, pAtK84b conferred transport of agrocinopines A and B on its host bacteria despite the absence of detectable sequence homology with the pTiC58-derived acc probe. However, unlike pTiC58, pAtK84b failed to confer sensitivity to or uptake of agrocin 84 on its bacterial host. These results indicate that at least four distinguishable systems exist for catabolism of the two agrocinopine opine families with the prototype locus, exemplified by acc from pTiC58, being strongly conserved among nopaline-type Ti plasmids.

Topics: Adenine Nucleotides; Anti-Bacterial Agents; Blotting, Southern; Conjugation, Genetic; Plasmids; Rhizobium; Sugar Phosphates

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.

Topics: Adenine Nucleotides; DNA Mutational Analysis; DNA Transposable Elements; DNA, Bacterial; Genes, Bacterial; Genetic Complementation Test; Plasmids; Restriction Mapping; Rhizobium; Sugar Phosphates; Transduction, Genetic

Overlapping segments of pTiC58 inserted into cosmid vectors were used to characterize the agrocinopine-agrocin 84 locus from the nopaline/agrocinopine A and B Agrobacterium tumefaciens strain C58. All of the clones conferring agrocin 84 sensitivity on agrobacteria also conferred uptake of agrocin 84 and agrocinopines A and B. Transposon Tn3-HoHo1 insertion mutations of one such clone were generated that simultaneously abolished agrocin 84 sensitivity and transport of agrocinopines A and B and agrocin 84. Such insertions were found to cluster within a 4.4-kilobase region. Analysis of beta-galactosidase activity in these insertion mutants suggested a single transcriptional unit regulated at the transcriptional level by agrocinopines A and B. The smallest DNA fragment subcloned from the region to confer all three activities was 8.5 kilobases long. This subclone was still properly regulated, indicating that the regulatory gene is closely linked to the locus. The data are consistent with a single operon encoding catabolism of agrocinopines A and B and conferring sensitivity to agrocin 84. Based on these results, we support the locus name acc, for agrocinopine catabolism.

Topics: Adenine Nucleotides; Arginine; Bacteriocins; Cloning, Molecular; Cosmids; DNA Restriction Enzymes; Escherichia coli; Gene Expression Regulation; Genes, Bacterial; Mutation; Plasmids; Rhizobium; Sugar Phosphates; Transcription, Genetic

Mutations affecting agrocin production on the 48-kilobase (kb) plasmid, pAgK84, can be complemented in trans with cloned portions of the plasmid. Five complementation groups ranging in minimum size from 1.2 to 5.6 kb were identified within a 14-kb segment. Plasmid pAgK84-encoded immunity to agrocin 84 was located to two separate regions of the plasmid. Either region alone was sufficient to protect sensitive strains, and both loci mapped to the agrocin 84 biosynthesis region. One region is located within complementation group I, while the other forms a part of complementation group IV. Production of agrocin 84 was unaffected by nopaline, agrocinopine A, acetosyringone, or low or high levels of ferric iron. Agrocin 84 production was greatly suppressed when the strain also contained a Ti plasmid nutritionally or mutationally derepressed for agrocinopine A catabolism. RNA dot-blot analysis indicated that decreased agrocin 84 production by such strains was not due to transcriptional repression of agrocin 84 biosynthetic loci. In strains also harboring pAtK84b, the opine catabolic plasmid of Agrobacterium radiobacter K84, induction of the agrocinopine A catabolic locus of this plasmid had no such effect on agrocin 84 production.

Topics: Adenine Nucleotides; Anti-Bacterial Agents; Bacteriocins; DNA Transposable Elements; DNA, Bacterial; Genes, Bacterial; Genetic Complementation Test; Mutation; Nucleic Acid Hybridization; Plasmids; Rhizobium; RNA, Bacterial; Sugar Phosphates; Transcription, Genetic

Opines are unusual compounds found specifically in plant crown gall tumors. Genes for their synthesis and catabolism reside in agrobacteria as tumor-inducing (Ti) plasmid DNA. Only a small Ti-plasmid segment (24 kilobase pairs), the T-DNA, is transferred to the plant cell where it commonly codes for enzymes involved in the biosynthesis of nitrogenous opines such as nopaline (N2-(1,3-D-dicarboxypropyl)-L-arginine) as well as the tumor phenotype. Ellis and Murphy, (Ellis, J.G., and Murphy, P.J. (1981) Mol. Gen. Genet. 181, 36-43) reported the existence of the phosphorylated opines, agrocinopines A and B in tumors containing nopaline. Pure agrocinopine A has now been isolated in a yield of 0.05-0.06 g/100 g, fresh weight, from such tumors. Physical, chemical, and biological data establish the structure of agrocinopine A as an unusual non-nitrogenous opine of sucrose and L-arabinose with a phosphodiester linkage from the 2-hydroxyl of the arabinose to the 4-hydroxyl of the fructose moiety in sucrose. Agrocinopine B is the corresponding phosphodiester, in which the glucose has been hydrolyzed from the sucrose portion of agrocinopine A. Borohydride reduction of the free L-arabinose anomeric carbon of agrocinopine A, to the corresponding arabinitol derivative eliminates the characteristic inhibition zone enhancement produced by both agrocinopines A and B in the agrocin 84 (a fraudulent adenine nucleotide) bioassay. Because of the limited number of genes in the T-DNA, a generalization is proposed, whereby all opines will be found to comprise two common plant cell constituents linked in an uncommon manner by the minimum number of enzymes.

Topics: Adenine Nucleotides; Arabinose; Biological Assay; Electrophoresis, Polyacrylamide Gel; Fructose; Magnetic Resonance Spectroscopy; Plants; Plasmids; Sugar Phosphates