N2-(1-carboxyethyl)methionine. A 'pseudo-opine' in octopine-type crown-gall tumours.
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ABSTRACT:
SUBMITTER: Firmin JL
PROVIDER: S-EPMC1152897 | biostudies-other | 1985 Dec
REPOSITORIES: biostudies-other
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Project description:An amine, after dansylation, has been isolated from Nicotiana tabacum crown gall tumours for the first time and characterized as 4-hydroxy-3-methoxy-beta-phenylethylamine (3-methoxytyramine). The compound cannot be detected in differentiated N. tabacum tissues but appears in the corresponding callus controls. Its concentration is further increased 5-42-fold when N. tabacum is transformed with all strains of Agrobacterium tumefaciens so far tested.
| S-EPMC1144120 | biostudies-other
Project description:Plasmids play a crucial role in the ecology of agrobacteria. In this study, we sequenced tumor-inducing (Ti) and opine-catabolic (OC) plasmids in three Rhizobium rhizogenes (Agrobacterium biovar 2) strains isolated from the same crown gall tumor on "Colt" cherry rootstock and conducted comparative genomic analyses. Tumorigenic strains C5.7 and C6.5 carry nopaline-type Ti plasmids pTiC5.7/pTiC6.5, whereas the nonpathogenic strain Colt5.8 carries the nopaline-type OC plasmid pOC-Colt5.8. Overall, comparative genomic analysis indicated that pTiC5.7/pTiC6.5 and related Ti plasmids described before (pTiC58 and pTi-SAKURA) originate from a common ancestor, although they have diverged during evolution. On the other hand, plasmid pOC-Colt5.8 was most closely related to the well-known OC plasmid pAtK84b; however, analysis suggested that they had different evolutionary histories and seem to share a more distant common ancestor. Although the reconstruction of the evolutionary history of Ti and OC plasmids is still speculative, we hypothesized that nopaline-type Ti plasmid might originate from the nopaline-type OC plasmid. Our results suggested that OC plasmids are widespread and closely associated with crown gall tumors. Finally, we proposed a thorough scheme for classification of Ti and OC plasmids that is based on separate comparative analysis of each functional element of the plasmid studied.
| S-EPMC6546132 | biostudies-literature
Project description:Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.
| S-EPMC178081 | biostudies-other
Project description:Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.
| S-EPMC3169888 | biostudies-literature
Project description:Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen.
| S-EPMC3359404 | biostudies-literature
Project description:BACKGROUND: Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens. RESULTS: Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines. CONCLUSIONS: CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.
| S-EPMC3852553 | biostudies-literature
Project description:Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.
| S-EPMC3446853 | biostudies-literature
Project description:The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases. Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the presence of NAD+. The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP. The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase. These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP. The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.
| S-EPMC178082 | biostudies-other
Project description:Using Illumina and Nanopore reads, we assembled a high-quality draft genome sequence of Allorhizobium vitis K309T (= ATCC 49767T, = NCPPB 3554T), a phytopathogenic strain isolated from a grapevine in Australia. The hybrid approach generated 50% fewer contigs and a 3-fold increase in the N 50 value compared with the previous Illumina-only assembly.
| S-EPMC6256525 | biostudies-literature
Project description:BACKGROUND:Crown gall disease, caused by the pathogenic bacterium Agrobacterium tumefaciens, is responsible for extensive economic losses in orchards. Cherry rootstock 'CDR-1' (Prunus mahaleb) shows high resistance but the mechanism remains unclear. Here, we examined the morphology of pathogen-infected root neck surface, determined the activity of 10 defense-related enzymes and the content of salicylic acid (SA) and jasmonic acid (JA), and also applied transcriptome analysis, transient expression and transgenic verification to explore the crown gall resistance genes in 'CDR-1' plants. RESULTS:In our study, peroxidase increased in the first 10?days, while phenylalanine ammonialyase and lipoxygenase increased in the first 15?days post-infection. Four key enzymes in the AsA-GSH cycle also responded, to a certain extent; although JA content increased significantly after the treatment, the SA content did not. In a follow-up transcriptome analysis, the differentially expressed genes Pm4CL2, PmCYP450, PmHCT1, PmHCT2, and PmCAD were up-regulated. Based on the above results, we focused on the lignin biosynthetic pathway, and further measured lignin content, and found it increased significantly. The Pm4CL2 gene was used to conduct transient expression and transgenic experiments to verify its function in crown gall disease resistance. It showed the relative expression of the treatment group was almost 14-fold that of the control group at 12?h post-treatment. After the infection treatment, clear signs of resistance were found in the transgenic lines; this indicated that under the higher expression level and earlier activation of Pm4CL2, plant resistance was enhanced. CONCLUSIONS:The crown gall resistance of 'CDR-1' is likely related to the lignin biosynthetic pathway, in which Pm4CL2 functions crucially during the plant defense response to the pathogen A. tumefaciens. The results thus offer novel insights into the defense responses and resistance mechanism of cherry rootstock 'CDR-1' against crown gall disease.
| S-EPMC7661173 | biostudies-literature