Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens

Abstract

Two nopaline-type strains ofAgrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine.The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs.[14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized[14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14Cjmannopine, was in the form of agropine.Crown gall and hairy root are plant diseases caused by Agrobacterium tumefaciens and Agrobacterium rhizogenes, respectively. Pathogenic bacteria harbor either a Ti (tumorinducing) or a Ri (root-inducing) plasmid. The bacteria mediate the transfer to the plant cell of a portion of the plasmid, called transferred DNA (T-DNA). The T-DNA becomes incorporated into the plant nuclear DNA, where its expression results in abnormal patterns of phytohormone metabolism and proliferation of the transformed plant cells.

Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp

“…The AGR resulting from MOP cyclase activity accumulates inside the cells and then is slowly degraded. The reverse activity, conversion of AGR to MOP by MOP cyclase, was detected in vitro, although the levels of MOP produced by this activity were very low (9,19). Consistent with this, ['4C]MOP was detected in cells of NT1 (pYDH208) Analysis of intracellular products indicates that catabolism of MOP or AGR produces a new compound that migrates faster than MOP or AGR at pH 1.8.…”

“…Using ['4C]mannose-glutamine as the substrate, LaPointe et al (19) detected radiolabeled mannose and fructose in cell extracts of wild-type strain Ach5. When mannose-['4C]glutamine was used, glutamine and glutamate were detected.…”

“…The assay with radiolabeled opines was performed as described by LaPointe et al (19). Bacteria were grown in ATN minimal medium with MOP as the sole carbon source.…”

Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event?

“…The appearance of nonpathogenic mutant bacteria was also observed when Agrobacterium strains were cultured in media supplemented with substances that are naturally present in plant wounds and that favor the activation of pathogenicity genes (13). The molecular bases of the spontaneous loss of pathogenicity in A. tumefaciens are not well understood, although previous work reported changes in the Ti plasmid, in either the regions involved in virulence, the transferred DNA (T-DNA) region, or the vir regions, due to point mutations or to the insertion of mobile elements (10,13,14,21). However, it was not entirely clear that the nonpathogenic isolates were necessarily mutants derived from the inoculated strain rather than naturally occurring nonpathogenic A. tumefaciens strains.…”

A metabolic pathway leading to mannosylfructose biosynthesis in Agrobacterium tumefaciens uncovers a family of mannosyltransferases

“…A. tumefaciens is a unique plant pathogen that can genetically manipulate its host, which does not compete with the bacterium for the mannose pool. Although mannose appears to be important in the bacterium cells in response to an environmental condition (6) and as a carbohydrate source necessary for tumor establishment (18,19), it has a toxic effect on most plants (20,21). Thus, it can be speculated that this aspect of the plant-microbe interaction might have contributed to the acquisition of MF biosynthesis in an apparently narrow ecological niche.…”