Molecular, genetic, and biochemical studies of mannityl opine utilization by Agrobacterium tumefaciens: Its evolutionary implications

Agrobacterium strain NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grows well with agropine (AGR). However, this strain grows slowly with mannopine (MOP) as sole carbon source, but gives rise to spontaneous mutants which grow as fast on this opine as does wild type cells. DNA sequence analysis of the region necessary for utilization of these opines revealed seven putative genes; moc (manniyl opine catabolism) D and E, oriented from right to left, and $mocRCBAR\sp\prime$, from left to right. MocD is a homologue of the anabolic conjugase encoded by mas2$\sp\prime$ required for MOP biosynthesis encoded on the T$\sb{\rm R}$ region of pTi15955. MocE and MocC have homologies with the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis encoded by mas1$\sp\prime$. MocR and MocR$\sp\prime$ are related to each other, and to the putative repressor for the AGR degradation system encoded by the agropine-type rhizogenic plasmid pRiA4. MocB and MocA are homologues of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively.mocC, mocD, and mocE were cloned and expressed in E. coli. A cell lysate prepared from E. coli strain DH5$\alpha$ expressing mocC contained an enzymatic activity that, in the presence of NAD$\sp+$, converted MOP to Santhopine (SOP). Biochemical and genetic studies suggest that both MocD and MocE are required for the degradation of SOP. These results indicate that the biochemical pathway for degradation of MOP is the reverse of that for biosynthesis of the opine by plant tumors, suggesting that the moc genes and the mas genes evolved from common ancestors. Molecular genetic studies indicated that the bacterial chromosome as well as the 450-kb megaplasmid pAtC58 of Agrobacterium encode genes that provide an alternate pathway for utilization of SOP.We also found that MOP is toxic to cells when it accumulates intracellularly, suggesting that the opine may assist MOP-utilizing agrobacteria to colonize tumors by inhibiting other related soil bacteria that can take up but cannot degrade this opine.U of I OnlyETDs are only available to UIUC Users without author permissio