FUNCTIONAL-ROLE OF THE TI PLASMID-ENCODED CATABOLIC MANNOPINE CYCLASEIN MANNITYL OPINE CATABOLISM BY AGROBACTERIUM SPP

Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactoni zes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pT i15955 encoding the catabolic MOP cyclase enzyme previously was locali zed to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone con taining only this region complemented the AGR catabolism-negative phen otype conferred by a derivative of the octopine type plasmid pTiB6S3 c ontaining a Tn7 insertion in the region encoding the MOP cyclase enzym e. Uptake assays of strains harboring pRiA4 or pArA4a, along with comp lementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transp ort system. To determine the requirement for MOP cyclase in opine cata bolism unequivocally, a site-specific, nonpolar deletion mutation abol ishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA) . Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP. Growth on AGR was re stored in this strain upon introduction of clones encoding the pTi1595 5-derived catabolic or anabolic MOP cyclase genes. The induction patte rn of MOA catabolism shown by strain NT1 harboring the MOP cyclase-def icient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA. Genetic and bio chemical analyses of MOP and AGR metabolism suggest that only the conv ersion of AGR to MOP is directly involved in catabolism of AGR, even t hough the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR.