Analysis of trans-acting elements for regulation of moc operons of pTi15955 in Agrobacterium tumefaciens

Two putative regulator genes, mocR and mocS, of the moc (mannityl opine catabolism) operons in pTi15955 of the octopine-/mannityl opine-type Agrobacterium tumefaciens strain 15955, were tested for their possible roles as repressors in the moc operons. The regions upstream of mocC and mocD, the first structural genes in the two divergently oriented moc operons, were transcriptionally fused into the promoterless lacZ reporter gene. Each of the IacZ-fusions was introduced into Agrobacterium strain UIA5, a Ti plasmid-cured derivative, harboring either a mocR or a mocS clone. The resulting strains were grown in media containing various sugar sources, and the beta-galactosidase activities were quantitatively measured. The results suggested that MocR repressed the expression of mocC and mocD. The expression of the fused beta-galactosidase was not induced by mannopine (MOP) or possible catabolic intermediates of the opine, e.g. santhopine (SOP), glucose, mannose, or glutamine. However, the repression was significantly relieved by the supplementation of MOP and the concomitant introduction of the agcA gene encoding MOP cyclase that catalyzes the lactonization of MOP to agropine (AGR). These results suggested that AGR, rather than MOP or the other catabolic intermediates, is the inducer for the expression of the operon. On the contrary to previous report showing that the induction levels of mocC and mocD were lowered by the supplementation of inorganic nitrogen in media, the expression of these genes was not affected by the level of nitrogen in our reporter system. MocS did not strongly repress the expressions of mocC and mocD. It is possible that MocS may be involved in the regulation of the operons present downstream of the moc operon, which are responsible for the utilization of mannopinic acid and agropinic acid.