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

Abstract

Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme. Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system. To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA 244-7830. While the degradative scheme of the two arginyl opines octopine and nopaline is known (26,27), the pathways involved in catabolism of the mannityl opines are not completely understood. A specific MOA degradative pathway produces mannose and probably glutamate (10). However, nothing is known about the pathways by which MOP, AGR, and AGA are catabolized. Although a MOA degrading activity that produces mannose is detectable in crude cell extracts (10), the lactonization of MOP into AGR is the only well-characterized enzymatic activity directly involved in mannityl opine catabolism (9, 15

A T-DNA gene required for agropine biosynthesis by transformed plants is functionally and evolutionarily related to a Ti plasmid gene required for catabolism of agropine by Agrobacterium strains

“…This activity did not require pregrowth with the opine. These two plasmids also expressed mannopine cyclase activity when tested in NT1 harboring pYDPH208, a cosmid clone that contains the genes for a mannopine transport system and two genes specifying putative transcriptional repressors (20) but contains a nonpolar deletion mutation in agcA (18). However, the levels of lactonizing activity detectable from pKS129⌬Sm and pKS129⌬H were substantially lower in this strain compared to the levels observed in a derivative strain of NT1 lacking pYDPH208 (Fig.…”

“…Plasmid pYDH208 is a cosmid clone containing an insert of DNA from pTi15955 that encodes enzymes involved in the catabolism of mannopine and agropine as well as mannopine cyclase (9). Plasmid pYDPH208 is a derivative of pYDH208 that contains a nonpolar deletion mutation in agcA (18). The plasmid confers the ability to grow on mannopine but not on agropine.…”

“…The plasmid confers the ability to grow on mannopine but not on agropine. The other bacteria and plasmids and the culture conditions and media have been described previously (17)(18)(19).…”

“…agcA is expressed from its own regulated promoter. Previous analysis suggested that agcA can be expressed from a promoter directly upstream of the ATG start codon (18). To confirm this, we constructed three plasmids in which agcA is preceded by increasing amounts of upstream sequence (Fig.…”

“…Bacterial cells harboring pYDH208 express catabolic mannopine cyclase activity (9,17). This clone, which contains a 21-kb insert mapping to the 4-o'clock region of the octopine-type Ti plasmid pTi15955 (9), confers all of the transport and enzymatic activities required for catabolism of mannopine and agropine (9,17,18,20,21) (Fig. 3).…”