RHIZOBIUM SP STRAIN NGR234 NODZ PROTEIN IS A FUCOSYL-TRANSFERASE

Rhizobium sp. strain NGR234 produces a large family of lipochitooligos accharide Nod factors carrying specific substituents. Among them are 3 -O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-m ethylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl su bstitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fuco se as the fucose donor show that fucosyltransferase activity is associ ated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells, Together with extra copies of the n odD1 gene, the nodZ gene and its associated nod box were introduced in to ANU265, which is NGR234 cured of the symbiotic plasmid, Crude extra cts of this transconjugant possess fucosyltransferase activity. Fusion of a His, tag to the NodZ protein expressed in Escherichia coli yield ed a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosa mine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyse s showed that the NodZ protein is more active on oligomers of chitin t han on nonfucosylated NodNGR factors. Pentameric chitin is the preferr ed substrate. These data suggest that fucosylation occurs before acyla tion of the Nod factors.