NEW AZOTOBACTER-VINELANDII MANNURONAN C-5-EPIMERASE GENE (ALGG) IS PART OF AN ALG GENE-CLUSTER PHYSICALLY ORGANIZED IN A MANNER SIMILAR TO THAT IN PSEUDOMONAS-AERUGINOSA

Alginate is an unbranched polysaccharide composed of the two sugar res idues beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). The M /G ratio and sequence distribution in alginates vary and are of both b iological and commercial significance, We have previously shown that a family of highly related mannuronan C-5-epimerase genes (algE1 to -E5 ) controls these parameters in Azotobacter vinelandii, by catalyzing t he Ca2+-dependent conversion of M to G at the polymer level, In this r eport, we describe the cloning and expression of a new A, vinelandii e pimerase gene (here designated algG), localized 29 nucleotides downstr eam of the previously described gene algJ. Sequence alignments show th at algG does not belong to the same class of genes as algE1 to -E5 but that it shares 66% sequence identity with a previously described mann uronan C-5-epimerase gene (also designated algG) from Pseudomonas aeru ginosa, A. vinelandii algG was expressed in Escherichia coli, and the enzyme was found to catalyze epimerization in the absence of Ca2+, alt hough the presence of the cation stimulated the activity moderately. S urprisingly, all activity was blocked by Zn2+, P. aeruginosa AlgG has been reported to contain an N-terminal export signal sequence which is cleaved off during expression in E. coli. This does not happen mith A . vinelandii AlgG, which appears to be produced at least partly in an insoluble form when expressed at high levels in E. coli. DNA sequencin g analyses of the regions flanking algG suggest that the gene is local ized in a cluster of genes putatively involved in alginate biosynthesi s, and the organization of this cluster appears to be the same as prev iously described for P. aeruginosa.