ISOLATION AND NUCLEOTIDE-SEQUENCE OF THE GDP-MANNOSE-CELLOBIOSYL-DIPHOSPHOPOLYPRENOL ALPHA-MANNOSYLTRANSFERASE GENE FROM ACETOBACTER-XYLINUM

A genetic locus from Acctobacter xylinum involved in acetan polysaccha ride synthesis has been characterized. The chromosomal region was iden tified by screening a genomic library of A. xylinum in a Xanthomonas c ampestri is mutant defective in xanthan polysaccharide synthesis. The A. xylinum cosmid clone can functionally complement a xanthan-negative mutant. The polymer produced by the recombinant strain was found to b e indistinguishable from xanthan. Insertion mutagenesis and subcloning of the cosmid clone combined with complementation studies allowed the identification of a 2,3-kb fragment of A. xylinum chromosomal DNA. Th e nucleotide sequence of this fragment was analyzed and found to conta in an open reading frame (accA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest t hat the accA gene encodes the GDF-mannose:cellobiosyl-diphosphopolyren ol alpha-mannosyltransferase enzyme, which is responsible for the tran sfer of an alpha-mannosyl residue from GDP-Man to cellohiosyl-diphosph opolyprenol. A search for similarities with other known mannosyltransf erases revealed that all bacterial alpha-mannosyltransferases leave a short COOH-terminal amino acid sequence in common.