Isolation and nucleotide sequence of the GDP-mannose:cellobiosyl-diphosphopolyprenol alpha-mannosyltransferase gene from Acetobacter xylinum

Abstract

A genetic locus from Acetobacter xylinum involved in acetan polysaccharide synthesis has been characterized. The chromosomal region was identified by screening a genomic library of A. xylinum in a Xanthomonas campestris 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 be 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. The nucleotide sequence of this fragment was analyzed and found to contain an open reading frame (aceA) of 1,182 bp encoding a protein of 43.2 kDa. Results from biochemical and genetic analyses strongly suggest that the aceA gene encodes the GDP-mannose:cellobiosyl-diphosphopolyprenol ␣-mannosyltransferase enzyme, which is responsible for the transfer of an ␣-mannosyl residue from GDP-Man to cellobiosyl-diphosphopolyprenol. A search for similarities with other known mannosyltransferases revealed that all bacterial ␣-mannosyltransferases have a short COOH-terminal amino acid sequence in common.

Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase

“…The same may be true for the glycosyltransferases from families 4, 5, and 15 of Campbell's classification, although in these cases, experimental confirmation would be required. This type of evidence has been obtained for Ace-A (35,46), an ␣-mannosyltransferase that belongs to family 4. Geremia et al (42) found an E-X 7 -E motif similar to that described here in a group of prokaryotic ␣-mannosyltransferases and proposed that both conserved Glu residues were important for catalysis.…”

Identification of Essential Amino Acids in the Bacterial α-Mannosyltransferase AceA

“…Since E295A produced very little amounts of polysaccharide, we have used the strain XcH E295G that produces slightly more EPS than XcH E295A. 2 The EPSs produced by strains D109A, H127A, S162A, L270A, and E295G were viscous, indicating the presence of a high molecular weight polysaccharide. The 1 H NMR spectra of D109A, H127A, S162A, and L270A display the characteristic signals of xanthan gum, in particular at ␦ 1.8, 2.1, and 5.2, which correspond to the acetyl, ketal pyruvate and ␣(1-3)-Man moieties (Fig.…”

“…A large number of genes thought to encode glycosyltransferases involved in the assembly of repeat units have been identified, reflecting the extreme structural diversity of these bacterial polysaccharides. AceA is a non-processive ␣-1,3 mannosyltransferase from Acetobacter xylinum, which transfers mannose from GDP-mannose (GDP-Man) 1 to polyprenyl-pyrophosphate-linked cellobiose (Glc 2 -PP-Lip) during the assembly of the heptasaccharide repeat unit of the exopolysaccharide acetan (2,3). Acetan is structurally related to the widely used exopolysaccharide xanthan gum from Xanthomonas campestris (see Fig.…”

“…AceA is homologous to the X. campestris ␣-1,3 mannosyltransferase GumH, which catalyzes the analogous reaction in the biosynthesis of the repeat unit of xanthan gum. Indeed, the aceA gene was cloned by functional complementation of a X. campestris gumH mutant strain (2). AceA and GumH are both retaining glycosyltransferases as they catalyze the formation of a glycosidic linkage with retention of stereochemistry about the donor sugar anomeric carbon.…”

Effects of Acetan on Production of Bacterial Cellulose by Acetobacter xylinum

“…Genetic analysis of BC showed that the bcs operon is associated with cellulose synthesis. 8) In contrast, the genetics of the acetan biosynthetic process has not been clariˆed 9,10) although the biochemical pathway has been elucidated. [11][12][13][14] Because the production patterns during culture of the two extracellular polysaccharides are similar, we have speculated that there is a relationship between BC and acetan in their productivity; however, to our knowledge, there has been no report on a relationship between acetan and BC biosynthesis.…”