Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12

Abstract

GDP-L-fucose, the substrate for fucosyltransferases for addition of fucose to polysaccharides or glycoproteins in both procaryotes and eucaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.

FIG. 1

Pathway for the synthesis of GDP–l-fucose from GDP–d-mannose.

FIG. 2

Organization of the GDP–l-fucose biosynthetic pathway genes of the K-12 CA gene cluster. The potential Shine-Dalgarno sequence upstream of gmd is shown as a black dot, and the insert of plasmid pPR1875 is also indicated. The arrows indicate the direction of transcription.

FIG. 3

Alignment of FX protein with amino acid sequences of Fcl proteins from the E. coli K-12 CA gene cluster (Ec_CA [WcaG]), the E. coli O157 O-antigen gene cluster (Ec_O157 [Orf8]), and the O-antigen cluster of Y. enterocolitica O8 (Ye_O8 [WbcJ]). Positions where more than 50% of the sequences have identical amino acids are shaded.

FIG. 4

Identification of ¹⁴C-labelled GDP–l-fucose (peak 3) and GDP–d-mannose (peak 2) by HPLC on a C₁₈ column. A, reaction product obtained by using the cell lysate of P5282. B, reaction product obtained by using the cell lysate of P5470. C and D, ¹⁴C-labelled GDP–d-mannose and GDP–l-fucose treated the same way as in A and B but without addition of the cell lysate. The data presented is from a representative experiment.

FIG. 5

TLC analysis of the ¹⁴C-labelled monosaccharides obtained by acid hydrolysis of the nucleoside diphosphate sugars in the reaction products of P5282 (A) and P5470 (B). ¹⁴C-labelled GDP–d-mannose (C) and GDP–l-fucose (D) were hydrolyzed and used as standards. Authentic unlabelled fucose and mannose were also used as standards (data not shown). TLC was carried out on a Merck silica plate (20 by 20 cm) with a solvent comprising 50 ml of n-butanol, 30 ml of pyridine, and 20 ml of 0.1 M HCl. The plate was then dried, the distribution of radioactivity was quantified by using a PhosphorImager 400B (Molecular Dynamics), and the data was analyzed by using the Molecular Dynamic ImageQuant software package. The plots show relative pixel values for radioactively labelled sugars obtained from the PhosphorImager.