Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1

Abstract

The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.