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 influence through modification of other region 2 proteins.

The Group B Streptococcal Sialic Acid O-Acetyltransferase Is Encoded by neuD, a Conserved Component of Bacterial Sialic Acid Biosynthetic Gene Clusters

“…E. coli K1 strain ATCC23511 neuD was amplified using primers 5Ј-cgtctagacgatgagtaaaaagttaataatatttggtgcggg-3Ј and 5Ј-cgggatcccgttcattccccctaattaatcttgttgg-3Ј based on published sequence (26) and cloned into pDCerm to produce pEcK1-NeuD. Sequencing of neuD from strain ATCC23511 revealed 99.5% identity with the published neuD sequence of E. coli K1 RS218 (NCBI accession number AAC43301), differing only at residue 164, where cysteine replaces valine.…”

Expression of sialic acid and polysialic acid in serogroup B Neisseria meningitidis: divergent transcription of biosynthesis and transport operons through a common promoter region

“…Using a separate approach, Tn916 mutagenesis, we defined the first gene of the biosynthetic operon (synX), showed that this gene was either the N-acetyl-D-glucosamine-6-phosphate 2-epimerase or the N-acetyl-D-mannosamine-6-phosphate phosphatase, demonstrated that a mutation in synX produced a capsule-defective (Cap Ϫ ) phenotype, and clarified the nucleotide sequence of synB (45,47). Recently, Ganguli et al (18) reported the cloning and analysis of genes for group B polysialic acid biosynthesis in a third group B meningococcal strain and suggested, on the basis of amino acid homology with E. coli NeuB (1), that the protein encoded by synC (neuB) was the putative NANA synthetase. Edwards et al (10) later showed that a cloned copy of synC was capable of NANA synthesis in vitro.…”

Structural and Genetic Characterization of Enterohemorrhagic Escherichia coli O145 O Antigen and Development of an O145 Serogroup-Specific PCR Assay

“…ORFs 1, 2, 3, and 4 shared, respectively, 59, 70, 51, and 60% identity to NnaD (NeuD), NnaB (NeuB), NnaC (NeuA), and NnaA (NeuC), encoded by the E. coli K1 capsule gene cluster (GenBank entries AAC43301, AAC43302, AAA24210, and AAA24211, respectively) (6). NnaA, NnaB, and NnaC of E. coli K1 synthesize CMP-Neu5Ac when they are cloned into E. coli K-12 on a plasmid (6), and NnaD has a role in the synthesis of CMPNeu5Ac by interacting with NnaB (17,18). NnaA is the GlcNAc 2-epimerase that converts GlcNAc to ManNAc.…”