Wendy J. Keenleyside

A Novel Pathway for O-Polysaccharide Biosynthesis in Salmonella enterica Serovar Borreze

The plasmid-encoded gene cluster for O:54 O-polysaccharide synthesis in Salmonella enterica serovar Borreze (rfbO:54) contains three genes that direct synthesis of a ManNAc homopolymer with alternating β1,3 and β1,4 linkages. In Escherichia coli K-12, RfbAO:54 adds the first ManNAc residue to the Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1-phosphate transferase)- modified lipopolysaccharide core. Hydrophobic cluster analysis of RfbAO:54 indicates this protein belongs to the ExoU family of nonprocessive β-glycosyltransferases. Two putative catalytic residues and a potential substrate-binding motif were identified in RfbAO:54. Topological analysis of RfbBO:54 predicts four transmembrane domains and a large central cytoplasmic domain. The latter shares homology with a similar domain in the processive β-glycosyltransferases Cps3S of Streptococcus pneumoniae and HasA of Streptococcus pyogenes. Hydrophobic cluster analysis of RfbBO:54 and Cps3S indicates both possess the structural features characteristic of the HasA family of processive β-glycosyltransferases. Four potential catalytic residues and a putative substrate-binding motif were identified in RfbBO:54. In Δrfb E. coli K-12, RfbAO:54 and RfbBO:54 direct synthesis of smooth O:54 lipopolysaccharide, indicating that this O-polysaccharide involves a novel pathway for O-antigen transport. Based on sequence and structural conservation, 15 new ExoU-related and 17 new HasA-related transferases were identified.

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography

A high-performance liquid chromatography (HPLC) method with pulsed-amperometric detection (PAD) was developed for the compositional analysis of the acidic, neutral, and basic monosaccharides recovered from the acid hydrolysis of bacterial cell wall polysaccharides. This HPLC-PAD method involved the chromatography of the acid hydrolysis products on a CarboPac PA-1 anion-exchange column of pellicular resin, with PAD detection following postcolumn addition of alkali. Complete resolution of a mixture of 19 monosaccharides, comprising 9 neutral, 3 basic, and 7 acidic sugars, frequently found in bacterial polysaccharides was achieved within 60 min by the system. The presence of amino acids in the mixture was shown not to affect the analysis. This protocol was applied to the compositional analysis of 2 extracellular polysaccharides produced by Escherichia coli, colanic acid, and K30 antigen, which share constituent monosaccharides. The overproduction of extracellular polysaccharide in E. coli CWG56 was shown to be a consequence of deregulation of K30 biosynthesis and not of coexpression of an additional polymer.

A plasmid‐encoded rfbO:54 gene cluster is required for biosynthesis of the O:54 antigen in Salmonella enterica serovar Borreze

Previous studies demonstrated that the presence of a 7–8 kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in several Salmonella enterica serovars. In this study, a 6.7 kb plasmid from a field isolate of S. enterica serovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O‐polysaccharide‐substituted LPS molecules and O:54 serotype. SDS‐PAGE analysis of other 0:54 isolates indicated that the O:54 O‐polysaccharide can be co‐expressed with an additional O‐polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N‐acetylmannosamine (D‐ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure:

Identification of Residues Involved in Catalytic Activity of the Inverting Glycosyl Transferase WbbE from Salmonella enterica Serovar Borreze

Synthesis of the O:54 O antigen of Salmonella enterica is initiated by the nonprocessive glycosyl transferase WbbE, assigned to family 2 of the glycosyl transferase enzymes (GT2). GT2 enzymes possess a characteristic N-terminal domain, domain A. Based on structural data from the GT2 representative SpsA (S. J. Charnock and G. J. Davies, Biochemistry 38:6380-6385, 1999), this domain is responsible for nucleotide binding. It possesses two invariant Asp residues, the first forming a hydrogen bond to uracil and the second coordinating a Mn(2+) ion. Site-directed replacement of Asp41 (D41A) of WbbE, the analogue of the first Asp residue of SpsA, revealed that this is not required for activity. WbbE possesses three Asp residues near the position analogous to the second conserved residue. Whereas D95A reduced WbbE activity, activity in D93A and D96A mutants was abrogated, suggesting that either D93 or D96 may coordinate the Mn(2+) ion. Our studies also identified a C-terminal region of sequence conservation in 22 GT2 members, including WbbE. SpsA was not among these. This region is characterized by an ED(Y) motif. The Glu and Asp residues of this motif were individually replaced in WbbE. E180D in WbbE had greatly reduced activity, and an E180Q replacement completely abrogated activity; however, D181E had no effect. E180 is predicted to reside on a turn. Combined with the alignment of the motif with potential catalytic residues in the GT2 enzymes ExoM and SpsA, we speculate that E180 is the catalytic residue of WbbE. Sequence and predicted structural divergence in the catalytic region of GT2 members suggests that this is not a homogeneous family.

[16] Identification of rcs Genes in Escherichia coli O9:K30:H12 and Involvement in Regulation of Expression of Group IA K30 Capsular Polysaccharide

Publisher Summary This chapter reviews the strategies that are used to isolate and characterize the role of rcs regulatory genes involved in the expression of the group IA capsular polysaccharide capsular polysaccharide (CPS) in Escherichia coli O9:K30:H12. In E. coli K-12, colanic acid is synthesized at low temperature (below 30˚C), or when grown on nitrogen-poor, carbon-rich media. Colanic acid production is also insignificant in E. coli strains with group IB and group II CPSs, when the bacteria are grown on rich media at 37°C. In contrast, the K30 and other group IA CPSs are produced and form a capsular structure at 37°C, despite the fact that these CPSs are regulated by the same system as colanic acid. In E. coli 09:K30 and K-12 derivatives, high levels of CPS expression require functional Rcs proteins. Mutations in E. coli K-12 affecting lon and rcsC (rcsC137) increase the transcription of two different cps::lacZ fusions by 10- to 46-fold and 116- to 226-fold, respectively. When colanic acid polymer formation is measured instead, this equates to 13- and 28-fold increases in synthesis in lon and rcsC137 mutants, respectively.