Peter Richard Reeves

Most Gram-negative bacteria synthesize a plethora of cell surface polysaccharides that play key roles in immune evasion, cell envelope structural integrity and host–pathogen interactions. In the predominant polysaccharide Wzx/Wzy-dependent pathway, synthesis is divided between the cytoplasmic and periplasmic faces of the membrane. Initially, an oligosaccharide composed of 3–8 sugars is synthesized on a membrane-embedded lipid carrier, undecaprenyl pyrophosphate, within the cytoplasmic face of the membrane. This lipid-linked oligosaccharide is then translocated to the periplasmic face by the Wzx flippase, where it is polymerized into a repeat-unit polysaccharide. Structural alterations to the O-antigen repeating oligosaccharide significantly reduce polysaccharide yield and lead to cell death or morphological abnormalities. These effects are attributed to the substrate recognition function of the Wzx flippase, which we postulated to act as a gatekeeper to ensure that only complete substrates are translocated to the periplasmic face. Here, we labelled Salmonella enterica serovar Typhimurium group B1 with [¹⁴C] d-galactose. Our results showed that strains unable to synthesize the full O-antigen repeat unit accumulate significantly higher levels of Und-P-linked material (~10-fold). Importantly, this sequestration is alleviated by membrane disruption which opens the lipid-linked oligosaccharide at the cytosolic face to periplasmic ligation to support accumulation occurs at the cytosolic face of the membrane.

In the predominant Wzx/Wzy-dependent bacterial surface polysaccharide biosynthetic pathway, synthesis is divided between the cytoplasmic and periplasmic faces of the membrane. Initially, an oligosaccharide composed of 3-8 sugars is synthesized on a membrane-embedded lipid carrier, Und-P, within the cytoplasmic face of the membrane. This Und-P-linked oligosaccharide is then translocated to the periplasmic face by the Wzx flippase, where it is polymerized into a repeat-unit polysaccharide. Structural alterations to the repeating oligosaccharide significantly reduce polysaccharide yield and lead to cell death or morphological abnormalities. These effects are attributed to the substrate recognition function of the Wzx flippase, which we postulated to act as a gatekeeper to ensure only complete substrates are translocated to the periplasmic face. Here, we labelled Salmonella enterica serovar Typhimurium Group B1 with [ ¹⁴ C] D-galactose. Our results showed that strains unable to synthesize the full O-antigen repeat unit accumulate significantly higher levels of Und-P-linked material (∼10-fold). Importantly, this sequestration is alleviated by mild membrane disruption which opens the cytosolic face Und-PP-linked material to O-antigen ligation that supports the accumulation to occur at the cytosolic face of the membrane.

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway.

The group A O antigen is the major surface polysaccharide of Salmonella enterica serovar Paratyphi A (SPA), and the focal point for most current vaccine development efforts. The SPA O-antigen repeat (O unit) is structurally similar to the group D1 O unit of S. enterica serovar Typhi, differing only in the presence of a terminal side-branch paratose (Par) in place of tyvelose (Tyv), both of which are attached by the glycosyltransferase WbaV. The two O-antigen gene clusters are also highly similar, but with a loss-of-function mutation in the group A tyv gene and the tandem amplification of wbaV in most SPA strains. In this study, we show that SPA strains consistently produce less O antigen than their group D1 counterparts and use an artificial group A strain (D1 Δtyv) to show this is due to inefficient Par attachment by WbaV. We also demonstrate that group A O-antigen production can be increased by overexpression of the wbaV gene in both the D1 Δtyv strain and two multi-wbaV SPA strains. These findings should be broadly applicable in ongoing vaccine development pipelines, where efficient isolation and purification of large quantities of O antigen is of critical importance.

Bacterial and archaeal flagellins are remarkable in having a shared region with variation in housekeeping proteins and a region with extreme diversity, perhaps greater than for any other protein. Analysis of the 113,285 available full-gene sequences of flagellin genes from published bacterial and archaeal sequences revealed the nature and enormous extent of flagellin diversity. There were 35,898 unique amino acid sequences that were resolved into 187 clusters. Analysis of the Escherichia coli and Salmonella enterica flagellins revealed that the variation occurs at two levels. The first is the division of the variable regions into sequence forms that are so divergent that there is no meaningful alignment even within species, and these corresponded to the E. coli or S. enterica H-antigen groups. The second level is variation within these groups, which is extensive in both species. Shared sequence would allow PCR of the variable regions and thus strain-level analysis of microbiome DNA.

Capsules are a critical virulence factor in many pathogenic Escherichia coli, of which groups 2 and 3 capsules are synthesised by the ABC transporter pathway. The well-studied forms are in group 2 and much of our knowledge of group 3 is inferred from our understanding of group 2. We analyse six group 3 gene clusters including representatives of K10, K11 and K96, and find unexpected diversity. Groups 2 and 3 both have gene clusters with terminal regions 1 and 3 containing mostly genes shared by all members of both groups, plus a central region 2, that in group 2 has the genes for synthesising the serotype-specific repeat unit. We find that in all but one case group 3 gene clusters include, in addition to serotype-specific genes, a previously unrecognised set of shared genes in region 2 that probably codes for an additional structural element. Also, the six shared genes in regions 1 and 3 of group 3 exist in two very different sequence forms. It appears that the E. coli ABC transporter capsules have a very long history, with more fundamental diversity present in group 3, but greater diversity in the exposed strongly antigenic serotype-specific component encoded by region 2.

Cloning of whole polysaccharide biosynthesis gene clusters for expression in a common Escherichia coli tester strain has the major advantage of enabling direct functional comparisons between gene clusters that are normally found in different strains, where their expression is potentially under differential regulatory control. However, due to the large size of many of these gene clusters, classical cloning methods are highly inefficient, time-consuming, and/or labor-intensive. Here we describe a recently developed system, called the operon assembly protocol (OAP), in which yeast homologous recombination pathways are used to assemble overlapping PCR fragments onto a specially engineered yeast E. coli shuttle vector, resulting in full-length customizable gene cluster clones on single-copy plasmids. Multiple versions of the same gene cluster can also be assembled in parallel with genes deleted, replaced, or rearranged, allowing the function and/or specificity of individual genes to be examined. Since the vector can be easily modified to include other bacterial replicons, it can also be broadly applied to the functional analysis of a wide range of bacterial gene clusters and operons.

The Wzx flippase is a critical component of the O‐antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram‐negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O‐unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O‐unit main‐chain, despite both being able to translocate several other structurally‐divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O‐unit residues are equally important determinants of translocation efficiency. We propose a new “Structure‐Specific Triggering” model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high‐frequency translocation only being triggered by Wzx interacting with one or more preferred O‐unit structural elements found on its cognate O unit(s).

Translocation of lipid-linked oligosaccharides is a common theme across prokaryotes and eukaryotes. For bacteria, such activity is used in cell wall construction, polysaccharide synthesis, and the relatively recently discovered protein glycosylation. To the best of our knowledge, the Gram-negative inner membrane flippase Wzx was the first protein identified as being involved in oligosaccharide translocation, and yet we still have only a limited understanding of this protein after 3 decades of research. At present, Wzx is known to be a multitransmembrane protein with enormous sequence diversity that flips oligosaccharide substrates with varied degrees of preference. In this review, we provide an overview of the major findings for this protein, with a particular focus on substrate preference.