Chris Whitfield

Bacterial polysaccharide synthesis and gene nomenclature

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Structure, assembly and regulation of expression of capsules in Escherichia coli

Many Escherichia coli strains are covered in a layer of surface‐associated polysaccharide called the capsule. Capsular polysaccharides represent a major surface antigen, the K antigen, and more than 80 distinct K serotypes result from structural diversity in these polymers. However, not all capsules consist of K antigen. Some are due to production of an extensive layer of a polymer structurally identical to a lipopolysaccharide O antigen, but distinguished from lipopolysaccharide by the absence of terminal lipid A‐core. Recent research has provided insight into the manner in which capsules are organized on the Gram‐negative cell surface, the pathways used for their assembly, and the regulatory processes used to control their expression. A limited repertoire of capsule expression systems are available, despite the fact that the producing bacteria occupy a variety of ecological niches and possess diverse physiologies. All of the known capsule assembly systems seen in Gram‐negative bacteria are represented in E. coli, as are the majority of the regulatory strategies. Escherichia coli therefore provides a variety of working models on which studies in other bacteria are (or can be) based. In this review, we present an overview of the current molecular and biochemical models for capsule expression in E. coli. By taking into account the organization of capsule gene clusters, details of the assembly pathway, and regulatory features that dictate capsule expression, we provide a new classification system that separates the known capsules of E. coli into four distinct groups.

Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram‐negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O‐polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O‐antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K‐12 have served as prototypes for studies on the LPS and lipo‐oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.

Biosynthesis of lipopolysaccharide O antigens

Lipopolysaccharide O antigens are important virulence determinants for many bacteria. O-antigen synthesis is an interesting problem in cell-surface assembly. There are two known assembly pathways, which differ in the cellular location of their polymerization steps and in the direction of chain polymerization. Some reactions are shared with those for other surface polymers, such as capsular polysaccharides, and may be potential targets for therapeutic intervention.

Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein

Many types of bacteria produce extracellular polysaccharides (EPSs). Some are secreted polymers and show only limited association with the cell surface, whereas others are firmly attached to the cell surface and form a discrete structural layer, the capsule, which envelopes the cell and allows the bacteria to evade or counteract the host immune system. EPSs have critical roles in bacterial colonization of surfaces, such as epithelia and medical implants; in addition some EPSs have important industrial and biomedical applications in their own right. Here we describe the 2.26 Å resolution structure of the 340 kDa octamer of Wza, an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. The bulk of the Wza structure is located in the periplasm and comprises three novel domains forming a large central cavity. Wza is open to the extracellular environment but closed to the periplasm. We propose a route and mechanism for translocation of the capsular polysaccharide. This work may provide insight into the export of other large polar molecules such as DNA and proteins.

Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria.

Publisher Summary This chapter provides an overview of the molecular mechanisms involved in synthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Biosynthesis of many cell-surface components, including polysaccharides, involves enzymes and enzyme complexes found in the cytoplasmic membrane. The peptidoglycan layer is located immediately external to the cytoplasmic membrane and this layer is required for cell shape and rigidity. Gram-negative bacteria possess a periplasm that contains a variety of proteins and enzymes, including some involved in import and export of macromolecules. Biosynthesis of bacterial cell-surface polysaccharides involves a series of sequential processes: (1) biosynthesis of activated precursors in the cytoplasm, (2) formation of repeating units, (3) polymerization of repeating units, and (d) export of polysaccharides to the cell surface. The assembly of polysaccharide repeating units and subsequent polymerization reactions occur at the cytoplasmic membrane, using precursors synthesized in the cytoplasm. Genes for biosynthesis of cell-surface polysaccharides are chromosomal and are arranged in clusters of one or more transcriptional units. The synthesis of lipopolysaccharide (LPS) may be subject to complex regulation, but on-off switching is not possible due to the essential structural requirement for the lipid A-core LPS molecule. Most bacteria use extracellular polysaccharides (EPSs) for protection, and many regulatory strategies are directed to modulating EPS synthesis in response to appropriate environmental cues. Application of genetic and biochemical approaches has facilitated detailed analysis of complex, multicomponent systems, such as those involved in synthesis of cell-surface polysaccharides.

Biosynthesis and Export of Bacterial Lipopolysaccharides

Lipopolysaccharide molecules represent a unique family of glycolipids based on a highly conserved lipid moiety known as lipid A. These molecules are produced by most gram-negative bacteria, in which they play important roles in the integrity of the outer-membrane permeability barrier and participate extensively in host-pathogen interplay. Few bacteria contain lipopolysaccharide molecules composed only of lipid A. In most forms, lipid A is glycosylated by addition of the core oligosaccharide that, in some bacteria, provides an attachment site for a long-chain O-antigenic polysaccharide. The complexity of lipopolysaccharide structures is reflected in the processes used for their biosynthesis and export. Rapid growth and cell division depend on the bacterial cells capacity to synthesize and export lipopolysaccharide efficiently and in large amounts. We review recent advances in those processes, emphasizing the reactions that are essential for viability.

Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria

SUMMARY Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology.

Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane

Surface expression of the group 1 K30 capsular polysaccharide of Escherichia coli strain E69 (O9a:K30) requires WzaK30, a member of the outer membrane auxiliary (OMA) protein family. A mutation in wzaK30 severely restricts the formation of the K30 capsular structure on the cell surface, but does not interfere with the biosynthesis or polymerization of the K30 repeat unit. Here we show that WzaK30 is a surface‐exposed outer membrane lipoprotein. WzaK30 multimers form ring‐like structures in the outer membrane that are reminiscent of the secretins of type II and III protein translocation systems. We propose that WzaK30 forms an outer membrane pore through which the K30‐capsular antigen is translocated. This is the first evidence of a potential mechanism for translocation of high molecular weight polysaccharide across the outer membrane. The broad distribution of the OMA protein family suggests a similar process for polysaccharide export in diverse Gram‐negative bacteria.

Modulation of the surface architecture of Gram-negative bacteria by the action of surface polymer:lipid A–core ligase and by determinants of polymer chain length

Lipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram‐negative bacteria. The lipid A–core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A–core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A–core‐linked and ‐unlinked forms. Analysis by SDS–PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A–core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram‐negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A–core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell‐surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.