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.

Many Gram-positive and Gram-negative bacteria utilize polysaccharide surface layers called capsules to evade the immune system; consequently, the synthesis and export of the capsule are a potential therapeutic target. In Escherichia coli K-30, the integral membrane tyrosine autokinase Wzc and the cognate phosphatase Wzb have been shown to be key for both synthesis and assembly of capsular polysaccharides. In the Gram-positive bacterium Streptococcus pneumoniae, the CpsCD complex is analogous to Wzc and the phosphatase CpsB is the corresponding cognate phosphatase. The phosphatases are known to dephosphorylate their corresponding autokinases, yet despite their functional equivalence, they share no sequence homology. We present the structure of Wzb in complex with phosphate and high-resolution structures of apo-CpsB and a phosphate-complexed CpsB. We show that both proteins are active toward Wzc and thereby demonstrate that CpsB is not specific for CpsCD. CpsB is a novel enzyme and represents the first solved structure of a tyrosine phosphatase from a Gram-positive bacterium. Wzb and CpsB have completely different structures, suggesting that they must operate by very different mechanisms. Although the mechanism of Wzb can be inferred from previous studies, CpsB appears to have a tyrosine phosphatase mechanism not observed before. We propose a chemical mechanism for CpsB based on site-directed mutagenesis and structural data.

SummaryMany pathogenic bacteria encase themselves in a polysaccharide capsule that provides a barrier to the physical and immunological challenges of the host. The mechanism by which the capsule assembles around the bacterial cell is unknown. Wzi, an integral outer-membrane protein from Escherichia coli, has been implicated in the formation of group 1 capsules. The 2.6 Å resolution structure of Wzi reveals an 18-stranded β-barrel fold with a novel arrangement of long extracellular loops that blocks the extracellular entrance and a helical bundle that plugs the periplasmic end. Mutagenesis shows that specific extracellular loops are required for in vivo capsule assembly. The data show that Wzi binds the K30 carbohydrate polymer and, crucially, that mutants functionally deficient in vivo show no binding to K30 polymer in vitro. We conclude that Wzi is a novel outer-membrane lectin that assists in the formation of the bacterial capsule via direct interaction with capsular polysaccharides.

The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.

Lyme disease is a complex tick-borne zoonosis that poses an escalating public health threat in several parts of the world, despite sophisticated healthcare infrastructure and decades of effort to address the problem. Concepts like the true burden of the illness, from incidence rates to longstanding consequences of infection, and optimal case management, also remain shrouded in controversy. At the heart of this multidisciplinary issue are the causative spirochetal pathogens belonging to the Borrelia Lyme complex. Their unusual physiology and versatile lifestyle have challenged microbiologists, and may also hold the key to unlocking mysteries of the disease. The goal of this review is therefore to integrate established and emerging concepts of Borrelia biology and pathogenesis, and position them in the broader context of biomedical research and clinical practice. We begin by considering the conventions around diagnosing and characterizing Lyme disease that have served as a conceptual framework for the discipline. We then explore virulence from the perspective of both host (genetic and environmental predispositions) and pathogen (serotypes, dissemination, and immune modulation), as well as considering antimicrobial strategies (lab methodology, resistance, persistence, and clinical application), and borrelial adaptations of hypothesized medical significance (phenotypic plasticity or pleomorphy).