Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Abstract

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx EcO157 ) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletionfusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx EcO157 consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PPlinked saccharide substrate across the membrane.Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, plays critical roles in bacterial cell physiology (36) and in disease (53). The structure of LPS is complex and consists at a minimum of lipid A and core oligosaccharide (OS) (42). Many Gram-negative bacteria also have an O-specific antigen polysaccharide (or O antigen) attached to one of the terminal residues of the core OS (42). The O antigen is the most variable portion of the LPS molecule and arises from the polymerization of discrete oligosaccharide units (42, 54).The biosynthesis of LPS requires many enzymes and assembly proteins and generally involves two separate pathways. One pathway results in the synthesis of the lipid A-core OS (42), which is translocated across the inner membrane by the lipid A flippase MsbA, an ABC transporter (14, 15, 60). The other pathway involves the synthesis and assembly of the O-antigen polysaccharide, which also begins at the cytosolic side of the inner membrane resulting in the formation of a lipid-linked molecule that is further translocated across the inner membrane. The formation of a complete LPS molecule containing O antigen is catalyzed by ...

Mutation of a Salmonella Serogroup-C1-Specific Gene Abrogates O7-Antigen Biosynthesis and Triggers NaCl-Dependent Motility Deficiency

“…In this study, we also found that Wzx C1 shared limited deduced amino acid sequence identity with other Wzx flippases (BLASTp), however, the topological prediction algorithms revealed very high similarity between the Wzx C1 and Wzx from S. Typhimurium and E. coli O157:H7 (Fig. S1), whose topological profiles have been reported based on experimental data [21], [22]. It has been reported that O-antigen construction in S. Choleraesuis is made of repeating units of the identical mannose sugar (i.e., homopolymeric O antigens), while the O-antigen of S. Typhimurium or E. coli O157:H7 are heteropolymeric, being composed of various sugars such as mannose, rhamnose and galactose [13].…”

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

“…We used two complementary approaches to determine the topology of MurJ experimentally: reporter fusion technology and the substituted cysteine accessibility method (SCAM). Similar approaches have been used for the MOP exporter and O-antigen flippase Wzx (38,62,63). For the reporter strategy, we employed a dual-reporter fusion system (39).…”

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

“…Soluble or membranebound GTs complete the repeating-unit structure in a sequential manner. The repeating units are then flipped to the periplasmic side of the inner membrane or across the cytoplasmic membrane by a Wzx flippase (109). The Wzy polymerase builds up the CPS/ EPS structure by the assembly of several repeating units at the reducing end of the growing polymer (61).…”