Biosynthesis Of Enterobacterial Common Antigen Research Articles

Genome-wide identification of fitness-genes in aminoglycoside-resistant Escherichia coli during antibiotic stress

Resistance against aminoglycosides is widespread in bacteria. This study aimed to identify genes that are important for growth of E. coli during aminoglycoside exposure, since such genes may be targeted to re-sensitize resistant E. coli to treatment. We constructed three transposon mutant libraries each containing > 230.000 mutants in E. coli MG1655 strains harboring streptomycin (aph(3″)-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3″)-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS), a combination of transposon mutagenesis and high-throughput sequencing, identified 56 genes which were deemed important for growth during streptomycin, 39 during gentamicin and 32 during neomycin exposure. Most of these fitness-genes were membrane-located (n = 55) and involved in either cell division, ATP-synthesis or stress response in the streptomycin and gentamicin exposed libraries, and enterobacterial common antigen biosynthesis or magnesium sensing/transport in the neomycin exposed library. For validation, eight selected fitness-genes/gene-clusters were deleted (minCDE, hflCK, clsA and cpxR associated with streptomycin and gentamicin resistance, and phoPQ, wecA, lpp and pal associated with neomycin resistance), and all mutants were shown to be growth attenuated upon exposure to the corresponding antibiotics. In summary, we identified genes that are advantageous in aminoglycoside-resistant E. coli during antibiotic stress. In addition, we increased the understanding of how aminoglycoside-resistant E. coli respond to antibiotic exposure.

Response of Gut Microbiota, Digestive Enzyme Ability, and Immune Function to Starvation in the Oriental River Prawn Macrobrachium nipponense

In order to evaluate the interplay of nutritional status and the digestive capacity, immune function, and gut microbiota in the oriental river prawn Macrobrachium nipponense, a 14-day starvation trial was conducted to detect the effects of starvation on the activities of digestive enzymes and immune enzymes and the structure of prawn gut microbiota. The adult prawns were randomly assigned to a control group (F group) and starvation group (S group) with three replicates. The F group was normally fed with commercial diet, and the S group was starved for 14 days. The result showed that digestive enzyme activities of trypsin and lipase in the hepatopancreas of the S group were significantly lower than those of the F group after 4 d of starvation ( P < 0.05 ), while the activity of amylase significantly increased after 14 d of starvation ( P < 0.05 ). Moreover, the results of the histological analysis of prawn gut showed that both the height of epithelial cells and microvilli of intestine in the S group were obviously decreased than those in the F group ( P < 0.05 ) after 14 d of starvation. The activities of immune-related enzymes including superoxide dismutase, catalase, and acid phosphatase in the hepatopancreas of the S group significantly decreased after 4 d of starvation, and lysozyme activity of the F group was lower than that of the S group after 7 d of starvation ( P < 0.05 ). In addition, the results of Illumina high-throughput sequencing showed that a total of 14,285 OTUs were obtained and classified into 30 phyla, among which Actinobacteria and Proteobacteria were the predominant microflora in the intestinal microbial communities of both groups. However, the relative abundance of opportunistic pathogens was significantly increased in the S group, while the relative abundance of beneficial bacterium was decreased ( P < 0.05 ). The bacterial richness with the Chao estimator was significantly higher in the F group than in the S group ( P < 0.05 ). Both the results of principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) demonstrated that the intestinal microbiota of the F group was separate from those of the S group. The result of functional prediction of the metabolic pathways showed that the intestinal microbiota was enriched in the KEGG pathways of amino acid, carbohydrate, fatty acid, and lipid biosynthesis and degradation at level 2. This result implied that the microbiota of shrimp gut decreased nutrition metabolism under the stress of starvation. Meanwhile, comparing to the F group, the immune-related pathway of enterobacterial common antigen biosynthesis was markedly reduced in the S group ( P < 0.05 ). The result of redundancy analysis (RDA) further confirmed that the activities of digestive and immune enzymes were correlated with the microbial community structure. Finally, the structural equation modeling highlighted that changes in the activities of digestive and immune enzymes were directly related to the gut bacterial community and notably affected prawn growth.

WecB Gene of Salmonella Gallinarum Plays a Critical Role in Systemic Infection of Fowl Typhoid.

Salmonella enterica serovar Gallinarum (S. Gallinarum) is a host-specific pathogen causing fowl typhoid, a severe systemic infection in poultry, which leads to substantial economic losses due to high morbidity and mortality in many developing countries. However, less is known about the pathogenic characteristics and mechanism of S. Gallinarum-induced systemic infection in chickens. In this study, we deleted the S. Gallinarum UDP-N-acetylglucosamine-1-phosphate transferase gene, which contributes to the biosynthesis of enterobacterial common antigen (ECA), and studied the pathogenicity of this wecB::Cm strain in a chicken model of systemic infection. The wecB::Cm mutant strain showed comparable growth but lower resistance to bile acid and nalidixic acid than the wild-type strain in vitro. In the oral infection model of chickens, the virulence of the wecB::Cm strain was significantly attenuated in vivo. Chickens infected with wild-type strain showed typical clinical signs and pathological changes of fowl typhoid and died between 6 and 9 days post-infection, and the bacteria rapidly disseminated to systemic organs and increased in the livers and spleens. In contrast, the wecB::Cm mutant strain did not cause chicken death, there were no significant clinical changes, and the bacterial numbers in the liver and spleen of the chickens were significantly lower than those of the chickens infected with the wild-type strain. In addition, the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and CXCLi1 in the livers of wecB::Cm-infected chickens was significantly lower than that of the chickens infected with the wild-type strain. Furthermore, the attenuated wecB::Cm strain could persistently colonize the liver and spleen at low levels for up to 25 days post-infection and could induce a protective immune response in the chickens. These results indicate that the wecB gene is an important virulence factor of S. Gallinarum in the chicken model of systemic infection, and the avirulent wecB::Cm mutant could possibly be used as a live-attenuated vaccine strain for controlling fowl typhoid.

Topology of the Shigella flexneri Enterobacterial Common Antigen polymerase WzyE

Enterobacteriales have evolved a specialized outer membrane polysaccharide [Enterobacterial Common Antigen (ECA)] which allows them to persist in various environmental niches. Biosynthesis of ECA initiates on the cytoplasmic leaflet of the inner membrane (IM) where glycosyltransferases assemble ECA repeat units (RUs). Complete RUs are then translocated across the IM and assembled into polymers by ECA-specific homologues of the Wzy-dependent pathway. Consisting of the membrane proteins Wzx, Wzy and Wzz, the Wzy-dependent pathway is the most common polysaccharide biosynthetic pathway in Gram-negative bacteria where it is most notably involved in LPS O antigen (Oag) biosynthesis. As such, the majority of research directed towards these proteins has been orientated towards Oag biosynthetic homologues with little directed towards ECA homologues. Belonging to the Shape, Elongation, Division and Sporulation (SEDS) protein family, Wzy proteins are polymerases, and are characterized as possessing little or no peptide homology among homologues as well as being polytopic membrane proteins with functionally relevant residues within periplasmic loops, as defined by C-terminal reporter fusion topology mapping. Here, we present the first the first major study into the ECA polymerase WzyE. Multiple sequence alignments and topology mapping showed that WzyE is unlike WzyB proteins involved with Oag biosynthesis WzyE displays high peptide conservation across Enterobacteriales. In silico structures and reporter mapping allowed us to identify possible functionally conserved residues with WzyESF's periplasmic loops, which we showed were crucial for its function. This work provides novel insight into Wzy proteins and suggests that WzyE is an optimal model to investigate Wzy proteins and the Wzy-dependent pathway.

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

Advances in the research of enterobacterial common antigen

The enterobacterial common antigen (ECA) is a polysaccharide composed of polysaccharide repeats that are located in the outer membrane of almost all Enterobacteriaceae bacteria and has diverse biological functions. ECA is synthesized by the synergistic action of multiple genes that are present in clusters on the genome of Enterobacteriaceae bacteria, forming the ECA antigen gene cluster, an important virulence factor that plays a role in host invasion and survival of Enterobacteriaceae in vivo. ECA also plays an important role in the maintenance of the bacterial outer membrane permeability barrier, flagella gene expression, swarming motility, and bile salts resistance. In addition, ECALPS, anchored in the core region of bacterial lipopolysaccharide, is an important surface antigen for bacteria, stimulating high levels of antibody production in the host and could be a target for vaccine research. This review summarizes ECA purification, genes involved in ECA biosynthesis, its immunological characteristics, biological functions and clinical applications.

Construction and Application of an Escherichia coli Strain Lacking 62 Genes Responsible for the Biosynthesis of Enterobacterial Common Antigen and Flagella.

The biosynthesis of the enterobacterial common antigen and flagella in Escherichia coli consumes lots of substrates and energy. In this study, 12 genes responsible for the biosynthesis of the enterobacterial common antigen were deleted in E. coli MG1655, resulting in WQM021. WQM021 grew better than MG1655 in both rich LB medium and minimum M9 medium. Compared with MG1655, WQM021 showed higher membrane permeability and higher production efficiency for recombinant proteins, polyhydroxyalkanoate, and l-threonine. Transcriptome analysis revealed that genes relevant to glucose consumption, glycolysis, and flagellar synthesis were significantly upregulated in WQM021. Therefore, 50 genes responsible for flagellar biosynthesis were further deleted in WQM021, resulting in WQM022. WQM022 grew better and could synthesize more polyhydroxyalkanoate and l-threonine than WQM021. The results demonstrate that the productivity of E. coli can be efficiently improved when the enterobacterial common antigen and flagella are eliminated. This strategy has guiding significance in the optimization of other industrial products and microorganisms.

Biosynthesis of glycolipid MPIase (membrane protein integrase) is independent of the genes for ECA (enterobacterial common antigen).

MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same. A series of biosynthetic genes for ECA, including ones for the corresponding nucleotide sugars, have been identified and extensively characterized. In this study, we found that knockouts as to the respective genes for ECA biosynthesis can grow in the minimum medium with the normal expression level of MPIase, indicating that MPIase can be biosynthesized de novo without the utilization of any compounds generated through ECA biosynthesis. Conversely, ECA was expressed normally upon MPIase depletion. From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.

Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier of Escherichia coli in a Manner Controlled by YhdP

ABSTRACTGram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.

Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate.

Bacteria use long-chain isoprenoids like undecaprenyl phosphate (Und-P) as lipid carriers to assemble numerous glycan polymers that comprise the cell envelope. In any one bacterium, multiple oligosaccharide biosynthetic pathways compete for a common pool of Und-P, which means that disruptions in one pathway may produce secondary consequences that affect the others. Using the Gram-negative bacterium Escherichia coli as a model, we demonstrate that interruption of the biogenesis of O antigen, a major outer membrane component, indirectly impairs peptidoglycan synthesis by sequestering Und-P into dead-end intermediates. These results strongly argue that the functions of many Und-P-utilizing pathways must be reevaluated, because much of our current understanding is based on experiments that did not control for these unintended secondary effects.

Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

Bacterial morphology is determined primarily by the architecture of the peptidoglycan (PG) cell wall, a mesh-like layer that encases the cell. To identify novel mechanisms that create or maintain cell shape in Escherichia coli, we used flow cytometry to screen a transposon insertion library and identified a wecE mutant that altered cell shape, causing cells to filament and swell. WecE is a sugar aminotransferase involved in the biosynthesis of enterobacterial common antigen (ECA), a non-essential outer membrane glycolipid of the Enterobacteriaceae. Loss of wecE interrupts biosynthesis of ECA and causes the accumulation of the undecaprenyl pyrophosphate-linked intermediate ECA-lipid II. The wecE shape defects were reversed by: (i) preventing initiation of ECA biosynthesis, (ii) increasing the synthesis of the lipid carrier undecaprenyl phosphate (Und-P), (iii) diverting Und-P to PG synthesis or (iv) promoting Und-P recycling. The results argue that the buildup of ECA-lipid II sequesters part of the pool of Und-P, which, in turn, adversely affects PG synthesis. The data strongly suggest there is competition for a common pool of Und-P, whose proper distribution to alternate metabolic pathways is required to maintain normal cell shape in E. coli.

LPS modification promotes maintenance of Yersinia pestis in fleas.

Yersinia pestis, the causative agent of plague, can be transmitted by fleas by two different mechanisms: by early-phase transmission (EPT), which occurs shortly after flea infection, or by blocked fleas following long-term infection. Efficient flea-borne transmission is predicated upon the ability of Y. pestis to be maintained within the flea. Signature-tagged mutagenesis (STM) was used to identify genes required for Y. pestis maintenance in a genuine plague vector, Xenopsylla cheopis. The STM screen identified seven mutants that displayed markedly reduced fitness in fleas after 4 days, the time during which EPT occurs. Two of the mutants contained insertions in genes encoding glucose 1-phosphate uridylyltransferase (galU) and UDP-4-amino-4-deoxy-l-arabinose-oxoglutarate aminotransferase (arnB), which are involved in the modification of lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N) and resistance to cationic antimicrobial peptides (CAMPs). These Y. pestis mutants were more susceptible to the CAMPs cecropin A and polymyxin B, and produced lipid A lacking Ara4N modifications. Surprisingly, an in-frame deletion of arnB retained modest levels of CAMP resistance and Ara4N modification, indicating the presence of compensatory factors. It was determined that WecE, an aminotransferase involved in biosynthesis of enterobacterial common antigen, plays a novel role in Y. pestis Ara4N modification by partially offsetting the loss of arnB. These results indicated that mechanisms of Ara4N modification of lipid A are more complex than previously thought, and these modifications, as well as several factors yet to be elucidated, play an important role in early survival and transmission of Y. pestis in the flea vector.

Progress in understanding the assembly process of bacterial O-antigen

The discovery that the surfaces of Gram-negative bacteria often carry unique polysaccharide signatures pre-dates most seminal discoveries of molecular biology and biochemistry of the 20th century. The O-antigen component of the lipopolysaccharide has been one of the most intensely studied bacterial polysaccharide surface structures for over 80years. Yet, many questions about the mechanism of biosynthesis of the O-antigen and its transport to the cell surface remain unanswered. In this review we provide an overview of how the molecular basis of the O-antigen assembly and trafficking were unraveled in a historical context. We pay particular attention to the emergence of novel technological approaches and how they fueled the elucidation of the O-antigen maturation process. Moreover, we provide a brief perspective on the biosynthesis of enterobacterial common antigen and underline the similarities and differences between the pathways used to assemble these two surface polysaccharides. Finally, we highlight key discoveries that led to the understanding of the mechanistic basis of bacteriophage-induced O-antigen modifications. We place special emphasis on the regulation of the length of O-antigen polymers and provide a detailed overview of the models explaining the O-antigen length determination. Finally, we highlight outstanding questions that need to be addressed both structurally and functionally to advance our understanding of the O-antigen assembly, trafficking and export within cellular and molecular contexts.

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

Identification of three oligo‐/polysaccharide‐specific ligases in Yersinia enterocolitica

In lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria the lipid A-core oligosaccharide (LA-core) and O-polysaccharide (O-PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL-encoded ligase joins O-PS onto LA-core. Enterobacterial common antigen (ECA) biosynthesis follows that of O-PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA-core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA-inner core (IC) onto which a homopolymeric O-PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O-PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O-PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single, double and triple ligase mutants indicated that two of ligases, named as WaaL(os) and WaaL(ps) , had a preferred substrate specificity for OC and O-PS, respectively, although with some promiscuity between the ligases; the third ligase named as WaaL(xs) was not involved in LPS or ECA biosynthesis. In Y. enterocolitica O:8 the WaaL(os) homologue (Ye1727) ligated a single pentasaccharide O-unit to LA-IC suggesting that in both Y. enterocolitica O:3 and O:8 WaaL(os) is an oligosaccharide (OS)-specific ligase. Finally, Yersinia pestis and Y. pseudotuberculosis carry only the waaL(ps) gene, while either waaL(os) or waaL(xs) or both are additionally present in other Yersinia species. This is the first report on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.

The Rcs Signal Transduction Pathway Is Triggered by Enterobacterial Common Antigen Structure Alterations inSerratia marcescens

The enterobacterial common antigen (ECA) is a highly conserved exopolysaccharide in Gram-negative bacteria whose role remains largely uncharacterized. In a previous work, we have demonstrated that disrupting the integrity of the ECA biosynthetic pathway imposed severe deficiencies to the Serratia marcescens motile (swimming and swarming) capacity. In this work, we show that alterations in the ECA structure activate the Rcs phosphorelay, which results in the repression of the flagellar biogenesis regulatory cascade. In addition, a detailed analysis of wec cluster mutant strains, which provoke the disruption of the ECA biosynthesis at different levels of the pathway, suggests that the absence of the periplasmic ECA cyclic structure could constitute a potential signal detected by the RcsF-RcsCDB phosphorelay. We also identify SMA1167 as a member of the S. marcescens Rcs regulon and show that high osmolarity induces Rcs activity in this bacterium. These results provide a new perspective from which to understand the phylogenetic conservation of ECA among enterobacteria and the basis for the virulence attenuation detected in wec mutant strains in other pathogenic bacteria.

The Klebsiella pneumoniae O2a Antigen Defines a Second Mechanism for O Antigen ATP-binding Cassette Transporters

An ATP-binding cassette (ABC) transporter-dependent mechanism is responsible for the biosynthesis of polysaccharide O antigens in the lipopolysaccharides of many Gram-negative bacteria. In the Escherichia coli O9a prototype, addition of a non-reducing terminal modification regulates chain length. The terminal residue is an export signal recognized by the nucleotide-binding domain component of the cognate ABC transporter. The Klebsiella pneumoniae O2a antigen lacks a capping residue, and the corresponding nucleotide-binding protein does not contain a separate substrate-binding domain. Unlike the O9a transporter, the O2a transporter can export heterologous O antigens. Export of the O2a antigen is obligatorily coupled to chain elongation, and the O2a transporter is essential for establishing O antigen chain length. The E. coli O9a transporter operates after chain length has been determined. Furthermore biosynthesis and export can be uncoupled. O antigen chain length is a critical element in the ability of lipopolysaccharides to confer resistance to complement-mediated killing. This study establishes that two distinctly different approaches are available for the regulation of O antigen chain length and export via an ABC transporter.

Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide.

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

Evidence That the wzxE Gene of Escherichia coli K-12 Encodes a Protein Involved in the Transbilayer Movement of a Trisaccharide-Lipid Intermediate in the Assembly of Enterobacterial Common Antigen

The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.

Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli.

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.