An Escherichia coli undecaprenyl-pyrophosphate phosphatase implicated in undecaprenyl phosphate recycling

Undecaprenyl phosphate (Und-P) is a universal lipid carrier of glycan biosynthetic intermediates for carbohydrate polymers that are exported to the bacterial cell envelope. Und-P arises from the dephosphorylation of undecaprenyl pyrophosphate (Und-PP) molecules produced by de novo synthesis and also from the recycling of released Und-PP after the transfer of the glycan component to other acceptor molecules. The latter reactions take place at the periplasmic side of the plasma membrane, while cytoplasmic enzymes catalyse the de novo synthesis. Four Und-PP pyrophosphatases were recently identified in Escherichia coli. One of these, UppP (formerly BacA), accounts for 75 % of the total cellular Und-PP pyrophosphatase activity and has been suggested to participate in the Und-P de novo synthesis pathway. Unlike UppP, the other three pyrophosphatases (YbjG, YeiU and PgpB) have a typical acid phosphatase motif also found in eukaryotic dolichyl-pyrophosphate-recycling pyrophosphatases. This study shows that double and triple deletion mutants in the genes uppP and ybjG, and uppP, ybjG and yeiU, respectively, are supersensitive to the Und-P de novo biosynthesis inhibitor fosmidomycin. In contrast, single or combined deletions including pgpB have no effect on fosmidomycin supersensitivity. Experimental evidence is also presented that the acid phosphatase motifs of YbjG and YeiU face the periplasmic space. Furthermore, the quadruple deletion mutant DeltauppP-DeltaybjG-DeltayeiU-DeltawaaL has a growth defect and abnormal cell morphology, suggesting that accumulation of unprocessed Und-PP-linked O antigen polysaccharides is toxic for these cells. Together, the results support the notion that YbjG, and to a lesser extent YeiU, exert their enzymic activity on the periplasmic side of the plasma membrane and are implicated in the recycling of periplasmic Und-PP molecules.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Functional analysis of the Campylobacter jejuni N-linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N-linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N-linked heptasaccharide. Furthermore, we show that C. jejuni- and E. coli-derived pathways can interact in the biosynthesis of N-linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP-GlcNAc: undecaprenylphosphate GlcNAc-1-phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N-linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni-derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N-linked glycan occurs on a lipid-linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N-linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LPS) biosynthesis, converge at the step in which PglB, the key enzyme of the C. jejuni N-glycosylation system, transfers O polysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PglB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PglB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PglB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.

Reconstitution of O-specific lipopolysaccharide expression in Burkholderia cenocepacia strain J2315, which is associated with transmissible infections in patients with cystic fibrosis

Burkholderia cenocepacia is an opportunistic bacterium that infects patients with cystic fibrosis. B. cenocepacia strains J2315, K56-2, C5424, and BC7 belong to the ET12 epidemic clone, which is transmissible among patients. We have previously shown that transposon mutants with insertions within the O antigen cluster of strain K56-2 are attenuated for survival in a rat model of lung infection. From the genomic DNA sequence of the O antigen-deficient strain J2315, we have identified an O antigen lipopolysaccharide (LPS) biosynthesis gene cluster that has an IS402 interrupting a predicted glycosyltransferase gene. A comparison with the other clonal isolates revealed that only strain K56-2, which produced O antigen and displayed serum resistance, lacked the insertion element inserted within the putative glycosyltransferase gene. We cloned the uninterrupted gene and additional flanking sequences from K56-2 and conjugated this plasmid into strains J2315, C5424, and BC7. All the exconjugants recovered the ability to form LPS O antigen. We also determined that the structure of the strain K56-2 O antigen repeat, which was absent from the LPS of strain J2315, consisted of a trisaccharide unit made of rhamnose and two N-acetylgalactosamine residues. The complexity of the gene organization of the K56-2 O antigen cluster was also investigated by reverse transcription-PCR, revealing several transcriptional units, one of which also contains genes involved in lipid A-core oligosaccharide biosynthesis.

Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279-282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279-282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.

Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein

We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A-core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild-type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane-impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes

Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.

Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli

Lipopolysaccharide (LPS), a glycolipid molecule found on the outer leaflet of outer membranes of gram-negative bacteria, consists of three moieties: lipid A, core oligosaccharide, and the O-specific polysaccharide chain. The O-specific side chain, which extends to the extracellular milieu, plays an important role in pathogenicity, especially during the initial stages of infection, because of its ability to interact with serum complement. In recent years, several laboratories have used recombinant DNA tools to determine, at the molecular level, the organization, expression, and regulation of genes involved in LPS biosynthesis in Salmonella and Escherichia coli. An increased understanding of the molecular aspects of the O-specific side-chain genes will shed light on the intimate details related with the formation of the O-specific side chain, its assembly onto the lipid A--core, and the translocation and insertion of the complete LPS molecule into the outer membrane. It will also contribute to the understanding of the evolution of these genes and the correlation of chemical diversity of O-specific side chains with the genetic diversity of O-specific side-chain genes. In addition, since the O-specific side chains are involved in the pathogenicity of medically important gram-negative bacteria, a basic understanding of the regulation and expression of O-specific side chain LPS genes will contribute to the field of molecular pathogenesis. This article provides an overview of the role of O-specific side chains in septicemic infections and also discusses the current status of molecular genetic studies on O-specific side-chain genes from E. coli.

Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

Molecular cloning, expression, and regulation in Escherichia coli K-12 of a chromosome-mediated aerobactin iron transport system from a human invasive isolate of E. coli K1

We have cloned chromosomal genes determining the aerobactin iron transport system from the Escherichia coli K1 strain VW187. Mapping and hybridization experiments showed that the VW187 aerobactin region was identical to that of the plasmid ColV-K30. However, in the E. coli K-12 background, the biosynthesis of both siderophore and ferric aerobactin receptor encoded by the VW187-derived recombinant plasmids was not repressed by iron to the same extent found when a recombinant plasmid derived from pColV-K30 was used. RNA-DNA dot-blot hybridization experiments demonstrated that the aerobactin-specific mRNA synthesized by the VW187-derived clones was not iron regulated in E. coli K-12. In contrast, the synthesis of aerobactin and its receptor in strain VW187 was completely repressed by iron regardless of whether the recombinant plasmids originated from VW187 or pColV-K30. Similar results were obtained with gene fusions in which a promoterless lac operon was placed under the control of aerobactin promoter regions of either chromosome- or plasmid-mediated aerobactin systems. DNA sequencing of the chromosomal aerobactin promoter region showed changes in bases located immediately upstream to the -35 region compared with the corresponding region in pColV-K30, which is known to be part of the binding site for the Fur repressor protein.

Regulation of erythropoietin gene expression depends on two different oxygen-sensing mechanisms

Erythropoietin (Epo), a glycoprotein hormone produced principally in the fetal kidney and in the adult liver in response to hypoxia, is the prime regulator of growth and differentiation in erythroid progenitor cells. The regulation of Epo gene expression is not fully understood, but two mechanisms have been proposed. One involves the participation of a heme protein capable of reversible oxygenation and the other depends on the intracellular concentration of reactive oxygen species (ROS), assumed to be a function of pO2. We have investigated the production of Epo in response to three stimuli, hypoxia, cobalt chloride, and the iron chelator desferrioxamine, in Hep3B cells. As expected, hypoxia caused a marked rise in Epo production. When the cells were exposed to the paired stimuli of hypoxia and cobalt no further increase was found. In contrast, chelation of iron under hypoxic conditions markedly enhanced Epo production, suggesting that the two stimuli act by separate pathways. The addition of carbon monoxide inhibited hypoxia-induced Epo production, independent of desferrioxamine concentration. Taken together these data support the concept that pO2 and ROS are sensed independently.