Functional analysis of predicted coiled-coil regions in the Escherichia coli K-12 O-antigen polysaccharide chain length determinant Wzz

Wzz is a membrane protein that determines the chain length distribution of the O-antigen lipopolysaccharide by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with an extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils, we constructed amino acid replacement mutants of the E. coli K-12 Wzz protein, which were designed so that the coiled coils could be separate without compromising the helicity of the individual molecules. Mutations in one of the regions, spanning amino acids 108 to 130 (region I), were associated with a partial defect in O-antigen chain length distribution, while mutants with mutations in the region spanning amino acids 209 to 223 (region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153 to 173 (region II) eliminated the Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted alpha-helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted to be coiled coils are important for Wzz function because they maintain the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.

O-antigen modal chain length in Shigella flexneri 2a is growth-regulated through RfaH-mediated transcriptional control of the wzy gene

Shigella flexneri 2a 2457T produces lipopolysaccharide (LPS) with two O-antigen (OAg) chain lengths: a short (S-OAg) controlled by WzzB and a very long (VL-OAg) determined by Wzz(pHS-2). This study demonstrates that the synthesis and length distribution of the S. flexneri OAg are under growth-phase-dependent regulation. Quantitative electrophoretic analysis showed that the VL-OAg increased during growth while the S-OAg distribution remained constant. Increased production of VL-OAg correlated with the growth-phase-regulated expression of the transcription elongation factor RfaH, and was severely impaired in a DeltarfaH mutant, which synthesized only low-molecular-mass OAg molecules and a small amount of S-OAg. Real-time RT-PCR revealed a drastic reduction of wzy polymerase gene expression in the DeltarfaH mutant. Complementation of this mutant with the wzy gene cloned into a high-copy-number plasmid restored the bimodal OAg distribution, suggesting that cellular levels of Wzy influence not only OAg polymerization but also chain-length distribution. Accordingly, overexpression of wzy in the wild-type strain resulted in production of a large amount of high-molecular-mass OAg molecules. An increased dosage of either wzzB or wzz(pHS-2) also altered OAg chain-length distribution. Transcription of wzzB and wzz(pHS-2) genes was regulated during bacterial growth but in an RfaH-independent manner. Overall, these findings indicate that expression of the wzy, wzzB and wzz(pHS-2) genes is finely regulated to determine an appropriate balance between the proteins responsible for polymerization and chain-length distribution of S. flexneri OAg.

Interplay of the Wzx translocase and the corresponding polymerase and chain length regulator proteins in the translocation and periplasmic assembly of lipopolysaccharide o antigen

Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, WzxO16 and WzxE, and also corresponding polymerases (WzyO16 and WzyE) and O-antigen chain-length regulators (WzzO16 and WzzE), respectively. In this study, we show that the E. coli wzxE gene can fully complement a wzxO16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the WzyE polymerase or the WzzE chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of WzxE. We also show that this property is not unique to WzxE, since WzxO16 and WzxO7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.

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.

Relatedness of O-specific lipopolysaccharide side chain genes from strains of Shigella boydii type 12 belonging to two clonal groups and from Escherichia coli O7:K1

The O-specific lipopolysaccharide side chains of Escherichia coli O7 and Shigella boydii type 12 possess similar but not identical chemical structures. We investigated the genetic relatedness between the O-specific side chain genes in members of these two species. Examination of outer membrane protein and lipopolysaccharide (LPS) banding patterns demonstrated that five strains which had been identified as S. boydii type 12 fell into two clonal groups, SB1 and SB2. Hybridizations with O7-specific radiolabeled probes derived from the chromosomal DNA of an E. coli O7 strain detected identical fragments among the three SB1 strains of S. boydii type 12 and the two E. coli O7 reference isolates. The two other S. boydii type 12 strains, which belonged to the SB2 clone, did not show homologies with the O7 probe under high-stringency conditions of hybridization. The homology between the O7 and type 12 LPS gene regions from the SB1 strains was further confirmed by the construction of O-specific side chain-deficient mutations in these strains by homologous recombination of a suicide plasmid containing O7-specific DNA sequences. Immunoblot experiments with O7 antiserum gave a weak cross-reaction with LPS purified from the SB2 strains but a very strong cross-reaction with the LPS from SB1 isolates. Antiserum raised to one of the SB2 strains cross-reacted only with S. boydii type 12 LPS from the SB1 clone but failed to react with O7 LPS.

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexaeri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz(pHS2). Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

FhCaBP4: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

In trematodes, there is a family of proteins which combine EF-hand-containing domains with dynein light chain (DLC)-like domains. A member of this family from the liver fluke, Fasciola hepatica-FhCaBP4-has been identified and characterised biochemically. FhCaBP4 has an N-terminal domain containing two imperfect EF-hand sequences and a C-terminal dynein light chain-like domain. Molecular modelling predicted that the two domains are joined by a flexible linker. Native gel electrophoresis demonstrated that FhCaBP4 binds to calcium, manganese, barium and strontium ions, but not to magnesium or zinc ions. The hydrophobic, fluorescent probe 8-anilinonaphthalene-1-sulphonate bound more tightly to FhCaBP4 in the presence of calcium ions. This suggests that the protein undergoes a conformational change on ion binding which increases the number of non-polar residues on the surface. FhCaBP4 was protected from limited proteolysis by the calmodulin antagonist W7, but not by trifluoperazine or praziquantel. Protein-protein cross-linking experiments showed that FhCaBP4 underwent calcium ion-dependent dimerisation. Since DLCs are commonly dimeric, it is likely that FhCaBP4 dimerises through this domain. The molecular model reveals that the calcium ion-binding site is located close to a key sequence in the DLC-like domain, suggesting a plausible mechanism for calcium-dependent dimerisation.

Examining acute and chronic effects of short- and long-chain fatty acids on peptide YY (PYY) gene expression, cellular storage and secretion in STC-1 cells

PURPOSE: Peptide YY (PYY) is a gastrointestinal hormone with physiological actions regulating appetite and energy homoeostasis. The cellular mechanisms by which nutrients stimulate PYY secretion from intestinal enteroendocrine cells are still being elucidated.

METHODS: This study comprehensively evaluated the suitability of intestinal STC-1 cells as an in vitro model of PYY secretion. PYY concentrations (both intracellular and in culture media) with other intestinal peptides (CCK, GLP-1 and GIP) demonstrated that PYY is a prominent product of STC-1 cells. Furthermore, acute and chronic PYY responses to 15 short (SCFAs)- and long-chain (LCFAs) dietary fatty acids were measured alongside parameters for DNA synthesis, cell viability and cytotoxicity.

RESULTS: We found STC-1 cells to be reliable secretors of PYY constitutively releasing PYY into cell culture media (but not into non-stimulatory buffer). We demonstrate for the first time that STC-1 cells produce PYY mRNA transcripts; that STC-1 cells produce specific time- and concentration-dependent PYY secretory responses to valeric acid; that linoleic acid and conjugated linoleic acid 9,11 (CLA 9,11) are potent PYY secretagogues; and that chronic exposure of SCFAs and LCFAs can be detrimental to STC-1 cells.

CONCLUSIONS: Our studies demonstrate the potential usefulness of STC-1 cells as an in vitro model for investigating nutrient-stimulated PYY secretion in an acute setting. Furthermore, our discovery that CLA directly stimulates L-cells to secrete PYY indicates another possible mechanism contributing to the observed effects of dietary CLA on weight loss.

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the ß-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.

Plasma chain-breaking antioxidants in Alzheimer's disease, vascular dementia and Parkinson's disease

We studied the plasma chain-breaking antioxidants alpha carotene, beta carotene, lycopene, Vitamin A, Vitamin C, Vitamin E and a measure of total antioxidant capacity, TAC, in 79 patients with Alzheimer's disease (AD), 37 patients with vascular dementia (VaD), 18 patients with Parkinson's disease and dementia (PDem), and 58 matching controls, together with 41 patients with Parkinson's disease (PD) and 41 matching controls. Significant reductions in individual antioxidants were observed in all dementia groups. When compared to controls, the following were reduced: Vitamin A in AD (p <0.01) and VaD (p <0.001); Vitamin C in AD (p <0.001), VaD (p <0.001) and PDem (p <0.01); Vitamin E in AD (p <0.01) and VaD (p <0.001); beta carotene in VaD (p = 0.01); lycopene in PDem (p <0.001). Lycopene was also reduced in PDem compared to AD (p <0.001) and VaD (p <0.001). Antioxidant levels in PD were not depleted. No significant change in TAC was seen in any group. The reduction in plasma chain-breaking antioxidants in patients with dementia may reflect an increased free-radical activity, and a common role in cognitive impairment in these conditions. Increased free-radical activity in VaD and PDem could be associated with concomitant AD pathology. Individual antioxidant changes are not reflected in TAC.

Chain confinement promotes β-phase formation in polyfluorene-based photoluminescent ionogels

The synthesis of photoluminescent conjugated polymer silica ionogels using sol–gel chemistry is described. Cooperative self-assembly of an ionic liquid, the silica precursor and poly(9,9-dioctylfluorene) (PFO) via hydrogen bonding and p-stacking interactions drives formation of the PFO ß-phase.

Using vulnerability performance indicators to attain food supply chain robustness

High effectiveness and leanness of modern supply chains (SCs) increase their vulnerability, i.e. susceptibility to disturbances reflected in non-robust SC performances. Both the SC management literature and SC professionals indicate the need for the development of SC vulnerability assessment tools. In this article, a new method for vulnerability assessment, the VULA method, is presented. The VULA method helps to identify how much a company would underperform on a specific Key Performance Indicator in the case of a disturbance, how often this would happen and how long it would last. It ultimately informs the decision about whether process redesign is appropriate and what kind of redesign strategies should be used in order to increase the SC's robustness. The applicability of the VULA method is demonstrated in the context of a meat SC using discrete-event simulation to conduct the performance analysis.

The Bright Side of Manufacturing–Remanufacturing Conflict in a Decentralised Closed-Loop Supply Chain

Researchers and managers broadly agree that original equipment manufacturers (OEMs), which have opportunities to produce both new and remanufactured products, are better off by centrally controlling their manufacturing and remanufacturing activities. Thus, OEMs should not remanufacture used products until the remanufacturing cost is sufficiently low to overcome the negative impact of new product cannibalisation. In this paper, we present a contrasting view of the manufacturing–remanufacturing conflict: OEMs sometimes benefit from the decentralised control mode under which they ignore the internal cannibalisation rather than the remanufacturing option. We consider a decentralised closed-loop supply chain in which one OEM can purchase new components from one supplier to produce new products and collect used products from consumers to produce remanufactured products. The key feature of our model is that the OEM can select a centralised or decentralised control mode to manage its manufacturing and remanufacturing activities before the supplier prices the new component. In a steady state period setting, we analyse the players’ optimal decisions and compare the OEM's profits under centralised and decentralised control modes. Our analytic results reveal that the decentralised control within the OEM can outperform the centralised control when the cost structure of producing new and remanufactured products satisfies certain conditions. Finally, the key findings are distilled in a conceptual framework and its managerial implications are discussed.