A novel alpha-galactosidase from I'ifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of a parts per thousand 243 kDa and a subunit size of a parts per thousand 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (a parts per thousand 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) a parts per thousand yen3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

BACKGROUND Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal 'Peptidase_M75' domain of 251 residues. The C-terminal domain contains a highly conserved 'HXXE' motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or 'Psyr_3370') encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M(W) 27,772Da). Circular dichroism spectroscopy of EfeM indicated a mainly alpha-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6A. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.

A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies

We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Overproduction, purification and preliminary X-ray diffraction analysis of YncE, an iron-regulated Sec-dependent periplasmic protein from Escherichia coli

The yncE gene of Escherichia coli encodes a predicted periplasmic protein of unknown function. The gene is de-repressed under iron restriction through the action of the global iron regulator Fur. This suggests a role in iron acquisition, which is supported by the presence of the adjacent yncD gene encoding a potential TonB-dependent outer-membrane transporter. Here, the preliminary crystallographic structure of YncE is reported, revealing that it consists of a seven-bladed beta-propeller which resembles the corresponding domain of the `surface-layer protein' of Methanosarcina mazei. A full structure determination is under way in order to provide insight into the function of this protein.

EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E-coli O157 : H7

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn(-)) for utilization of methyl phosphonate (MePn) and that Phn(+) variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn(+) variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn(+) revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn(+) and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Comparative effects of fatty acids on endothelial inflammatory gene expression

Background: Endothelial dysfunction may be related to adverse effects of some dietary fatty acids (FAs). Although in vitro studies have failed to show consistent findings, this may reflect the diverse experimental protocols employed and the limited range of FAs and end points studied. Aims: To investigate the effect of dietary FA type (saturated, monounsaturated, n-6 and n-3 polyunsaturated fatty acids), concentration, incubation time and cell stimulation state, on a broad spectrum of endothelial inflammatory gene expression. Methods: Using human umbilical vein endothelial cells, with and without stimulation (+/- 10 ng/ml TNF alpha), the effects of arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA), linoleic (LA), oleic (OA) and palmitic acids (PA) (10, 25 and 100 mu M), on the expression of genes encoding a number of inflammatory proteins and transcription factors were assessed by quantitative real time RT-PCR. Results: Individual FAs differentially affect endothelial inflammatory gene expression in a gene-specific manner. EPA, LA and OA significantly up-regulated MCP-1 gene expression compared to AA (p = 0.001, 0.013, 0.008, respectively) and DHA (p < 0.0005, = 0.004, 0.002, respectively). Furthermore, cell stimulation state and FA incubation time significantly influenced reported FA effects on gene expression. Conclusions: The comparative effects of saturated, monounsaturated, n-6 and n-3 polyunsaturated FAs on endothelial gene expression depend on the specific FA investigated, its length of incubation, cell stimulation state and the gene investigated. These findings may explain existing disparity in the literature. This work was funded by the EC, Framework Programme 6 via the LIPGENE project (FOOD-CT-2003-505944).

Dietary vitamin E modulates differential gene expression in the rat hippocampus: potential implications for its neuroprotective properties

A wide range of cell culture, animal and human epidemiological studies are suggestive of a role of vitamin E (VE) in brain function and in the prevention of neurodegeneration. However, the underlying molecular mechanisms remain largely unknown. In the current investigation Affymetrix gene chip technology was utilised to establish the impact of chronic VE deficiency on hippocampal genes expression. Male albino rats were fed either a VE deficient or standard diet (60 mg/kg feed) for a period of 9 months. Rats were sacrificed, the hippocampus removed and genes expression established in individual animals. VE deficiency showed to have a strong impact on genes expression in the hippocampus. An important number of genes found to be regulated by VE was associated with hormones and hormone metabolism, nerve growth factor, apoptosis, dopaminergic neurotransmission, and clearance of amyloid-beta and advanced glycated endproducts. In particular, VE strongly affected the expression of an array of genes encoding for proteins directly or indirectly involved in the clearance of amyloid beta, changes which are consistent with a protective effect of VE on Alzheimer's disease progression.

ms1, a novel stress-responsive, muscle-specific gene that is up-regulated in the early stages of pressure overload-induced left ventricular hypertrophy

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that rust may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Modulation of interleukin signalling and gene expression in cardiac myocytes by endothelin-1

The related inflammatory cytokines, interleukin- (IL-) 1β and IL-33, are both implicated in the response of the heart to injury. They also activate mitogen-activated protein kinases (MAPKs) in cardiac myocytes. The hypertrophic Gq protein-coupled receptor agonist endothelin-1 is a potentially cardioprotective peptide and may modulate the inflammatory response. Endothelin-1 also stimulates (MAPKs) in cardiac myocytes and promotes rapid changes in expression of mRNAs encoding intercellular and intracellular signalling components including receptors for IL-33 (ST2) and phosphoprotein phosphatases. Prior exposure to endothelin-1 may specifically modulate the response to IL-33 and, more globally, influence MAPK activation by different stimuli. Neonatal rat ventricular myocytes were exposed to IL-1β or IL-33 with or without pre-exposure to endothelin-1 (5 h) and MAPK activation assessed. IL-33 activated ERK1/2, JNKs and p38-MAPK, but to a lesser degree than IL-1β. Endothelin-1 increased expression of soluble IL-33 receptors (sST2 receptors) which may prevent binding of IL-33 to the cell-surface receptors. However, pretreatment with endothelin-1 only inhibited activation of p38-MAPK by IL-33 with no significant influence on ERK1/2 and a small increase in activation of JNKs. Inhibition of p38-MAPK signalling following pretreatment with endothelin-1 was also detected with IL-1β, H2O2 or tumour necrosis factor α (TNFα) indicating an effect intrinsic to the signalling pathway. Endothelin-1 pretreatment suppressed the increase in expression of IL-6 mRNA induced by IL-1β and decreased the duration of expression of TNFα mRNA. Coupled with the general decrease in p38-MAPK signalling, we conclude that endothelin-1 attenuates the cardiac myocyte inflammatory response, potentially to confer cardioprotection.