Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR.

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio).

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Challenging the model for induction of metallothionein gene expression.

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.

Genotoxic and stress inductive potential of cadmium in Xenopus laevis larvae.

The present investigation evaluates the toxic potential of Cd in larvae of the frog Xenopus laevis after 12 days of exposure to environmentally relevant contamination levels, close to those measured in the river Lot (France). Several genotoxic and detoxification mechanisms were analyzed in the larvae: clastogenic and/or aneugenic effects in the circulating blood by micronucleus (MN) induction, metallothionein (MT) production in whole larvae, gene analyses and Cd content in the liver and also in the whole larvae. The results show: (i) micronucleus induction at environmental levels of Cd contamination (2, 10, 30 microgL(-1)); (ii) an increased and concentration-dependent quantity of MT in the whole organism after contamination with 10 and 30 microgCdL(-1) (a three- and six-fold increase, respectively) although no significant difference was observed after contamination with 2 microgCdL(-1); (iii) Cd uptake by the whole organism and by the liver as a response to Cd exposure conditions; (4) up-regulation of the genes involved in detoxification processes and response to oxidative stress, while genes involved in DNA repair and apoptosis were repressed. The results confirm the relevance of the amphibian model and highlight the complementarity between a marker of genotoxicity, MT production, bioaccumulation and genetic analysis in the evaluation of the ecotoxicological impact.

Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio).

Effects of dietary methylmercury (MeHg) on gene expression were examined in three organs (liver, skeletal muscle, and brain) of the zebrafish (Danio rerio). Adult male fish were fed over 7, 21, and 63 days on three different diets: one control diet (C0: 0.08 microg of Hg g(-1), dry wt) and two diets (C1 and C2) contaminated by MeHg at 5 and 13.5 microg of Hg g(-1), dry wt. Total Hg and MeHg concentrations were determined in the three organs after each exposure duration, and a demethylation process was evidenced only in the liver. Thirteen genes known to be involved in antioxidant defenses, metal chelation, active efflux of organic compounds, mitochondrial metabolism, DNA repair, and apoptosis were investigated by quantitative real-time RT-PCR and normalized according to actin gene expression. Surprisingly, no change in the expression levels of these genes was observed in contaminated brain samples, although this organ accumulated the highest mercury concentration (63.5 +/- 4.4 microg g(-1), dry wt after 63 days). This lack of genetic response could explain the high neurotoxicity of MeHg. coxI and cytoplasmic and mitochondrial sod gene expressions were induced early in skeletal muscle and later in liver, indicating an impact on the mitochondrial metabolism and production of reactive oxygen species. Results demonstrated that skeletal muscle was not only an important storage reservoir but was also affected by MeHg contamination. The expression of the metallothionein mt2 and the DNA repair rad51 genes was up-regulated in liver between 21 and 63 days, whereas in skeletal muscle, mt2 remained uninduced, and gadd and rad51 were found to be repressed.

Induction of a multixenobiotic resistance protein (MXR) in the Asiatic clam Corbicula fluminea after heavy metals exposure.

Multixenobiotic resistance mechanisms (MXR) related to the mammalian P-glycoprotein multidrug transporter protein (P-gp) are known to occur in several marine invertebrates. In the present work, we report on the induction of an MXR protein by various heavy metals in the gills of the freshwater clam Corbicula fluminea. The evaluation of the MXR protein level was assessed by Western blot using a specific monoclonal antibody raised against the human P-gp (C219). A field transplantation experiment, where clams were caged in a gradient relative to an industrial site, demonstrated a positive relationship between MXR levels and (a) metal pollution (Cd and Zn) in the environment and (b) metal bioaccumulation in the gills. To establish this correlative relationship, clams were exposed to different levels of cadmium (15-60 microg l(-1)) for up to 15 days in a controlled laboratory experiment. MXR protein levels increased in time for all treatments (including the control). However, the highest levels of MXR protein titer were expressed in clams that had been exposed to the lowest dose of cadmium. The causes for this observed inverse relationship between the exposure dose and the MXR induction is discussed. MXR protein titer was also shown to be induced by other heavy metals (zinc, inorganic mercury, and copper).

A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine.

The wine lactic acid bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, non-optimal growth temperatures, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins. We here describe characterisation and cloning of the O. oeni omrA gene encoding a protein belonging to the ATP-binding cassette superfamily of transporters. The OmrA protein displays the highest sequence similarity with the subfamily of ATP-dependent multidrug resistance (MDR) proteins, most notably the bacterial Lactococcus lactis LmrA homologue of the human MDR1 P-glycoprotein. The omrA gene proved to be a stress-responsive gene since its expression was increased at high temperature or under osmotic shock. The OmrA protein function was tested in Escherichia coli, and consistent with the omrA gene expression pattern, OmrA conferred protection to bacteria grown on a high salt medium. OmrA also triggered bacterial resistance to sodium laurate, wine and ethanol toxicity. The homologous LmrA protein featured the same stress-protective pattern than OmrA when expressed in E. coli, and the contribution to resistance of both OmrA and LmrA transporters was decreased by verapamil, a well-known inhibitor of the human MDR1 protein. Genes homologous to omrA were detected in other wine lactic acid bacteria, suggesting that this type of genes might constitute a well-conserved stress-protective molecular device.

The arcABC gene cluster encoding the arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene.

Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.

At acidic pH, the GPA2-cAMP pathway is necessary to counteract the ORD1-mediated repression of the hypoxic SRP1/TIR1 yeast gene.

The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein and this gene is downregulated at acidic pH. The stress-responsive HOG pathway is necessary to maintain hypoxic TIR1 expression, but only at acidic pH. However, unlike known HOG pathway-dependent genes, TIR1 is under positive cAMP control and this effect is mediated by GPA2 but not by RAS2. Genetic analysis showed that ord1 mutation was epistatic to the gpa2 mutation, thereby indicating that Gpa2p is needed to counteract the Ord1 factor, which is involved in the repression of hypoxic TIR1 expression, while the HOG pathway appears to be independent from Ord1 repression. In addition, an increased ORD1 gene expression was observed in the Deltagpa2 mutant cells, meaning that GPA2 maintains a low basal level of ORD1 transcripts. Thus, cAMP allows partial relief of the TIR1 repression exerted by Ord1p. However, this is contradicted at acidic pH by the HOG pathway requirement because Hog1p is activated under stress conditions when the cAMP cellular content is low. The opposite effects of the GPA2-cAMP and HOG pathways are likely to explain the diminished hypoxic expression of TIR1 at acidic pH.

A Rox1-independent hypoxic pathway in yeast. Antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene.

Hypoxic SRP1/TIR1 gene expression depends on the absence of haem but is independent of Rox1-mediated repression. We have found a new hypoxic pathway involving an antagonistic interaction between the Ixr1/Ord1 repressor and the Yap1 factor, a transcriptional activator involved in oxidative stress response. Here, we show that Ord1 repressed SRP1 gene expression under normoxia and hypoxia, whereas Yap1 activated it. Ord1 and Yap1 have been shown to bind the SRP1 promoter in a region extending from -299 to -156 bp upstream of the start codon. A typical AP-1 responsive element lying from -247 to -240 bp allows Yap1 binding. Internal deletion of sequences within the SRP1 promoter were introduced. Two regions were characterized at positions -299/-251 and -218/-156 that, once removed, resulted in a constitutive expression of SRP1 in a wild-type strain under normoxic conditions. Deletion of both these two sequences allowed the bypass of YAP1 requirement in a Deltayap1 strain, whereas these two internal deletions did not yield increased expression in a Deltaord1 strain compared with the full-length promoter. Both a single Deltaord1 mutant and a doubly disrupted Deltayap1 Deltaord1 strain yielded normoxic constitutive SRP1 expression and increased hypoxic SRP1 induction, thereby demonstrating that ord1 is epistatic to yap1. Thus, Yap1 is not directly involved in SRP1 induction by hypoxia, but is necessary to counteract the Ord1 effect.

At acidic pH, the diminished hypoxic expression of the SRP1/TIR1 yeast gene depends on the GPA2-cAMP and HOG pathways.

The hypoxic SRP1/TIR1 gene encodes a stress-response cell wall mannoprotein, which is shown to be necessary for yeast growth at acidic pH in the presence of sodium dodecyl sulfate. However, the hypoxic expression of SRP1 is shown to be downregulated at acidic pH. The stress-responsive HOG pathway appeared necessary to maintain hypoxic SRP1 expression, but only at acidic pH. However, unlike known HOG pathway-dependent genes, SRP1 was under positive cAMP control and was positively modulated by protein kinase A at neutral and acidic pH. In addition, the HOG-independent hypoxic HEM13 gene was also positively regulated by cAMP levels. Therefore, the positive cAMP control of the hypoxic SRP1 and HEM13 genes was uncoupled from the HOG pathway. Surprisingly, this positive cAMP control was found to be mediated by GPA2 but not by RAS2, so the Gpa2p requirement appears critical at acidic pH. Although RAS2 is not involved in the regulation of SRP1 expression, the guanine nucleotide exchange factor Cdc25, which is known to control the GTP/GDP ratio on the Ras proteins, was nevertheless required for hypoxic SRP1 expression. Furthermore, the Ras proteins did not compensate for Gpa2p requirement in a delta gpa2 mutated strain. These results suggest that the Cdc25 factor might also control Gpa2p.

A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting.

GPI anchors are widely represented among organisms and have several cellular functions. It has been proposed that in yeast there are two groups of GPI proteins: plasma membrane-resident proteins, such as Gas1p or Yap3p, and cell wall-targeted proteins, such as Tir1p or alpha-agglutinin. A model has been proposed for the plasma membrane retention of proteins from the first group because of a dibasic motif located just upstream of the GPI-anchoring signal. The results we report here are not in agreement with such a model as we show that constructs containing the C-terminal parts of Gas1p and Yap3p are also targeted to the cell wall. We also detect the genuine Gas1p after cell wall treatment with Quantazyme or Glucanex glycanases. In addition, we show that the GPI-anchoring signal from the human placental alkaline phosphatase (PLAP) is not compatible with the yeast machinery unless the human transamidase hGpi8p is co-expressed. In this condition, this human signal is able to target a protein to the cell wall. Moreover, TIR1 proved to be a multicopy suppressor of Deltagas1 mutation. The present findings suggest a constitutive role for GPI anchors in yeast: the cell wall targeting of proteins.

Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae.

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.

Regulation by low temperatures and anaerobiosis of a yeast gene specifying a putative GPI-anchored plasma membrane protein [corrected].

Expression of the yeast Saccharomyces cerevisiae SRP1 (Serine-rich Protein) gene is shown here to be induced both by low temperature and anaerobic growth conditions. We show that anaerobic SRP1 expression is haem-dependent; however, haem influence does not operate through the action of the hypoxic-gene ROX1 repressor. The SRP1 promoter region displaying the stress-responsive elements is restricted to its first 551 bp, upstream of the initiation codon, although an upstream activation site contained in upstream sequences is required for full promoter activity. In addition, we demonstrate that the TIP1 gene, sharing similar nucleotide and polypeptide structure with SRP1, and previously reported to be a cold-shock-inducible gene, is also a hypoxic gene. Srp1 protein production is similarly induced by low temperature and anaerobic growth conditions. This protein, detected in the plasma membrane fraction, is shown to be exposed on the cell surface via a glycosyl-phosphatidylinositol membrane anchoring.

Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti.

In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the 35S-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulphate ([35S]PAPS) from ATP and [35S]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM. Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.

Characterization of the arcD arginine:ornithine exchanger of Pseudomonas aeruginosa. Localization in the cytoplasmic membrane and a topological model.

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins.

Involvement of OmpF during reception and translocation steps of colicin N entry.

[125I]-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C. Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross-react with various monoclonal antibodies directed against colicin A.

Cytoplasmic and periplasmic expression of a synthetic gene for ferredoxin in Escherichia coli.

A synthetic gene coding for a modified ferredoxin II of Desulfovibrio desulfuricans Norway strain was assembled from 10 oligonucleotides. This gene was cloned into various expression vectors allowing either cytoplasmic expression or export to the periplasmic space. In the latter case, two different constructs were made, each of which contained the OmpA signal peptide: one of these constructs contained 3 additional N-terminal amino acids as compared to the wild-type ferredoxin (56 amino acid residues). The expression of proteins encoded by the 3 constructs was assayed in E coli and the proteins were localized by cell fractionation and immunogold labelling. A low percentage of the periplasmic ferredoxin (approximately 5%) was secreted to the medium in the absence of cell lysis. The recombinant ferredoxin was purified and found to be correctly processed by the leader peptidase. However, due to the high cysteine content intramolecular and intermolecular disulfide bonds were formed and prevented binding of [4Fe-4S] clusters. Reconstitution experiments using these recombinant proteins are in progress.

In vivo properties of colicin A: channel activity is voltage dependent but translocation may be voltage independent.

The kinetics of K+ efflux caused by colicin A in Escherichia coli-sensitive cells have been investigated by using a K(+)-selective electrode. The order of magnitude of the rate of K+ efflux per colicin molecule was comparable to that of ion channels. The dependence of K+ efflux upon multiplicity, pH, temperature, and membrane potential (delta psi) was determined. The translocation of colicin A from the outer membrane receptor to the inner membrane and insertion into the inner membrane required a fluid membrane, but once inserted, the channel properties showed little dependence upon the state of the lipids. At a given multiplicity, the lag time before the onset of K+ efflux was found to reflect the time required for translocation and/or insertion of colicin into the cytoplasmic membrane. Opening of the channel only occurred above a threshold value of delta psi of 85 +/- 10 and 110 +/- 5 mV at pH 6.8 and 7.8, respectively. Conditions were designed for closing and reopening of the channel in vivo. These conditions allowed us to test separately the delta psi requirements for translocation and channel opening: translocation and/or insertion did not appear to require delta psi. The channel formed in vivo featured properties similar to that of the channel in lipid planar bilayers.

Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E coli.

Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells. Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-forming colicins. In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described. The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently. This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies. The role of the membrane potential in channel functioning is also discussed.