Massive diversification in aging colonies of Escherichia coli.

The evolutionary success of bacteria depends greatly on their capacity to continually generate phenotypic diversity. Structured environments are particularly favorable for diversification because of attenuated clonal interference, which renders selective sweeps nearly impossible and enhances opportunities for adaptive radiation. We examined at the microscale level the emergence and the spatial and temporal dynamics of phenotypic diversity and their underlying causes in Escherichia coli colonies. An important dynamic heterogeneity in the growth, metabolic activity, morphology, gene expression patterns, stress response induction, and death patterns among cells within colonies was observed. Genetic analysis indicated that the phenotypic variation resulted mostly from mutations and that indole production, oxidative stress, and the RpoS-regulated general stress response played an important role in the generation of diversity. We observed the emergence and persistence of phenotypic variants within single colonies that exhibited variable fitness compared to the parental strain. Some variants showed improved capacity to produce biofilms, whereas others were able to use different nutrients or to tolerate antibiotics or oxidative stress. Taken together, our data show that bacterial colonies provide an ecological opportunity for the generation and maintenance of vast phenotypic diversity, which may increase the probability of population survival in unpredictable environments.

Identification of Food Spoilage Fungi Using MALDI-TOF MS: Spectral Database Development and Application to Species Complex.

Fungi, including filamentous fungi and yeasts, are major contributors to global food losses and waste due to their ability to colonize a very large diversity of food raw materials and processed foods throughout the food chain. In addition, numerous fungal species are mycotoxin producers and can also be responsible for opportunistic infections. In recent years, MALDI-TOF MS has emerged as a valuable, rapid and reliable asset for fungal identification in order to ensure food safety and quality. In this context, this study aimed at expanding the VITEK® MS database with food-relevant fungal species and evaluate its performance, with a specific emphasis on species differentiation within species complexes. To this end, a total of 380 yeast and mold strains belonging to 51 genera and 133 species were added into the spectral database including species from five species complexes corresponding to Colletotrichum acutatum, Colletotrichum gloeosporioides, Fusarium dimerum, Mucor circinelloides complexes and Aspergillus series nigri. Database performances were evaluated by cross-validation and external validation using 78 fungal isolates with 96.55% and 90.48% correct identification, respectively. This study also showed the capacity of MALDI-TOF MS to differentiate closely related species within species complexes and further demonstrated the potential of this technique for the routine identification of fungi in an industrial context.

Effects of cytoprotective antioxidants on lymphocytes from representative mitochondrial neurodegenerative diseases.

Two new aza analogues of the neuroprotective agent idebenone have been synthesized and characterized. Their antioxidant activity, and ability to augment ATP levels have been evaluated in several different cell lines having suboptimal mitochondrial function. Both compounds were found to be good ROS scavengers, and to protect the cells from oxidative stress induced by glutathione depletion. The compounds were more effective than idebenone in neurodegenerative disease cells. These novel pyrimidinol derivatives were also shown to augment ATP levels in coenzyme Q(10)-deficient human lymphocytes. The more lipophilic side chains attached to the pyrimidinol redox core in these compounds resulted in less inhibition of the electron transport chain and improved antioxidant activity.

Effects of alkyl side chain modification of coenzyme Q10 on mitochondrial respiratory chain function and cytoprotection.

The effect of the alkyl side chain length of coenzyme Q10 on mitochondrial respiratory chain function has been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were identified that exhibited significantly improved effects on mitochondrial oxygen consumption and mitochondrial membrane potential, and also conferred significant cytoprotection on cultured mammalian cells in which glutathione had been depleted by treatment with diethyl maleate. The analogues also exhibited lesser inhibition of the electron transport chain than idebenone. The results obtained provide guidance for the design of CoQ10 analogues with improved activity compared to that of idebenone (1), the latter of which is undergoing evaluation in the clinic as a therapeutic agent.

One-pot regio- and stereoselective synthesis of α'-methoxy-γ-pyrones: biological evaluation as mitochondrial respiratory complex inhibitors.

The one-pot construction of functionalized α'-methoxy-γ-pyrones is detailed. Starting from α,α'-dimethoxy-γ-pyrone, molecular diversity is attained by a regio- and stereoselective desymmetrization using allyllithium followed by vinylogous aldol reaction. Mechanistic considerations including density functional theory calculations and insightful experiments have been gathered to shed light on this complex multistep process. To illustrate the versatility of this methodology, some of the molecules prepared were evaluated for their ability to inhibit NADH-oxidase and NADH-ubiquinone oxidoreductase. In the process, a potent new inihibitor of NADH-oxidase activity (IC(50) 44 nM) was identified.

Nomenclatural issues concerning cultured yeasts and other fungi: why it is important to avoid unneeded name changes.

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate.

Thioredoxin targets in Arabidopsis roots.

The importance of redox-regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol-disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y-type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy-two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid-dependent biosynthesis of isoprenoids as a new redox-mediated process. The redox-regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia-lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT-reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.

A simple phenotypic test for detecting the contribution of outer membrane permeability to carbapenem resistance.

Introduction. The worldwide emergence of carbapenem resistance in Gram-negative bacteria makes the development of simple tests mandatory to identify antimicrobial resistance mechanisms. Enzymatic and membrane barriers are the prominent resistance mechanisms described in these bacteria. Several tests are currently used to detect carbapenemase activities.Aim. However, a simple test for the identification of membrane-associated mechanisms of resistance is not yet available and this mechanism is often inferred after the exclusion of a carbapenemase in carbapenem-resistant Gram-negative bacteria.Methodology. Different media (liquid and solid) containing a membrane permeabilizer were tested to identify the existence of a membrane barrier. Here, polymyxin B nonapeptide (PMBN) was selected to bypass the role of impermeability in clinical carbapenem-resistant Enterobacteriaceae, including Escherichia coli, Enterobacter cloacae , Klebsiella pneumoniae and Klebsiella aerogenes isolates. In parallel, the expression of porins (OmpC and OmpF types) was checked in the various bacterial strains in order to search for a correlation between the restoration of susceptibility and the expression of porin.Results. Using a large number of clinical isolates, PMBN associated with a carbapenem allowed us to detect porin-deficient isolates with a sensitivity ranging from 89 to 93 % and a specificity ranging from 86 to 100 %.Conclusion. This paves the way for a diagnostic assay allowing the detection of this membrane-associated mechanism of resistance in Enterobacteriaceae.

Fosfomycin and Staphylococcus aureus: transcriptomic approach to assess effect on biofilm, and fate of unattached cells.

Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

Strong increase in the autofluorescence of cells signals struggle for survival.

Prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence due to the presence of fluorescent cellular structural components and metabolites. Therefore, cellular autofluorescence (AF) is expected to vary with the metabolic states of cells. We examined how exposure to the different stressors changes the AF of Escherichia coli cells. We observed that bactericidal treatments increased green cellular AF, and that de novo protein synthesis was required for the observed AF increase. Excitation and emission spectra and increased expression of the genes from the flavin biosynthesis pathway, strongly suggested that flavins are major contributors to the increased AF. An increased expression of genes encoding diverse flavoproteins which are involved in energy production and ROS detoxification, indicates a cellular strategy to cope with severe stresses. An observed increase in AF under stress is an evolutionary conserved phenomenon as it occurs not only in cells from different bacterial species, but also in yeast and human cells.

Genome Sequence of a Clinical Staphylococcus aureus Strain from a Prosthetic Joint Infection.

Here, we report the genome sequence ofStaphylococcus aureusLYO-S2, an isolate with sequence type (ST) 45 that was isolated in 2001 from a prosthetic joint infection.

Effects of antibiotics on biofilm and unattached cells of a clinical Staphylococcus aureus isolate from bone and joint infection.

Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51,200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected. Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections.

Characterization of Arabidopsis Mutants for the Variable Subunit of Ferredoxin:thioredoxin Reductase.

The ferredoxin/thioredoxin reductase (FTR) is the key enzyme of a light dependent redox regulatory system controlling enzyme activities in oxygenic photosynthetic cells. It is composed of two dissimilar subunits. The catalytic subunit contains a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site. The function of the second subunit, named the variable subunit because it has less conserved primary sequence and length, is not yet known. In order to get insights into the physiological role and importance of FTR, we studied two Arabidopsis mutant lines in which one of two genes encoding FTRA subunit was disrupted by T-DNA insertion. In FTRA1 mutants, the absence of the corresponding transcript was not compensated by the increase in the level of FTRA2 mRNA. Mutant plants exhibited phenotypic perturbations when compared with wild-type plants. Disruptants were found significantly more sensitive to oxidative stress as imposed under high light or in the presence of paraquat. Mutants were further characterized at the biochemical level. Despite the fact that no difference was found by immunodetection of FTR polypeptides, evidence for an impaired FTR system occurring in the mutants was obtained by measuring the endogenous activation rate of one of its targets. In the leaves of mutants placed under normal culture conditions, NADP-dependent malate dehydrogenase (NADP-MDH) activation rate was abnormally low. A partially compensating increase of the enzyme activity was found as well as a higher amount of 2-cys-peroxiredoxin. Our results provide in planta confirmation of the antioxidant role previously proposed for some of the plastidial thioredoxins from Arabidopsis thaliana. The variable subunit of the FTR proved to be important, but its precise role remains to be established.

Comparison of two approaches for the classification of 16S rRNA gene sequences.

The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

Prompt and easy activation by specific thioredoxins of calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex.

The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A(4), B(4), and B(8) isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A(4)-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B(4)- and B(8)-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A(4)-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B(4)- or B(8)-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.

Vitamin E is essential for the tolerance of Arabidopsis thaliana to metal-induced oxidative stress.

Arabidopsis (Arabidopsis thaliana) plants were grown in a hydroponic culture system for 7 to 14 d in the absence or presence of 75 microM Cd or 75 microM Cu. The Cu treatment resulted in visual leaf symptoms, together with anthocyanin accumulation and loss of turgor. Pronounced lipid peroxidation, which was detected by autoluminescence imaging and malondialdehyde titration, was observed in Cu-treated leaves. The Cd treatment also resulted in loss of leaf pigments but lipid peroxidation and oxidative stress were less pronounced than in the leaves exposed to Cu. Analysis of low-molecular-weight chloroplast and cytosolic antioxidants (ascorbate, glutathione, tocopherols, carotenoids) and antioxidant enzymes (thiol-based reductases and peroxidases) revealed relatively few responses to metal exposure. However, there was a marked increase in vitamin E (alpha-tocopherol) in response to Cd and Cu treatments. Ascorbate increased significantly in Cu-exposed leaves. Other antioxidants either remained stable or decreased in response to metal stress. Transcripts encoding enzymes of the vitamin E biosynthetic pathway were increased in response to metal exposure. In particular, VTE2 mRNA was enhanced in Cu- and Cd-treated plants, while VTE5 and hydroxylpyruvate dioxygenase (HPPD) mRNAs were only up-regulated in Cd-treated plants. Consistent increases in HPPD transcripts and protein were observed. The vitamin E-deficient (vte1) mutant exhibited an enhanced sensitivity towards both metals relative to the wild-type (WT) control. Unlike the vte1 mutants, which showed enhanced lipid peroxidation and oxidative stress in the presence of Cu or Cd, the ascorbate-deficient (vtc2) mutant showed WT responses to metal exposure. Taken together, these results demonstrate that vitamin E plays a crucial role in the tolerance of Arabidopsis to oxidative stress induced by heavy metals such as Cu and Cd.

Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses.

We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of -295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.

Peroxiredoxin Q of Arabidopsis thaliana is attached to the thylakoids and functions in context of photosynthesis.

Peroxiredoxin Q (Prx Q) is one out of 10 peroxiredoxins encoded in the genome of Arabidopsis thaliana, and one out of four that are targeted to plastids. Peroxiredoxin Q functions as a monomeric protein and represents about 0.3% of chloroplast proteins. It attaches to the thylakoid membrane and is detected in preparations enriched in photosystem II complexes. Peroxiredoxin Q decomposes peroxides using thioredoxin as an electron donor with a substrate preference of H(2)O(2) > cumene hydroperoxide >> butyl hydroperoxide >> linoleoyl hydroperoxide and insignificant affinity towards complex phospholipid hydroperoxide. Plants with decreased levels of Prx Q did not have an apparently different phenotype from wildtype at the plant level. However, similar to antisense 2-cysteine (2-Cys) Prx plants [Baier, M. et al. (2000)Plant Physiol., 124, 823-832], Prx Q-deficient plants had a decreased sensitivity to oxidants in a leaf slice test as indicated by chlorophyll a fluorescence measurements. Increased fluorescence ratios of photosystem II to I at 77 K and modified transcript levels of plastid- and nuclear-encoded proteins show that regulatory mechanisms are at work to compensate for the lack of Prx Q. Apparently Prx Q attaches to photosystem II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absence causes metabolic changes that are sensed and trigger appropriate compensatory responses.

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants.

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions.

Characterization of plastidial thioredoxins from Arabidopsis belonging to the new y-type.

The plant plastidial thioredoxins (Trx) are involved in the light-dependent regulation of many enzymatic activities, owing to their thiol-disulfide interchange activity. Three different types of plastidial Trx have been identified and characterized so far: the m-, f-, and x-types. Recently, a new putative plastidial type, the y-type, was found. In this work the two isoforms of Trx y encoded by the nuclear genome of Arabidopsis (Arabidopsis thaliana) were characterized. The plastidial targeting of Trx y has been established by the expression of a TrxGFP fusion protein. Then both isoforms were produced as recombinant proteins in their putative mature forms and purified to characterize them by a biochemical approach. Their ability to activate two plastidial light-regulated enzymes, NADP-malate dehydrogenase (NADP-MDH) and fructose-1,6-bisphosphatase, was tested. Both Trx y were poor activators of fructose-1,6-bisphosphatase and NADP-MDH; however, a detailed study of the activation of NADP-MDH using site-directed mutants of its regulatory cysteines suggested that Trx y was able to reduce the less negative regulatory disulfide but not the more negative regulatory disulfide. This property probably results from the fact that Trx y has a less negative redox midpoint potential (-337 mV at pH 7.9) than thioredoxins f and m. The y-type Trxs were also the best substrate for the plastidial peroxiredoxin Q. Gene expression analysis showed that Trx y2 was mainly expressed in leaves and induced by light, whereas Trx y1 was mainly expressed in nonphotosynthetic organs, especially in seeds at a stage of major accumulation of storage lipids.