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The performance of on-site wastewater treatment systems (OWTSs) can be improved by altering digester design and by manipulating environmental variables that affect microbial community composition. Community composition can potentially be assessed using high-throughput DNA sequencing, but the two most common methods of community DNA sequencing (16S and shotgun sequencing) generally yield different taxonomic identification profiles and can perform differently according to the sampled environment. To evaluate the use of these two approaches in monitoring OWTS operation, we conducted a comparative parallel analysis using both 16S rDNA and shotgun sequencing in a controlled field study. Results indicate that when examining microorganisms above 0.1% relative abundance, 16S and shotgun sequencing produced similar results in terms of individual sample species richness and between-sample community similarity. However, shotgun sequencing provided comparatively higher taxonomic richness for the bacterial communities with lower abundance in the OWTSs. In addition, 16S sequencing resolved only 48 out of 188 bacterial communities identified by shotgun sequencing (using a 0.1% abundance cutoff). Three key bacterial genera (Desulfomicrobium, Simplicispira, and Phenylobacterium) in anaerobic digestion processes were differentially abundant for both sequencing methods. These data indicate that both sequencing methods provide similar overall profiles for bacterial communities in anaerobic digestor systems. However, shotgun sequencing provides significantly (p-value < 0.01) higher taxonomic richness overall. Thus, shotgun sequencing provides a more robust taxonomic and functional profile that can be used for the optimization of anaerobic digestor systems.

Residents in rural communities across Canada collect potable water from aquifers. Fecal contaminants from sewage and agricultural runoffs can penetrate aquifers, posing a public health risk. Standard methods for detecting fecal contamination test for fecal indicator bacteria (FIB), but the presence of these do not identify sources of contamination. In contrast, DNA-based diagnostic tools can achieve this important objective. We employed quantitative polymerase chain reaction (qPCR) and high-throughput DNA sequencing to trace fecal contamination sources in Wainfleet, a rural Ontario township that has been under the longest active boil water advisory in Canada due to FIB contamination in groundwater wells. Using traditional methods, we identified FIBs indicating persistent fecal pollution in well waters. We used 16S rRNA sequencing to profile groundwater microbial communities and identified Campylobacteraceae as a fecal contamination DNA marker in septic tank effluents (STEs). We also identified Turicibacter and Gallicola as a potential cow and chicken fecal contamination marker, respectively. Using human specific Bacteroidales markers, we identified leaking septic tanks as the likely primary fecal contamination source in some of Wainfleet's groundwater. Overall, the results support the use of sequencing-based methods to augment traditional water quality testing methods and help end-users assess fecal contamination levels and identify point and non-point pollution sources. This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.

This study proposes injectable polymeric filters to prevent diesel migration during its bioremediation. Pseudomonas aeruginosa (Gram negative bacterium) was isolated from forest soil and used to biodegrade diesel. P. aeruginosa produced biosurfactants that emulsified diesel in water while degrading it, by decreasing the interfacial tension and forming skins around diesel droplets. In lab experiments, emulsified diesel droplets could migrate through model soil columns (sand and limestone) saturated with water (to mimic aquifers). These results indicate that clean aquifers downstream of diesel spills are at risk of becoming contaminated during diesel bioremediation by P. aeruginosa. Injectable filters were developed to mitigate this risk. Injectable filters were obtained by injecting into model soils aqueous solutions of quaternized hydroxyethylcellulose (HEC+, with a positive electrostatic charge). HEC + adsorbed onto model soils, yielding positively charged surfaces. Diesel droplets stabilized by P. aeruginosa had a negative electrostatic charge (as demonstrated through zeta potential measurements) and adhered onto HEC + coated soil, likely due to attractive electrostatic forces. As a result, diesel droplet mobility through soil columns decreased. Our proof of concept indicates that injectable HEC + filters can protect clean regions of aquifers during the bioremediation of diesel spills by P. aeruginosa.

Methylenetetrahydrofolate models (green substructure) reduce organohalides to the respective hydrocarbons under biomimetic conditions and mimic the activity of dehalohydrogenases., Halocarbons R–X are reduced to hydrocarbons R–H by folate model compounds under biomimetic conditions. The reactions correspond to a halide–hydride exchange with the methylenetetrahydrofolate (MTHF) models acting as hydride donors. The MTHF models are also functional equivalents of dehalohydrogenases but, unlike these enzymes, do not require a metal cofactor. The reactions suggest that halocarbons have the potential to act as endocrinological disruptors of biochemical pathways involving MTHF. As a case in point, we observe the rapid reaction of the MTHF models with the inhalation anaesthetic halothane. The ready synthetic accessibility of the MTHF models as well as their dehalogenation activity in the presence of air and moisture allow for the remediation of toxic, halogenated hydrocarbons.

Pseudomonas sp. bacteria were incorporated into a double emulsion system (water in oil in water, W/O/W), which can be injected in the subsurface to trap and biotreat naphthalene. The outermost phase of the W/O/W emulsion was an aqueous solution of chitosan (1.65 wt.%). Naphthalene could be dispersed in the aqueous chitosan phase of W/O/W emulsions. Two separate primary water in oil (W/O) emulsions were emulsified in chitosan, using Tween 20 as emulsifier. W/O emulsions were prepared with canola oil (containing ethylcellulose and calcium stearate as emulsifiers), and aqueous solutions of either Pseudomonas sp. in minimal broth or sodium citrate (crosslinker). The formation of W/O/W double emulsions was verified with optical and confocal microscopy. Without addition of triggers able to induce gelation on demand, W/O/W emulsions were stable and mainly viscous under shear. The shear elastic modulus (G’ = 1.90 ± 0.39 Pa) and the shear viscous modulus (G” = 5.76 ± 0.06 Pa) were low, to facilitate pumping in the subsurface. With addition of the surfactant cocamidopropyl betaine, W/O/W emulsions were destabilized on demand, thereby releasing citrate (crosslinker) and Pseudomonas sp. in the chitosan phase, where naphthalene was dispersed. Mixing chitosan with citrate led to its gelation, trapping naphthalene. Mixing naphthalene with Pseudomonas sp. led to its bioremediation. Gelation was demonstrated by the increase in the viscoelastic moduli, with G’ = 111.02 ± 1.67 Pa > G” = 8.66 ± 0.10 Pa. With naphthalene, Pseudomonas sp. survival in gelled chitosan was confirmed by plating bacteria sampled from the gel. Without naphthalene, Pseudomonas sp. did not survive in chitosan.

As bacterial pathogens develop resistance against most currently used antibiotics, novel alternatives for treatment of microbial infectious diseases are urgently needed. Targeting bacterial virulence functions in order to disarm pathogens represents a promising alternative to classical antibiotic therapy. Type IV secretion systems, which are multiprotein complexes in the cell envelope that translocate effectors into host cells, are critical bacterial virulence factors in many pathogens and excellent targets for such “antivirulence” drugs. The VirB8 protein from the mammalian pathogen Brucella was chosen as a specific target, since it is an essential type IV secretion system component, it participates in multiple protein-protein interactions, and it is essential for the assembly of this translocation machinery. The bacterial two-hybrid system was adapted to assay VirB8 interactions, and a high-throughput screen identified specific small-molecule inhibitors. VirB8 interaction inhibitors also reduced the levels of VirB8 and of other VirB proteins, and many of them inhibited virB gene transcription in Brucella abortus 2308, suggesting that targeting of the secretion system has complex regulatory effects in vivo . One compound strongly inhibited the intracellular proliferation of B. abortus 2308 in a J774 macrophage infection model. The results presented here show that in vivo screens with the bacterial two-hybrid assay are suited to the identification of inhibitors of Brucella type IV secretion system function.

The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K(m) being 25 micro m and the k(cat) 1.7 x 10(-3) s(-1). Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K(m) (47 micro m) but not significantly the k(cat) (2.1 x 10(-3) s(-1)). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.

NiFe-hydrogenases have an Ni-Fe site in which the iron has one CO and two CN groups as ligands. Synthesis of the CN ligands requires the activity of two hydrogenase maturation proteins: HypF and HypE. HypF is a carbamoyltransferase that transfers the carbamoyl moiety of carbamoyladenylate to the COOH-terminal cysteine of HypE and thus forms an enzyme-thiocarbamate. HypE dehydrates the S -carbamoyl moiety in an adenosine triphosphate–dependent process to yield the enzyme thiocyanate. Chemical model reactions corroborate the feasibility of this unprecedented biosynthetic route and show that thiocyanates can donate CN to iron. This finding underscores a striking parallel between biochemistry and organometallic chemistry in the formation of an iron-cyano complex.

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino-acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]-hydrogenases as a recognition motif.

An established inverse clinical correlation between serum adiponectin levels and renal cell carcinoma (RCC) aggressiveness exists. We have recently demonstrated that adiponectin suppresses clear cell RCC (ccRCC) progression through interaction with its receptor, adiponectin receptor 1 (AdipoR1). ERp46 has been shown to inhibit adiponectin signaling via interaction with AdipoR1 in HeLa cells. However, the expression of ERp46 in RCC has not been described thus far. The objectives of this study were to investigate ERp46 in RCC, its expression, its effects on RCC growth in a mouse model and whether it interacts with AdipoR1. We demonstrated a higher ERp46/AdipoR1 expression ratio in metastatic compared to non-metastatic ccRCC, as determined by immunohistochemistry of tissue microarrays and subsequent image analysis. When ERp46 was stably knocked down using shRNA or overexpressed in murine RCC RAG cells, RCC growth after subcutaneous injection in BALB/c nude mice was inhibited and accelerated, respectively. In vitro analysis to determine the molecular interaction between AdipoR1 and ERp46 included co-immunoprecipitation using human ccRCC 786-O cells and a bacterial adenylate cyclase-based two hybrid system and demonstrated no sustained AdipoR1-ERp46 interaction. This is the first report to suggest a role for ERp46 as a potential therapeutic target in RCC given its expression profile in human RCC samples and its effect on in vivo RCC growth. Since a stable interaction with AdipoR1 could not be established, we suggest that the tumorigenic properties of ERp46 in RCC cells are not related to an inhibitory modulation of AdipoR1.

SummarySecretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein–protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure–function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis , which identifies amino acid residues required for three protein–protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein–protein interactions were purified and characterized in vitro and in vivo . Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro . The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens . Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.

The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K(m) being 25 micro m and the k(cat) 1.7 x 10(-3) s(-1). Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K(m) (47 micro m) but not significantly the k(cat) (2.1 x 10(-3) s(-1)). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.

HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.

The iron of the binuclear active center of [NiFe]-hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O-carbamoyl-transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]-hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated

The hypoxic and glycolytic environment of malignant cells facilitates high levels of reactive oxygen species (ROS) production. Breast, ovarian and prostate cancer etiology is associated with the ROS production, which are thought to play an important role in regulating the cancers cell growth, survival and metastasis. A potent and major cellular, however functional limited characterized source of ROS, is the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (GPD2). GPD2 is an essential component of the glycerol-3-phosphate shuttle and necessarily expressed in all tissues. In several cancer cell lines, GPD2 is highly expressed and we estimated it accounts for up to 70% of the total ROS production within the prostate cancer cell line PC3. In this study we have utilized this knowledge, to complete a cell-based high throughput-screening (HTS) assay, aimed to identify small molecule inhibitors of the GPD2 dependent ROS formation in PC3 cell lines. We employed the amplex red hydrogen peroxidase detection assay and adapted it to the HTS format. 133 inhibitory compounds from the Canadian Compound Collection were identified to reduce the ROS production activity within the PC3 cells. In order to validate these hits and to determine their mode of action, we PCR amplified GPD2 form PC3-cell cDNA and purified recombinant human GPD2 from a bacterial overproducing strain to homogeneity by employing Strep-tactin affinity-chromatography. The heterologously expressed enzyme, devoid of its mitochondrial target sequence, displayed glycerol-3-phosphate dependent resazurin, dichlorophenolindophenol, as well as tetrazole/phenazine methosulfate oxidoreductase activity. Follow up enzymatic assays of the primary HTS hits with the artificial electron acceptors identified several GPD2 activity specific inhibitors. Subsequent concentration response studies of the HTS derived inhibitors elucidated their in vitro half maximal inhibitory concentration at the range of 10 μM. In order to map GPD2's ROS production site and determine the inhibitor binding sites within the enzyme, we bioinformatically derived the proteins tertiary structure. Considering that GPD2 dependent ROS formation may drive the prostate cancer proliferation, we aim by exploiting the results of this work to generate novel lead molecules as promising potential anti-proliferating cancer drug candidates. (This work was supported by a grant from the Canadian Institutes of Health Research to Gurmit Singh) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3694.