Browsing by Author "Lewenza, Shawn"

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    Biosensors for the Detection of Naphthenic Acids in Wastewater from Oil Sands Operations
    (2019-09-19) Shideler, Steven Mark; DeVinney, Rebekah; Lewenza, Shawn; Gieg, Lisa Marie; Savchenko, Alexei
    Naphthenic acids (NA) are a complex group of acyclic and cyclic alkyl-substituted carboxylic acids that are present in the bitumen mined from the oil sands. NAs accumulate in the tailings waste produced from processing bitumen, are toxic to living organisms and are difficult, both in terms of time and resources, to remediate. In this study, we established a high throughput pipeline using bacterial genomics and synthetic biology methods to build biosensor constructs using promoters from Pseudomonas synxantha, an organism isolated from oilsands process-affected water (OSPW). By observing the gene expression profiles of P. synxantha, we have been able to identify genes that are induced by NAs, and that likely play a role in the transport and catabolism of various NA species. We have identified a catabolic operon that is expressed in a dose-dependent manner, in response to NA exposure. In addition, we identified a TetR regulator that is divergent from this operon, that represses expression of the catabolic operon during normal conditions. The TetR regulator was purified and was shown to bind to the target promoter in electrophoretic mobility shift assays. In the presence of specific naphthenic acids, the repressor loses DNA binding affinity and no longer interacts with the promoter. Based on these findings, we have proposed a model of naphthenic acid sensing through a TetR repressor protein and have therefore identified all the components required to build an NA sensing biosensor using P. synxantha as a chassis. We have therefore identified all the components required to build an NA biosensor using P. synxantha as a chassis. The NA biosensor can be employed as a method to detect NA contamination in the environment, and also to aid in the discovery of novel genes for the purpose of supporting the bioremediation of the 1.2 trillion liters of NA contaminated water currently being stored in Northern Alberta.
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    Determining the Role of the Clr6 Histone Deacetylase Subunit Prw1 and Regulation of the Flocculin Gene gsf2+ in the Flocculation of Schizosaccharomyces pombe
    (2021-11-08) Panchal, Priyal; Chua, Gordon; Muench, Douglas; Shemanko, Carrie; Lewenza, Shawn
    Flocculation is the nonsexual aggregation of yeast cells in liquid medium under stressed environmental conditions. This process is used as a coping mechanism to survive in undesirable conditions such as lack of nutrients or drastic changes within the environment. Flocculation in Schizosaccharomyces pombe is primarily governed by the flocculin gene, gsf2+. Gsf2 is the primary flocculin as only the absence of this flocculin causes failure to flocculate. Several transcription factors have been identified that directly regulate gsf2+. However, there has not been studies showing the intracellular localization and temporal expression of Gsf2 under various growth conditions, stressed conditions or under control of known and potential transcription factors implicated in flocculation in live cells. In this study, the intracellular localization of Gsf2 was shown using a green fluorescent protein (GFP) tag. First, we found through fluorescence microscopy that Gsf2-GFP was expressed in cells during stationary phase in minimal medium (EMM), and throughout exponential and stationary phases in flocculation-inducing medium (FIM). Next, we looked at the ectopic expression of Gsf2-GFP in the presence and absence of transcriptional activators and repressors, respectively. Second, we explored alternate inducing conditions and found that high concentrations of ferrous chloride (FeCl2) induces flocculation. Further, we tested our gsf2-GFP strain in EMM + FeCl2 medium and detected Gsf2-GFP expression in cells during exponential and stationary phases. Finally, we explored the Clr6 histone deacetylase (HDAC) complex subunit Prw1 for its ability to repress flocculation. We confirmed by plasmid complementation that the constitutive flocculation phenotype was caused by loss of prw1+. Next, we determined that overexpression of prw1+ or clr6+ was not enough to repress flocculation completely in FIM. Further, we found through qPCR analysis that the prw1? strain shows upregulation of several flocculin genes including gsf2+, as well as cell wall remodeling genes, and the former is likely responsible for the constitutive flocculation phenotype. Collectively, these results substantially contribute to a better understanding of flocculation in S. pombe.
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    Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms
    (Public Library of Science, 2008-11-21) Mulcahy, Heidi; Charron-Mazenod, Laetitia; Lewenza, Shawn
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    Metabolite-Sensing Transcription Factors for Developing Whole Cell Naphthenic Acid Biosensor Technology
    (2022-09) Bookout, Tyson; Gieg, Lisa; Lewenza, Shawn; Chua, Gordon; Dunfield, Peter
    Naphthenic Acids (NA) are a complex group of acyclic and cyclic alkyl-substituted carboxylic acids. These compounds are naturally produced during the degradation of petroleum and are present in high concentrations in the waste produced during the bitumen extraction process. NAs are very toxic, and monitoring and treating the oil sands process-affected water (OSPW) is costly and time-consuming. Whole cell biosensors are a proven technology holding the potential for a rapid and low-cost method of monitoring NA in tailings ponds. We are exploiting the sensitive and reliable detection mechanisms present in bacterial cells for inducing appropriate responses to environmental changes, by engineering bacterial strains to produce a simple and quantitative output in proportion to NA. Using bacterial genomics and synthetic biology, we designed transcription factor-based biosensors using a novel Pseudomonas species isolated from the OSPW, by examining the upregulated genes in the presence of different NA mixtures. The promoters of these genes are then synthesized and cloned directly upstream from a bacterial lux operon, that is used as a bioluminescent reporter. We are particularly interested in those involved in transcriptional regulation, antibiotic efflux, or bioremediation. We have identified three strong promoters for NA-detection: a promoter driving the expression of a hypothetical gene, a divergent NA-inducible promoter from the atu operon regulated by a TetR family repressor, and a promoter for a toxic RND efflux system regulated by a MarR family regulator. These transcriptional regulators bind within the promoter sequence to repress gene expression, unless in the presence of a target analyte. Upon analyte binding, a confirmational change occurs to release the protein from the promoter region and allow transcription. Using electrophoretic mobility shift assays, this mechanism of promoter repression and NA detection was confirmed for both AtuR and MarR regulators. Through lux expression experiments, the hypothetical promoter, divergent atu promoter, and MarR regulated promoter were confirmed to sensitively respond in a dose-dependent manner to a custom mixture of NA compounds, a mixture of primarily acyclic NAs, and the complex OSPW mixture, respectively. These biosensors are sensitive within 2-30 parts per million, and the capability of these biosensors to uniquely respond to NA compounds and not other hydrocarbons such as alkanes and BTEX, demonstrates the potential for using a panel of these biosensors for detecting NA toxicity in the environment.
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    Microbial natural products that inhibit c-di-GMP signalling and biofilm formation in Pseudomonas aeruginosa
    (2017) McCartney, Nathan; Harrison, Joe; Derksen, Darren; Storey, Douglas; Parkins, Michael; Lewenza, Shawn
    Pseudomonas aeruginosa is a notorious opportunistic pathogen that forms highly antibiotic resistant biofilms to establish chronic infections. Antibiotic resistance of this organism is a significant contributor to mortality in immunocompromised patients. In this thesis, a high-throughput screening protocol was used to identify microbial spent media with inhibitory activity against P. aeruginosa c-di-GMP signalling. The biocontrol agent Pseudomonas protegens Pf-5 was identified to have significant inhibitory activity against the c-di-GMP signalling and biofilm forming capacity of P. aeruginosa. The natural product secreted by P. protegens was shown to be dependent on the PSL polysaccharide produced by P. aeruginosa and may exert its effect through the dipA pathway. Results demonstrated that this natural product had activity against a range of clinically-isolated P. aeruginosa and holds promising translational benefits to the eradication of chronic biofilm infections.
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    Plant defensin antibacterial mode of action against Pseudomonas species
    (2020-06-19) Sathoff, Andrew E; Lewenza, Shawn; Samac, Deborah A
    Abstract Background Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. Results Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. Conclusions MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.
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    Small Molecule Biofilm Inhibitors with Antivirulence Properties against Pseudomonas aeruginosa
    (2017) van Tilburg Bernardes, Erik; Lewenza, Shawn; DeVinney, Rebekah; Dong, Tao; Harrison, Joe
    The opportunistic pathogen Pseudomonas aeruginosa grows within biofilms in the Cystic Fibrosis airways, leading to chronic, life-threatening infections. Biofilms are dense communities of bacteria surrounded by a protective polymeric extracellular matrix comprised of exopolysaccharides (EPS), proteins and extracellular DNA. The two major EPS molecules produced by P. aeruginosa are the Pel and Psl. Considering the essential role of EPS in biofilm formation, antimicrobial resistance and immune evasion, we developed a high-throughput gene expression screen for the identification of small molecules that reduce both pel and psl gene expression. Testing of the identified pel/psl repressors demonstrated their antibiofilm activity against static and flow biofilm models. Moreover, these antibiofilm molecules also reduce PAO1 virulence in a nematode infection model, as well as increase P. aeruginosa biofilm susceptibility to antibiotic killing. These small molecules represent a novel anti-infective strategy for the possible adjuvant treatment of chronic P. aeruginosa infections.