Biology subjects

Lewenza, S.

Publications and source records attributed to Lewenza, S..

6 recordsLinked to original sources

bioRxiv2026

Biosensor monitoring of naphthenic acids remediation in mesocosms and constructed wetlands: a head-to-head comparison with orbitrap mass spectrometry.

Oil sands process-affected water (OSPW) contains complex mixtures of naphthenic acids (NA) that are the central targets for water treatment and reclamation. Here, we compared three whole-cell bacterial NA biosensors with Orbitrap mass spectrometry (MS) for quantifying NA remediation in greenhouse mesocosms and a pilot-scale constructed wetland. Solid-phase extracts from both systems were analyzed in parallel by Orbitrap MS and biosensor assays, enabling direct comparison of biosensor-derived NA estimates with MS-derived naphthenic acid fraction compounds (NAFC) concentrations. Across both treatment systems, biosensor outputs broadly tracked declines in NAFC measured by Orbitrap MS, and positive linear relationships were observed between methods. Biosensor 2 (3680-lux) and biosensor 3 (atuA-lux) showed, early rapid decreases in NA, whereas biosensor 1 (marR-lux) frequently remained elevated later in treatment. These differences are consistent with the distinct chemical response profiles of the biosensor panel and suggest that biosensor outputs reflect shifts in NA mixture composition as remediation proceeds. This interpretation is supported by the published Orbitrap analysis of the constructed wetland, which showed decreasing O2-NAFCs and increasing O3/O4-containing species, consistent with oxidative degradation. Together, these results support the use of NA-responsive biosensors as rapid and scalable complementary tools for tracking remediation trends, while Orbitrap MS remains the reference method for molecular-level characterization.

microbiology↗

bioRxiv2026

Narcosis biosensor for the detection of bacterial membrane disruption by naphthenic acids.

Naphthenic acids are amphipathic compounds whose toxicity has primarily been attributed to narcosis toxicity to cell membranes. However, few methods exist that specifically study the membrane disruption and toxicity of this complex family of cyclic, polycyclic and acyclic alkyl-substituted carboxylic acids. Here we describe a whole cell biosensor approach that relies on the ability of Pseudomonas aeruginosa, a ubiquitous environmental organism and opportunistic pathogen, to sense membrane damage (narcosis) and induce protective genes to repair and protect the outer membrane. Many classes of membrane disrupting antimicrobials induce the expression of two operons that encode protective defense systems against outer membrane (OM) damage, including antimicrobial peptides, chelators, and detergents. We demonstrate that the pmrF and spdE2 transcriptional lux reporters are induced by exposure to individual NA compounds with diverse structures, as well as mixtures and naphthenic acid fraction compounds (NAFCs). To further support the narcosis hypothesis, we demonstrated that NA permeabilizes the outer membrane to assist in lysozyme killing, and disrupts the inner membrane integrity, allowing uptake of the DNA binding dye propidium iodide. The conventional OM permeability assay that measures NPN fluorescence is not applicable to study NAs, because they stimulate NPN fluorescence in the absence of cells. This narcosis biosensor approach constitutes a rapid and simple method to measure narcosis and could be developed as a novel toxicity indicator of oil sands tailings.

microbiology↗

bioRxiv2025

AtuR and MarR transcriptional repressors that sense fatty or naphthenic acids and regulate degradation or antimicrobial resistance operons in Pseudomonas sp. OST1909.

Naphthenic acids (NA) are a complex family of acyclic and cyclic carboxylic acids that accumulate in tailings after bitumen extraction from oil sands in Alberta. In response to exposure to naphthenic acids, Pseudomonas sp. OST1909 induces the expression of operons regulated by TetR and MarR-type transcriptional repressors. This class of repressor releases from promoters after binding small molecules, leading to a de-repression of the target operons. We propose that these regulators sense fatty and naphthenic acids and control the expression of genes required to cope with their exposure. Plasmid-encoded transcriptional lux reporters to the atu degradation and mar antimicrobial resistance promoters were constructed that also included the corresponding repressor protein, controlled by various strength constitutive promoters. We provide evidence that the atuAR and marR promoters are directly repressed by AtuR and MarR, respectively. His-tagged MarR and AtuR proteins bound to their promoters containing palindromic binding sites in electrophoretic mobility shift assays (EMSA). Fatty and naphthenic acid compounds and mixtures prevented promoter DNA binding to the repressor proteins in EMSA assays, which suggested direct analyte binding to the protein. The marR-H biosensor construct was functional when introduced into E. coli, confirming the role of NA sensing and transcriptional repressor proteins. This study provides mechanistic insight into the use of NA-inducible promoters as bacterial biosensors for NA detection and identifies novel ligands for the MarR and TetR types of repressor proteins. ImportanceUnderstanding the gene expression responses to environmental pollutants is necessary to construct whole cell biosensors that can be employed as a monitoring technology. We have previously identified bacterial genes that were induced by exposure to naphthenic acids, a complex family of cyclic and polycyclic, alkyl substituted carboxylic acids that originate in oil sands but accumulate in the water used to extract bitumen from oil sands. In this study we focus on MarR and TetR-type regulatory proteins and their role in binding fatty acids and naphthenic acids, to further our understanding of how bacteria sense these compounds and mitigate a gene expression response.

microbiology↗

bioRxiv2024

Construction of whole cell bacterial biosensors as an alternative environmental monitoring technology to detect naphthenic acids in oil sands process-affected water.

After extraction of bitumen from oil sands deposits, the oil sand process-affected water (OSPW) is stored in tailings ponds. Naphthenic acids in tailings ponds have been identified as the primary contributor to toxicity to aquatic life. As an alternative to other analytical methods, here we identify bacterial genes induced after growth in naphthenic acids and use synthetic biology approaches to construct a panel of candidate biosensors for NA detection in water. The main promoters of interest were the atuAR promoters from a naphthenic acid degradation operon and upstream TetR regulator, the marR operon which includes a MarR regulator and downstream naphthenic acid resistance genes, and a hypothetical gene with a possible role in fatty acid biology. Promoters were printed and cloned as transcriptional lux reporter plasmids that were introduced into a tailings pond-derived Pseudomonas species. All candidate biosensor strains were tested for transcriptional responses to naphthenic acid mixtures and individual compounds. The three priority promoters respond in a dose-dependent manner, which allows semi-quantitative measurements, to simple, acyclic and complex NA mixtures, and each promoter has unique NA specificities. The limits of NA detection from the various NA mixtures ranged between 1.5 - 15 mg/L. The atuA and marR promoters also detected NA in small volumes of OSPW samples and were induced by extracts of the panel of OSPW samples. While biosensors have been constructed for other hydrocarbons, here we describe a biosensor approach that could be employed in environmental monitoring of naphthenic acids in oil sands mining wastewater.

microbiology↗

bioRxiv2019

Extracellular DNA controls expression of Pseudomonas aeruginosa genes involved in nutrient utilization, metal homeostasis, acid pH tolerance, and virulence.

Pseudomonas aeruginosa grows in extracellular DNA-enriched biofilms and infection sites. Extracellular DNA (eDNA) is generally considered a structural biofilm polymer required for aggregation and biofilm maturation. In addition, eDNA can sequester divalent metal cations, acidify the growth media, and serve as a nutrient source. Here we determine the transcriptome of planktonic P. aeruginosa grown in the presence of eDNA using RNA-seq. Transcriptome analysis identified 89 induced genes and 76 repressed genes in the presence of eDNA (FDR<0.05), and transcriptional lux fusions were used to confirm eDNA regulation. A large number of eDNA-induced genes appear to be involved in utilizing DNA as a nutrient. Several eDNA-induced genes are also induced by acidic pH 5.5, and growth in the presence of eDNA or acidic pH promoted an acid tolerance response in P. aeruginosa. The cyoABCDE terminal oxidase is induced at pH 5.5 and contributed to the acid tolerance phenotype. Quantitative metal analysis confirmed that DNA binds to diverse metals, which helps explain why many genes involved in a general uptake of metals were controlled by eDNA. Growth in the presence of eDNA also promoted intracellular bacterial survival and influenced virulence and during the acute infection model of fruit flies. The diverse functions of the eDNA-regulated genes underscore the important role of this extracellular polymer in promoting antibiotic resistance, virulence, acid tolerance, and nutrient utilization; phenotypes that contribute to long-term survival.

microbiology↗

bioRxiv2018

Pseudomonas aeruginosa displays a dormancy phenotype during long-term survival in water.

Pseudomonas aeruginosa is capable of long-term survival in water, which may serve as a reservoir for infection. Although viable cell counts of PAO1 incubated in water remain stable throughout 8 weeks, LIVE/DEAD(R) staining indicated a high proportion of cells stained with propidium iodide (PI). The proportion of PI-stained cells increased by 4 weeks, then decreased again by 8 weeks, suggesting an adaptive response. This was also evident in an observed shift in cell morphology from a rod to a coccoid shape after 8 weeks. Fluorescence-activated cell sorting (FACS) was used to recover PI-stained cells, which were plated and shown to be viable, indicating that PI-stained cells were membrane-compromised but still cultivable. PAO1 mid-log cells in water were labeled with the dsDNA-binding dye PicoGreen(R) to monitor viability as well as DNA integrity, which demonstrated that the population remains viable and transitions towards increased dsDNA staining. Metabolic activity was found to decrease significantly in water by 4 weeks. The PAO1 outer membrane became less permeable and more resistant to polymyxin B damage in water, and the profile of total membrane lipids changed over time. None of the individual mutants within a library of ~2500 mapped, mini-Tn5-lux transposon mutants were found to have decreased survival in water. Among the ~1400 transcriptional lux fusions, gene expression in water revealed that the majority of genes were repressed, but subsets of genes were induced at particular time points. In summary, these results indicate that P. aeruginosa is dormant in water and this adaptation involves a complex pattern of gene regulation and changes to the cell to promote long-term survival and antibiotic tolerance. The approach of P. aeruginosa incubated in water may be useful to study antibiotic tolerance and the mechanisms of dormancy and survival in nutrient limiting conditions.

microbiology↗