Biology subjects

Gallagher, J. E. G.

Publications and source records attributed to Gallagher, J. E. G..

5 recordsLinked to original sources

bioRxiv2020

Oxidative Stress Responses and Nutrient Starvation in MCHM Treated Saccharomyces cerevisiae

In 2014, the coal cleaning chemical 4-methylcyclohexane methanol (MCHM) spilled into the water supply for 300,000 West Virginians. Initial toxicology tests showed relatively mild results, but the underlying effects on cellular biology were underexplored. Treated wildtype yeast cells grew poorly, but there was only a small decrease in cell viability. Cell cycle analysis revealed an absence of cells in S phase within thirty minutes of treatment. Cells accumulated in G1 over a six-hour time course, indicating arrest instead of death. A genetic screen of the haploid knockout collection revealed 329 high confidence genes required for optimal growth in MCHM. These genes encode three major cell processes: mitochondrial gene expression/translation, the vacuolar ATPase, and aromatic amino acid biosynthesis. The transcriptome showed an upregulation of pleiotropic drug response genes and amino acid biosynthetic genes and downregulation in ribosome biosynthesis. Analysis of these datasets pointed to environmental stress response activation upon treatment. Overlap in datasets included the aromatic amino acid genes ARO1, ARO3, and four of the five TRP genes. This implicated nutrient deprivation as the signal for stress response. Excess supplementation of nutrients and amino acids did not improve growth on MCHM, so the source of nutrient deprivation signal is still unclear. Reactive oxygen species and DNA damage were directly detected with MCHM treatment, but timepoints showed these accumulated slower than cells arrested. We propose that wildtype cells arrest from nutrient deprivation and survive, accumulating oxidative damage through the implementation of robust environmental stress responses.

genomics↗

bioRxiv2019

Pick Your Poison: Benzalkonium Chloride and Copper Enable Nanocellulose Derivatives to Form Antimicrobial Properties Against a Spectrum of Microorganisms

TEMPO nanofibrillated cellulose (TNFC), nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and lignin were used as templates for the addition of two well-known antimicrobial substances, benzalkonium chloride (BZK) and copper. The resulting hybrid of cellulose and antimicrobial materials were analyzed for biocidal activity in three separate products. Assays of the nanocellulose-antimicrobials were assayed for viability against Escherichia coli in suspension, against Saccharomyces cerevisiae on PVA plastic, and against bacillus lincheniformis in paper additives. Instant biocidal activity was achieved with a minimal inhibitory concentration of 0.116 M BZK-TNFC hybrid suspension. BZK-Lignin and BZK-CMC suspensions demonstrated increased antimicrobial activity with longer exposure times during a 24-hour exposure which completely inhibited the bacteria. BZK was slowly released into the suspension, a desirable trait for long-term antimicrobial activity. PVA plastic incorporated with BZK/Cu-nanocellulose scaffolds created solid films that completely inhibited yeast growth by 270 seconds. Interestingly, lignin-BZK PVA films counteracted each other and showed no biocidal activity at all. The multiple combinations of nanocellulose and biocidal agents in the surface viability assay demonstrates the importance of synergy between both components in designing nanocellulose antimicrobials. TNFC-Cu was more suited to inhibit growth in paper than NFC-Cu as seen in a zone of inhibition assay. The most potent biocidal material in PVA was NFC-BZK. Here we show the diversity of the cellulosic derivatives and their impact on the antimicrobial additive. We employed a variety of assays to assess to biocidal of these nanoparticles against three species of bacteria and yeast relevant to food packaging and medical fields. From our study, there are many factors that play a role in the design of antimicrobial materials; cellulose derivative scaffold, antimicrobial agent, type of final material in which to be incorporated, target organism, and duration of application.

microbiology↗

bioRxiv2019

Thickening of the cell wall increases the resistance of S. cerevisiae to commercial formulations of glyphosate

The use of glyphosate-based herbicides is widespread and despite its extensive use, its effects are yet to be deciphered completely. The additives in commercial formulations of glyphosate, though labeled as inert when used individually, have adverse effects when used in combination with other additives and the active ingredient. As a species, Saccharomyces cerevisiae has a wide range of resistance to glyphosate-based herbicides. To investigate the underlying genetic differences between sensitive and resistant strains, global changes in gene expression were measured when yeast were exposed to a commercial formulation of glyphosate (CFG). Changes in gene expression involved in numerous pathways such as DNA replication, MAPK signaling, meiosis, and cell wall synthesis. Because so many diverse pathways were affected, these strains were then subjected to in-lab-evolutions (ILE) to select mutations that confer increased resistance. Common fragile sites were found to play a role in adaptation mechanisms used by cells to attain resistance with long-term exposure to CFG. The cell wall structure acts as a protective barrier in alleviating the stress caused by exposure to CFG. The thicker the cell wall, the more resistant the cell is against CFG. Hence, a detailed study of the changes occurring at the genome and transcriptome level is essential to better understand the possible effects of CFG on the cell as a whole.\n\nAuthor SummaryWe are exposed to various chemicals in the environment on a daily basis. Some of these chemicals are herbicides that come in direct contact with the food we consume. This makes the thorough investigation of these chemicals crucial. Some of the most commonly used herbicides around the world are glyphosate-based. Their mode of action effects a biosynthetic pathway that is absent in mammals and insects and so it is deemed safe for consumption. However, there are many additives to these herbicides that increase its effects. Thorough testing of these commercially available herbicides is essential to decipher all the potentially adverse effects that it could have on a cell. Saccharomyces cerevisiae has a wide range of genetic diversity, making it is suitable to test different chemicals and identify any harmful effects. In this study, we exposed yeast cells to some glyphosate-based herbicides available in the market, to understand what effects it could have on a cell. We found that the additives in the herbicides have an effect on the cell wall and the mode of entry of glyphosate into the cell.

genetics↗

bioRxiv2019

The polymorphic PolyQ tail protein of the Mediator Complex, Med15, regulates variable response to stress

The Mediator is a multi-protein complex composed of subunits called head, body, tail, and CDK that is conserved from yeast to humans and plays a central role in transcription. However, not all the components are required for basal transcription. Components of the tail are not essential but to varying degrees are required for growth in different stresses. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic, yet yeast can elicit a successful stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that a naturally occurring Med15 allele with polyQ (polyglutamine) expansion conferred MCHM sensitivity. Expansion in polyQ repeat can induce protein aggregation and in humans can cause neurodegenerative diseases. In yeast, the MCHM sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive and the homozygous null mutant was less sensitive than the hemizygous hybrids. This suggests that there is an incompatibility between Mediator components from genetic divergent yeast strains. Transcriptomics from yeast expressing the incompatible Med15 (longer polyQ repeats in the strain with fewer repeats) changed gene expression in diverse pathways. Med15 protein existed in multiple isoforms, mostly from likely post-translational modifications and different alleles have different patterns of isoforms. Stability of both alleles of Med15 was dependent on Ydj1, a J-type chaperone. The protein level of the incompatible Med15 allele was lower than the compatible allele and was turned over faster. Med15 is tethered to the rest of the Mediator complex via Med2 and 3. Deletion of either Med2 or Med3 changed the Med15 isoform patterns in a similar manner. Whereas deletion of Med5, a distal component of the Mediator tail, did not change the pattern. The med2 and med3 mutants were similarly sensitive to MCHM while med5 mutants were not. Differences in the phenotype of yeast carrying different Med15 alleles extend to other stresses. The incompatible allele of Med15 improved growth of yeast to chemicals that produce free radicals and the compatible allele of Med15 improved growth to reducing agents, caffeine, and hydroxyurea. Med15 directly interacts with Gcn4 and other TFs and in vitro form phase-separated droplets. This variation may reflect the positive and negative role that Med15 has in transcription. Genetic variation in transcriptional regulators can magnify differences in response to environmental changes, in contrast, genetic variation in a metabolic enzyme. These polymorphic control genes are master variators.

molecular biology↗

bioRxiv2019

MCHM acts as a hydrotrope, altering the balance of metals in yeast

While drugs and other industrial chemicals are routinely studied to assess risks, many widely-used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking-water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM and transcriptomics and mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein-interactions, while zinc-induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM.

biochemistry↗