Matching Items (31)
Description
When exposed to abiotic stresses, Escherichia coli responds by activating various stress-mitigating pathways. Initiation of stress responses partially relies on the RNA polymerase (RNAP) to transcribe genes necessary to tolerate various stresses, including nutritional deprivation and heat exposure. Consequently, RNAP mutations impacting transcription can have pleiotropic effects on the cell physiology and the ability to tolerate stress. Previously, while investigating antibiotic-resistant mutations arising in the absence of major antibiotic efflux pumps, four mutants containing alterations in the RNA polymerase beta subunit gene (rpoB) were isolated (Cho & Misra, 2021). Of the four mutants, one (RpoB58) was found to be thermotolerant, permitting homogenous, stable growth at temperatures up to 47°C, whereas the parental rpoB wildtype (RpoB-WT) was only able to do so up to 45°C. Additionally, RNA-Seq analysis indicated that the RpoB58 mutant had a ‘stringent’ profile that is normally seen under nutritionally deprived conditions. To better understand the regulatory pathways used to confer stress tolerance, this thesis sought to further characterize and investigate the intracellular mechanisms contributing to the thermotolerance conferred by the rpoB58 mutation. The RpoB58 mutant was found to be significantly more tolerant to both continuous heat stress (up to 47°C) and short-term heat (55°C) and ethanol (25%) exposure. Additionally, the RpoB58 mutant tolerated the absence or depletion of major heat shock chaperones DnaJ and DnaK that normally play key roles during temperature stresses by reducing protein misfolding. RNA-Seq data and reporter gene assays showed reduced expression of genes involved in protein synthesis. A similar reduction in the expression of protein synthesis genes was observed when cells were grown in growth-limiting minimal media. Interestingly, growth in minimal medium rescued the ΔdnaJ defect like the rpoB58 mutation. Based on these data, it was proposed that a decrease in protein synthesis, whether caused by rpoB58 or the growth medium, would result in less growth-inhibiting protein misfolding and aggregation, especially at higher growth temperatures where proteins are susceptible to denaturation and aggregation. As a result of these investigations, a possible mechanistic insight was provided as to how the rpoB58 mutation confers thermotolerance.
ContributorsYeh, Melody (Author) / Misra, Rajeev RM (Thesis advisor) / Wang, Xuan XW (Committee member) / Muralinath, Maneesha MM (Committee member) / Arizona State University (Publisher)
Created2021
Description
I studied the molecular mechanisms of ultraviolet radiation mitigation (UVR) in the terrestrial cyanobacterium Nostoc punctiforme ATCC 29133, which produces the indole-alkaloid sunscreen scytonemin and differentiates into motile filaments (hormogonia). While the early stages of scytonemin biosynthesis were known, the late stages were not. Gene deletion mutants were interrogated by metabolite analyses and confocal microscopy, demonstrating that the ebo gene cluster, was not only required for scytonemin biosynthesis, but was involved in the export of scytonemin monomers to the periplasm. Further, the product of gene scyE was also exported to the periplasm where it was responsible for terminal oxidative dimerization of the monomers. These results opened questions regarding the functional universality of the ebo cluster. To probe if it could play a similar role in organisms other than scytonemin producing cyanobacteria, I developed a bioinformatic pipeline (Functional Landscape And Neighbor Determining gEnomic Region Search; FLANDERS) and used it to scrutinize the neighboring regions of the ebo gene cluster in 90 different bacterial genomes for potentially informational features. Aside from the scytonemin operon and the edb cluster of Pseudomonas spp., responsible for nematode repellence, no known clusters were identified in genomic ebo neighbors, but many of the ebo adjacent regions were enriched in signal peptides for export, indicating a general functional connection between the ebo cluster and biosynthetic compartmentalization. Lastly, I investigated the regulatory span of the two-component regulator of the scytonemin operon (scyTCR) using RNAseq of scyTCR deletion mutants under UV induction. Surprisingly, the knockouts had decreased expression levels in many of the genes involved in hormogonia differentiation and in a putative multigene regulatory element, hcyA-D. This suggested that UV could be a cue for developmental motility responses in Nostoc, which I could confirm phenotypically. In fact, UV-A simultaneously elicited hormogonia differentiation and scytonemin production throughout a genetically homogenous population. I show through mutant analyses that the partner-switching mechanism coded for by hcyA-D acts as a hinge between the scytonemin and hormogonia based responses. Collectively, this dissertation contributes to the understanding of microbial adaptive responses to environmental stressors at the genetic and regulatory level, highlighting their phenomenological and mechanistic complexity.
ContributorsKlicki, Kevin (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Wilson, Melissa (Committee member) / Mukhopadhyay, Aindrila (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2021
Description
Persons with cystic fibrosis (CF) are highly susceptible to lung infections caused by the opportunistic pathogens Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). By age 20, ~16% of CF patients have co-infections with these two bacteria, and this number grows as the patients age1. PA-SA co-infections are associated with worsened clinical outcomes in CF patients, but the reasons are not well understood. One hypothesis is that SA influences the production of PA virulence factors and other chronic infection phenotypes. Previous work in our lab investigated the effects of SA on PA quorum-regulated phenotypes when they are grown as planktonic co-cultures. We are expanding on this result by testing whether SA can influence PA phenotypes without being in direct contact, and without being able to exchange soluble secreted factors. In this study, we hypothesized that SA produces volatile organic compounds (VOCs) that cause changes in PA phenotypes leading to a down-regulation of motility and protease production, and increased antibiotic resistance. To test this hypothesis, we exposed two laboratory strains of PA to the VOCs produced by pre-grown lawns of two strains of SA, and measured PA motility by conducting swarming, swimming, and twitching assays, measuring protease production, as well as antibiotic sensitivity. After exposing PA to a pre-grown lawn of SA, there was a significant difference in some phenotypes compared to controls. There were significant decreases in swarming motility, twitching motility, and protease production, and an increase in a bright green pigment (possibly siderophores) when PA was exposed to SA. The degree of phenotypic alterations was dependent on both the PA strain and the SA strain being tested. Exposure to SA VOCs also altered PA sensitivity to ciprofloxacin, though one strain caused an increase in susceptibility while the other SA strain caused an increase in resistance. These data demonstrate that SA VOCs can influence PA phenotypes in vitro, which may have relevance for CF patients who are co-infected with these two bacteria.
ContributorsLopez, Brianna Marie (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.
ContributorsYeh, Melody (Author) / Misra, Rajeev (Thesis director) / Wang, Xuan (Committee member) / Kelly, Keilen (Committee member) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Following the journey through the sewerage system, wastewater is subject to a series of purification procedures, prior to water reuse and disposal of the resultant sewage sludge. Biosolids, also known as treated sewage sludge, deemed fit for application on land, is a nutrient-rich, semisolid byproduct of biological wastewater treatment. Technological progression in metagenomics has allowed for large-scale analysis of complex viral communities in a number of samples, including wastewater. Members of the Microviridae family are non-enveloped, ssDNA bacteriophages, and are known to infect enterobacteria. Members of the Genomoviridae family similarly are non-enveloped, ssDNA viruses, but are presumed to infect fungi rather than eubacteria. As these two families of viruses are not relatively documented and their diversity poorly classified, this study aimed to analyze the presence of genomoviruses and the diversity of microviruses in nine samples representative of wastewater in Arizona and other regions of the United States. Using a metagenomic approach, the nucleic acids of genomoviruses and microviruses were isolated, assembled into complete genomes, and characterized through visual analysis: a heat chart showing percent coverage for genomoviruses and a circular phylogenetic tree showing diversity of microviruses. The heat map results for the genomoviruses showed a large presence of 99 novel sequences in all nine wastewater samples. Additionally, the 535 novel microviruses displayed great diversity in the cladogram, both in terms of sub-family and isolation source. Further research should be conducted in order to classify the taxonomy of microviruses and the diversity of genomoviruses. Finally, this study suggests future exploration of the viral host, prior to entering the wastewater system.
ContributorsSchreck, Joshua Reuben (Author) / Varsani, Arvind (Thesis director) / Rolf, Halden (Committee member) / Misra, Rajeev (Committee member) / School of Film, Dance and Theatre (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Each year, more and more multi-drug resistant bacterial strains emerge, thus complicating treatment and increasing the average stay in the intensive care unit. As antibiotics are being rendered inefficient, there is a need to look into ways of weakening the internal state of bacterial cells to make them more susceptible to antibiotics. For this, we first need to understand what methods bacteria employ to fight against antibiotics. In this work, we have reviewed how bacteria respond to antibiotics. There is a similarity in response to antibiotic exposure and starvation (stringent stress) which changes the metabolic state. We have delineated what metabolism changes take place and how they are associated with oxidative stress. For example, there is a common change in NADH concentration that is tied to both metabolism and oxidative stress. Finally, we have compared the findings in literature with our research on an antibiotic-resistant RNA polymerase mutant that alters the gene expression profile in the general areas of metabolism and oxidative stress. Based on this thesis, we have suggested a couple of strategies to make antibiotics more efficient; however, as antibiotic-mediated killing is very complex, researchers need to delve deeper to understand and manipulate the full cellular response.
ContributorsPredtechenskaya, Maria (Author) / Misra, Rajeev (Thesis director) / Varman, Arul Mozhy (Committee member) / Mhatre, Apurv (Committee member) / Computer Science and Engineering Program (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
When limited for iron, Escherichia coli secretes a siderophore, enterobactin, to solubilize and intake extracellular Fe3+ by a TonB-dependent high-affinity pathway. Consequently, E. coli tonB mutants grow poorly on a medium limited for iron. Upon longer incubation, however, faster growing colonies emerge and overcome this growth defect. The work presented in this paper reports and characterizes these faster growing colonies (revertants) in an attempt to dissect the mechanism by which they overcome the TonB deficiency. Genomic analysis revealed mutations in yejM, a putative inner-to-outer membrane cardiolipin transporter, which are responsible for the faster growth phenotype in a tonB mutant background. Further characterization of the revertants revealed that they display hypersensitivity to vancomycin, a large antibiotic that is normally precluded from entering E. coli cells, and leaked periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild type copy of yejM on a plasmid, suggesting that yejM mutations are solely responsible for the observed phenotypes. In the absence of wild type tonB, however, the deletion of all known of cardiolipin synthase genes (clsABC) did not produce the phenotype similar to mutations in the yejM gene, suggesting the absence of cardiolipin from the outer membrane per se is not responsible for the increased outer membrane permeability. These data show that a defect in lipid biogenesis and transport can compromise outer membrane permeability barrier to allow siderophore intake and that YejM may have additional roles other than transporting cardiolipin.
ContributorsQiu, Nan (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Yu, Julian (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The FOF1 ATP synthase is responsible for generating the majority of adenosine triphosphate (ATP) in almost all organisms on Earth. A major unresolved question is the mechanism of the FO motor that converts the transmembrane flow of protons into rotation that drives ATP synthesis. Using single-molecule gold nanorod experiments, rotation of individual FOF1 were observed to measure transient dwells (TDs). TDs occur when the FO momentarily halts the ATP hydrolysis rotation by the F1-ATPase. The work presented here showed increasing TDs with decreasing pH, with calculated pKa values of 5.6 and 7.5 for wild-type (WT) Escherichia coli (E. coli) subunit-a proton input and output half-channels, respectively. This is consistent with the conclusion that the periplasmic proton half-channel is more easily protonated than the cytoplasmic half-channel. Mutation in one proton half-channel affected the pKa values of both half-channels, suggesting that protons flow through the FO motor via the Grotthuss mechanism. The data revealed that 36° stepping of the E. coli FO subunit-c ring during ATP synthesis consists of an 11° step caused by proton translocations between subunit-a and the c-ring, and a 25° step caused by the electrostatic interaction between the unprotonated c-subunit and the aR210 residue in subunit-a. The occurrence of TDs fit to the sum of three Gaussian curves, which suggested that the asymmetry between the FO and F1 motors play a role in the mechanism behind the FOF1 rotation. Replacing the inner (N-terminal) helix of E. coli c10-ring with sequences derived from c8 to c17-ring sequences showed expression and full assembly of FOF1. Decrease in anticipated c-ring size resulted in increased ATP synthesis activity, while increase in c-ring size resulted in decreased ATP synthesis activity, loss of Δψ-dependence to synthesize ATP, decreased ATP hydrolysis activity, and decreased ACMA quenching activity. Low levels of ATP synthesis by the c12 and c15-ring chimeras are consistent with the role of the asymmetry between the FO and F1 motors that affects ATP synthesis rotation. Lack of a major trend in succinate-dependent growth rates of the chimeric E. coli suggest cellular mechanisms that compensates for the c-ring modification.
ContributorsYanagisawa, Seiga (Author) / Frasch, Wayne D (Thesis advisor) / Misra, Rajeev (Committee member) / Redding, Kevin (Committee member) / Singharoy, Abhishek (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
ContributorsDeb, Arpan (Author) / Johnson, William (Author) / Kline, Alexander (Author) / Scott, Boston (Author) / Meador, Lydia (Author) / Srinivas, Dustin (Author) / Martin Garcia, Jose Manuel (Author) / Dorner, Katerina (Author) / Borges, Chad (Author) / Misra, Rajeev (Author) / Hogue, Brenda (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / School of Molecular Sciences (Contributor) / Applied Structural Discovery (Contributor) / Personalized Diagnostics (Contributor)
Created2017-02-22
Description
Natural antibacterial clays, when hydrated and applied topically, kill human pathogens including antibiotic resistant strains proliferating worldwide. Only certain clays are bactericidal; those containing soluble reduced metals and expandable clay minerals that absorb cations, providing a capacity for extended metal release and production of toxic hydroxyl radicals. Here we show the critical antibacterial components are soluble Fe2+ and Al3+ that synergistically attack multiple cellular systems in pathogens normally growth-limited by Fe supply. This geochemical process is more effective than metal solutions alone and provides an alternative antibacterial strategy to traditional antibiotics. Advanced bioimaging methods and genetic show that Al3+ misfolds cell membrane proteins, while Fe2+ evokes membrane oxidation and enters the cytoplasm inflicting hydroxyl radical attack on intracellular proteins and DNA. The lethal reaction precipitates Fe3+-oxides as biomolecular damage proceeds. Discovery of this bactericidal mechanism demonstrated by natural clays should guide designs of new mineral-based antibacterial agents.
ContributorsMorrison, Keith D. (Author) / Misra, Rajeev (Author) / Williams, Lynda (Author) / College of Liberal Arts and Sciences (Contributor)
Created2016-01-08