Matching Items (39)
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- All Subjects: Microbiology
- Creators: School of Life Sciences
- Creators: Misra, Rajeev
- Resource Type: Text
- Status: Published
Description
Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold.
ContributorsWeeks, Jon William (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Shi, Yixin (Committee member) / Clark-Curtiss, Josephine (Committee member) / Arizona State University (Publisher)
Created2012
Description
The discovery and development of novel antibacterial agents is essential to address the rising health concern over antibiotic resistant bacteria. This research investigated the antibacterial activity of a natural clay deposit near Crater Lake, Oregon, that is effective at killing antibiotic resistant human pathogens. The primary rock types in the deposit are andesitic pyroclastic materials, which have been hydrothermally altered into argillic clay zones. High-sulfidation (acidic) alteration produced clay zones with elevated pyrite (18%), illite-smectite (I-S) (70% illite), elemental sulfur, kaolinite and carbonates. Low-sulfidation alteration at neutral pH generated clay zones with lower pyrite concentrations pyrite (4-6%), the mixed-layered I-S clay rectorite (R1, I-S) and quartz.
Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates.
The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack pathogens while generating hydrogen peroxide (H2O¬2). Results show that dissolved Fe2+ and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+-oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays.
Antibacterial susceptibility testing reveals that hydrated clays containing pyrite and I-S are effective at killing (100%) of the model pathogens tested (E. coli and S. epidermidis) when pH (< 4.2) and Eh (> 450 mV) promote pyrite oxidation and mineral dissolution, releasing > 1 mM concentrations of Fe2+, Fe3+ and Al3+. However, certain oxidized clay zones containing no pyrite still inhibited bacterial growth. These clays buffered solutions to low pH (< 4.7) and oxidizing Eh (> 400 mV) conditions, releasing lower amounts (< 1 mM) of Fe and Al. The presence of carbonate in the clays eliminated antibacterial activity due to increases in pH, which lower pyrite oxidation and mineral dissolution rates.
The antibacterial mechanism of these natural clays was explored using metal toxicity and genetic assays, along with advanced bioimaging techniques. Antibacterial clays provide a continuous reservoir of Fe2+, Fe3+ and Al3+ that synergistically attack pathogens while generating hydrogen peroxide (H2O¬2). Results show that dissolved Fe2+ and Al3+ are adsorbed to bacterial envelopes, causing protein misfolding and oxidation in the outer membrane. Only Fe2+ is taken up by the cells, generating oxidative stress that damages DNA and proteins. Excess Fe2+ oxidizes inside the cell and precipitates Fe3+-oxides, marking the sites of hydroxyl radical (•OH) generation. Recognition of this novel geochemical antibacterial process should inform designs of new mineral based antibacterial agents and could provide a new economic industry for such clays.
ContributorsMorrison, Keith D (Author) / Williams, Lynda B (Thesis advisor) / Williams, Stanley N (Thesis advisor) / Misra, Rajeev (Committee member) / Shock, Everett (Committee member) / Anbar, Ariel (Committee member) / Arizona State University (Publisher)
Created2015
Description
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.
ContributorsPetrovich Flynn, Chandler James (Author) / Misra, Rajeev (Thesis director) / Bean, Heather (Committee member) / Perkins, Kim (Committee member) / Mechanical and Aerospace Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Space microbiology, or the study of microorganisms in space, has significant applications for both human spaceflight and Earth-based medicine. This thesis traces the evolution of the field of space microbiology since its creation in 1935. Beginning with simple studies to determine if terrestrial life could survive spaceflight, the field of space microbiology has grown to encompass a substantial body of work that is now recognized as an essential component of NASA' research endeavors. Part one provides an overview of the early period of space microbiology, from high-altitude balloon and rocket studies to work conducted during the Apollo program. Part two summarizes the current state of the field, with a specific focus on the revolutionary contributions made by the Nickerson lab at the Biodesign Institute at ASU using the NASA-designed Rotating Wall Vessel (RWV) Bioreactor. Finally, part three highlights the research I've conducted in the Nickerson lab, as well as continuing studies within the field of space microbiology.
ContributorsMcCarthy, Breanne E. (Author) / Lynch, John (Thesis director) / Foy, Joseph (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Cystic Fibrosis (CF) is a genetic disorder that disrupts the hydration of mucous of the lungs, which promotes opportunistic bacterial infections that begin in the affected person’s childhood, and persist into adulthood. One of the bacteria that infect the CF lung is Pseudomonas aeruginosa. This gram-negative bacterium is acquired from the environment of the CF lung, changing the expression of phenotypes over the course of the infection. As P. aeruginosa infections become chronic, some phenotype changes are known to be linked with negative patient outcomes. An important exoproduct phenotype is rhamnolipid production, which is a glycolipid that P. aeruginosa produces as a surfactant for surface-mediated travel. Over time, the expression of this phenotype decreases in expression in the CF lung.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
ContributorsKiermayr, Jonathan Patrick (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Many bacteria actively import environmental DNA and incorporate it into their genomes. This behavior, referred to as transformation, has been described in many species from diverse taxonomic backgrounds. Transformation is expected to carry some selective advantages similar to those postulated for meiotic sex in eukaryotes. However, the accumulation of loss-of-function alleles at transformation loci and an increased mutational load from recombining with DNA from dead cells create additional costs to transformation. These costs have been shown to outweigh many of the benefits of recombination under a variety of likely parameters. We investigate an additional proposed benefit of sexual recombination, the Red Queen hypothesis, as it relates to bacterial transformation. Here we describe a computational model showing that host-pathogen coevolution may provide a large selective benefit to transformation and allow transforming cells to invade an environment dominated by otherwise equal non-transformers. Furthermore, we observe that host-pathogen dynamics cause the selection pressure on transformation to vary extensively in time, explaining the tight regulation and wide variety of rates observed in naturally competent bacteria. Host-pathogen dynamics may explain the evolution and maintenance of natural competence despite its associated costs.
ContributorsPalmer, Nathan David (Author) / Cartwright, Reed (Thesis director) / Wang, Xuan (Committee member) / Sievert, Chris (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Clean water for drinking, food preparation, and bathing is essential for astronaut health and safety during long duration habitation of the International Space Station (ISS), including future missions to Mars. Despite stringent water treatment and recycling efforts on the ISS, it is impossible to completely prevent microbial contamination of onboard water supplies. In this work, we used a spaceflight analogue culture system to better understand how the microgravity environment can influence the pathogenesis-related characteristics of Burkholderia cepacia complex (Bcc), an opportunistic pathogen previously recovered from the ISS water system. The results of the present study suggest that there may be important differences in how this pathogen can respond and adapt to spaceflight and other low fluid shear environments encountered during their natural life cycles. Future studies are aimed at understanding the underlying mechanisms responsible for these phenotypes.
ContributorsKang, Bianca Younseon (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, Mark (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Vaccinia virus (VV) is a prototype virus of the Orthopox viruses. The large dsDNA virus composed of 200kbp genome contains approximately 200 genes and replicates entirely in the cytosol. Since its use as a live vaccine against smallpox that leads to the successful eradication of smallpox, Vaccinia has been intensely studied as a vaccine vector since the large genome allows for the insertion of multiple genes. It is also studied as a molecular tool for gene therapy and gene functional study. Despite its success as a live vaccine, the vaccination causes some mild to serious bur rare adverse events in vaccinees such as generalized Vaccinia and encepharitis. Therefore, identification of virulence genes and removal of these genes to create a safer vaccine remain an important tasks. In this study, the author seeks to elucidate the possible relationship between immune evading proteins E3 and B19. VV did not allow double deletions of E3 and B19, indicating the existence of a relationship between the two genes.
ContributorsBarclay, Shizuka (Author) / Jacobs, Bertram (Thesis director) / Ugarova, Tatiana (Committee member) / Kibler, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.
ContributorsDufault, Matthew Elijah (Co-author, Co-author) / Wang, Xuan (Thesis director) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05