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- Genre: Masters Thesis
Description
Microbial mat communities that inhabit hot springs in Yellowstone National Park have been studied for their biodiversity, energetics and evolutionary history, yet little is know about how these communities cope with nutrient limitation. In the present study the changes in assimilatory gene expression levels for nitrogen (nrgA), phosphorus (phoA), and iron (yusV) were measured under various nutrient enrichment experiments. While results for nrgA and phoA were inconclusive, results for yusV showed an increase in expression with the addition of N and Fe. This is the first data that shows the impact of nutrients on siderophore uptake regulation in hot spring microbes.
ContributorsThorne, Michele (Author) / Elser, James J (Thesis advisor) / Touchman, Jeffrey (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2012
Description
Pathogenic Gram-negative bacteria employ a variety of molecular mechanisms to combat host defenses. Two-component regulatory systems (TCR systems) are the most ubiquitous signal transduction systems which regulate many genes required for virulence and survival of bacteria. In this study, I analyzed different TCR systems in two clinically-relevant Gram-negative bacteria, i.e., oral pathogen Porphyromonas gingivalis and enterobacterial Escherichia coli. P. gingivalis is a major causative agent of periodontal disease as well as systemic illnesses, like cardiovascular disease. A microarray study found that the putative PorY-PorX TCR system controls the secretion and maturation of virulence factors, as well as loci involved in the PorSS secretion system, which secretes proteinases, i.e., gingipains, responsible for periodontal disease. Proteomic analysis (SILAC) was used to improve the microarray data, reverse-transcription PCR to verify the proteomic data, and primer extension assay to determine the promoter regions of specific PorX regulated loci. I was able to characterize multiple genetic loci regulated by this TCR system, many of which play an essential role in hemagglutination and host-cell adhesion, and likely contribute to virulence in this bacterium. Enteric Gram-negative bacteria must withstand many host defenses such as digestive enzymes, low pH, and antimicrobial peptides (AMPs). The CpxR-CpxA TCR system of E. coli has been extensively characterized and shown to be required for protection against AMPs. Most recently, this TCR system has been shown to up-regulate the rfe-rff operon which encodes genes involved in the production of enterobacterial common antigen (ECA), and confers protection against a variety of AMPs. In this study, I utilized primer extension and DNase I footprinting to determine how CpxR regulates the ECA operon. My findings suggest that CpxR modulates transcription by directly binding to the rfe promoter. Multiple genetic and biochemical approaches were used to demonstrate that specific TCR systems contribute to regulation of virulence factors and resistance to host defenses in P. gingivalis and E. coli, respectively. Understanding these genetic circuits provides insight into strategies for pathogenesis and resistance to host defenses in Gram negative bacterial pathogens. Finally, these data provide compelling potential molecular targets for therapeutics to treat P. gingivalis and E. coli infections.
ContributorsLeonetti, Cori (Author) / Shi, Yixin (Thesis advisor) / Stout, Valerie (Committee member) / Nickerson, Cheryl (Committee member) / Sandrin, Todd (Committee member) / Arizona State University (Publisher)
Created2013
Description
The viscous lung mucus of cystic fibrosis (CF) patients is characterized by oxygen gradients, which creates a unique niche for bacterial growth. Pseudomonas aeruginosa and Staphylococcus aureus, two predominant microorganisms chronically infecting the airways of CF patients, typically localize in hypoxic regions of the mucus. While interspecies interactions between P. aeruginosa and S. aureus have been reported, little is known about the role of low oxygen in regulating these interactions. Studying interspecies interactions in CF lung disease is important as evidence suggests that microbial community composition governs disease progression. In this study, P. aeruginosa lab strain PAO1 and two primary clinical isolates from hypoxic tissues were cultured alone, or in combination, with methicillin resistant S. aureus (MRSA) strain N315 under hypoxic or normoxic conditions. Herein, it is shown for the first time that low oxygen conditions relevant to the CF lung affect the competitive behavior between P. aeruginosa and S. aureus. Specifically, S. aureus was able to better survive competition in hypoxic versus normoxic conditions. Competition data from different oxygen concentrations were consistent using PAO1 and clinical isolates even though differences in the level of competition were observed. PAO1 strains carrying mutations in virulence factors known to contribute to S. aureus competition (pyocyanin/phzS, elastase/lasA and lasI quorum sensing/lasI) were used to determine which genes play a role in the differential growth inhibition. The lasA and lasI mutants competed less effectively with S. aureus regardless of the oxygen level present in the culture compared to the isogenic wild type strain. These results are consistent with previous findings that elastase and lasI quorum sensing play a role in competitive behavior of P. aeruginosa and S. aureus. Interestingly, the phzS mutant competed less effectively in hypoxic conditions suggesting that pyocyanin may be important in microaerophilic conditions. This study demonstrates that oxygen plays a role in competition between P. aeruginosa and S. aureus and contributes to understanding CF environmental factors that may regulate microbial community dynamics important for disease progression with potential for development of therapeutic avenues.
ContributorsLedesma Barrera, Maria Alexandra (Author) / Nickerson, Cheryl A. (Thesis advisor) / Reyes del Valle, Jorge (Committee member) / Clark-Curtiss, Josephine (Committee member) / Stout, Valerie (Committee member) / Ott, C M (Committee member) / Arizona State University (Publisher)
Created2014
Description
With the aid of metabolic pathways engineering, microbes are finding increased use as biocatalysts to convert renewable biomass resources into fine chemicals, pharmaceuticals and other valuable compounds. These alternative, bio-based production routes offer distinct advantages over traditional synthesis methods, including lower energy requirements, rendering them as more "green" and "eco-friendly". Escherichia coli has recently been engineered to produce the aromatic chemicals (S)-styrene oxide and phenol directly from renewable glucose. Several factors, however, limit the viability of this approach, including low titers caused by product inhibition and/or low metabolic flux through the engineered pathways. This thesis focuses on addressing these concerns using magnetic mesoporous carbon powders as adsorbents for continuous, in-situ product removal as a means to alleviate such limitations. Using process engineering as a means to troubleshoot metabolic pathways by continuously removing products, increased yields are achieved from both pathways. By performing case studies in product toxicity and reaction equilibrium it was concluded that each step of a metabolic pathway can be optimized by the strategic use of in-situ adsorption as a process engineering tool.
ContributorsVasudevan, Anirudh (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2014
Description
Rhodoferax antarcticus strain ANT.BR, a purple nonsulfur bacterium isolated from a microbial mat in Ross Island, Antarctica, is the first described anoxygenic phototrophic bacterium that is adapted to cold habitats and is the first beta-proteobacterium to undergo complete genome sequencing. R. antarcticus has unique absorption spectra and there are no obvious intracytoplasmic membranes in cells grown phototrophically, even under low light intensity. Analysis of the finished genome sequence reveals a single chromosome (3,809,266 bp) and a large plasmid (198,615 bp) that together harbor 4,262 putative genes. The genome contains two types of Rubiscos, Form IAq and Form II, which are known to exhibit quite different kinetic properties in other bacteria. The presence of multiple Rubisco forms could give R. antarcticus high metabolic flexibility in diverse environments. Annotation of the complete genome sequence along with previous experimental results predict the presence of structural genes for three types of light-harvesting (LH) complexes, LH I (B875), LH II (B800/850), and LH III (B800/820). There is evidence that expression of genes for the LH II complex might be inhibited when R. antarcticus is under low temperature and/or low light intensity. These interesting condition-dependent light-harvesting apparatuses and the control of their expression are very valuable for the further understanding of photosynthesis in cold environments. Finally, R. antarcticus exhibits a highly motile lifestyle. The genome content and organization of all putative polar flagella genes are characterized and discussed.
ContributorsZhao, Tingting, M.S (Author) / Touchman, Jeffrey (Thesis advisor) / Rosenberg, Michael (Committee member) / Redding, Kevin (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.
ContributorsTao, Zenan (Author) / Shi, Yixin (Thesis advisor) / Wang, Xuan (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2018
Description
Peatlands represent 3% of the earth’s surface but have been estimated to contain up to 30% of all terrestrial soil organic carbon and release an estimated 40% of global atmospheric CH4 emissions. Contributors to the production of CH4 are methanogenic Archaea through a coupled metabolic dependency of end products released by heterotrophic bacteria within the soil in the absence of O2. To better understand how neighboring bacterial communities can influence methanogenesis, the isolation and physiological characterization of two novel isolates, one Methanoarchaeal isolate and one Acidobacterium isolate identified as QU12MR and R28S, respectively, were targeted in this present study. Co-culture growth in varying temperatures of the QU12MR isolate paired with an isolated Clostridium species labeled R32Q and the R28S isolate were also investigated for possible influences in CH4 production. Phylogenetic analysis of strain QU12MR was observed as a member of genus Methanobacterium sharing 98% identity similar to M. arcticum strain M2 and 99% identity similar to M. uliginosum strain P2St. Phylogenetic analysis of strain R28S was associated with genus Acidicapsa from the phylum Acidobacteria, sharing 97% identity to A. acidisoli strain SK-11 and 96% identity similarity to Occallatibacter savannae strain A2-1c. Bacterial co-culture growth and archaeal CH4 production was present in the five temperature ranges tested. However, bacterial growth and archaeal CH4 production was less than what was observed in pure culture analysis after 21 days of incubation.
ContributorsRamirez, Zeni Elizia (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Roberson, Robert (Thesis advisor) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2018
Description
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative enteric pathogen that causes self-limiting gastroenteritis in healthy individuals and can cause systemic infections in those who are immunocompromised. During its natural lifecycle, S. Typhimurium encounters a wide variety of stresses it must sense and respond to in a dynamic and coordinated fashion to induce resistance and ensure survival. Salmonella is subjected to a series of stresses that include temperature shifts, pH variability, detergent-like bile salts, oxidative environments and changes in fluid shear levels. Previously, our lab showed that cultures of S. Typhimurium grown under physiological low fluid shear (LFS) conditions similar to those encountered in the intestinal tract during infection uniquely regulates the virulence, gene expression and pathogenesis-related stress responses of this pathogen during log phase. Interestingly, the log phase Salmonella mechanosensitive responses to LFS were independent of the master stress response sigma factor, RpoS, departing from our conventional understanding of RpoS regulation. Since RpoS is a growth phase dependent regulator with increased stability in stationary phase, the current study investigated the role of RpoS in mediating pathogenesis-related stress responses in stationary phase S. Typhimurium grown under LFS and control conditions. Specifically, stationary phase responses to acid, thermal, bile and oxidative stress were assayed. To our knowledge the results from the current study demonstrate the first report that the mechanical force of LFS globally alters the S. Typhimurium χ3339 stationary phase stress response independently of RpoS to acid and bile stressors but dependently on RpoS to oxidative and thermal stress. This indicates that fluid shear-dependent differences in acid and bile stress responses are regulated by alternative pathway(s) in S. Typhimurium, were the oxidative and thermal stress responses are regulated through RpoS in LFS conditions. Results from this study further highlight how bacterial mechanosensation may be important in promoting niche recognition and adaptation in the mammalian host during infection, and may lead to characterization of previously unidentified pathogenesis strategies.
ContributorsCrenshaw, Keith (Author) / Nickerson, Cheryl A. (Thesis advisor) / Barrila, Jennifer (Thesis advisor) / Ott, C. (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2016
Description
Synthetic biology and metabolic engineering has aided the production of chemicals using renewable resources, thus offering a solution to our dependence on the dwindling petroleum resources. While a major portion of petroleum resources go towards production of fuels, a significant fraction also goes towards production of specialty chemicals. There has been a growing interest in recent years in commercializing bio-based production of such high value compounds. In this thesis the biosynthesis of aromatic esters has been explored, which have typical application as flavor and fragrance additive to food, drinks and cosmetics. Recent progress in pathway engineering has led to the construction of several aromatic alcohol producing pathways, the likes of which can be utilized to engineer aromatic ester biosynthesis by addition of a suitable enzyme from the acyltransferase class. Enzyme selection and screening done in this work has identified chloramphenicol O-acetyltransferase enzyme(CAT) as a potential candidate to complete the biosynthetic pathways for each of 2-phenethyl acetate, benzyl acetate, phenyl acetate and acetyl salicylate. In the end, E. coli strains capable of producing up to 60 mg/L 2-phenethyl acetate directly from glucose were successfully constructed by co-expressing CAT in a previously engineered 2-phenylethanol producing host.
ContributorsMadathil Soman Pillai, Karthika (Author) / Nielsen, David (Thesis advisor) / Wang, Xuan (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2016
Description
Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
ContributorsBlake, Mellecha (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016