Matching Items (46)
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
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
Teleosts have the most primitive adaptive immune system. However, in terms of functionality the teleost immune system is similar to birds and mammals. On the other hand, enteric bacterial pathogens of mammals and birds present conserved regulatory mechanisms that control virulence factors. In this context, deletion of conserved genes that control virulence factors have been successfully used as measure to construct live attenuated bacterial vaccines for mammals and birds. Here, I hypothesize that evolutionary conserved genes, which control virulence factors or are essential for bacterial physiology in Enterobacteriaceae, could be used as universal tools to design live attenuated recombinant bacterial vaccines from fish to mammals. The evolutionary conserved genes that control virulence factors, crp and fur, and the essential gene for the synthesis of the cell wall, asd, were studied in Edwardsiella ictaluri to develop a live recombinant vaccine for fish host. The genus Edwardsiella is one of the most ancient represent of the Enterobacteriaceae family. E. ictaluri, a host restricted pathogen of catfish (Ictalurus punctatus), is the causative agent of the enteric septicemia and one of the most important pathogens of this fish aquaculture. Although, crp and fur control different virulence factors in Edwardsiella, in comparison to other enterics, individual deletion of these genes triggered protective immune response at the systemic and mucosal level of the fish. Deletion of asdA gene allowed the creation of a balanced-lethal system to syntheses heterologous antigens. I concluded that crp, fur and asd could be universally used to develop live attenuate recombinant Enterobacteriaceae base vaccines for different hosts.
ContributorsSantander Morales, Javier Alonso (Author) / Curtiss, Roy Iii (Thesis advisor) / Chandler, Douglas (Committee member) / Chang, Yung (Committee member) / Shi, Yixin (Committee member) / Arizona State University (Publisher)
Created2012
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 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
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
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
Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain.
ContributorsBellefleur, Matthew Paul Allen (Author) / Curtiss, III, Roy (Thesis advisor) / Nielsen, David R (Committee member) / Wang, Xuan (Committee member) / Rittmann, Bruce E. (Committee member) / Arizona State University (Publisher)
Created2018
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
Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker methods can be used to remove and reuse marker genes as desired, resulting in markerless engineered strains and allowing for theoretically unlimited rounds of genetic modifications. The development of suitable counter-selection markers is vital for the development of model organisms such as cyanobacteria as biotechnological platforms.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
ContributorsNewell, Phoebe Quynh (Co-author) / Newell, Phoebe (Co-author) / Vermaas, Willem (Thesis director) / Wang, Xuan (Committee member) / Li, Shuqin (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05