Department: Microbiology ![[clear]](close-x.png "Remove this limiter")
30 matches in the database.
These are records: 1 - 30.
1.
Brock, Jay Edward.
Interactions between mRNA and Escherichia coli ribosomes that contribute to the formation of translation initiation complexes.
Degree: PhD, Microbiology, 2006, Miami University
► Initiation is the rate-limiting step of translation involving the assembly of stable…
(more)
▼ Initiation is the rate-limiting step of translation involving the assembly of stable ternary complexes (mRNA-tRNA-ribosomes) on the translation initiation region (TIR) of the mRNA. Evidence for many non-Shine-Dalgarno (SD)-led genes, including genes encoding leaderless mRNA, provides motive for studying mRNA features within the coding sequence that contribute to the formation of these complexes. Binding and expression assays reveal here that adenines downstream from a mRNA’s start codon contribute significantly to the rate and amount of ternary complex formed as well as the level of in vivo expression, even in the presence of a canonical SD sequence. Leaderless mRNA, which lack signals upstream of the start codon, requires a 5’-terminal triphosphate for efficient translation. A mutagenesis approach was developed to search for ribosome sites involved in recognizing a leaderless mRNA’s 5’-terminal triphosphate. Binding assays demonstrated tRNA-dependent ribosome binding to leaderless mRNA; however, site-specific crosslinks between a leaderless mRNA’s start codon, containing a 4-thiouridine at +2 position, and components of the ribosome formed independent of initiator tRNA. Several leaderless mRNA-r-protein contacts were dependent on the presence of a 5’-terminal AUG, suggesting that specific r-proteins might facilitate recognition of a leaderless mRNA’s start codon. In addition, initiation factors did not influence the initial interactions between leaderless mRNA and ribosomes identified in site-specific crosslinking assays. This work provides the first evidence that recognition and ribosomal positioning of a leaderless mRNA’s 5’-AUG is a function of the ribosome.
Advisors/Committee Members: Janssen, Gary R.
Subjects: Biology, Microbiology
Keywords: Translation Initiation; Ribosomes; Leaderless mRNA
More Like This
2.
Clark, Jason P.
Investigating Cellular DNA Damage Responses Induced During Adenovirus Early Region 4 Mutant Infection and Their Impact on Viral DNA Replication.
Degree: MS, Microbiology, 2010, Miami University
► DNA double-strand breaks elicit the DNA damage response (DDR) in eukaryotic cells.…
(more)
▼ DNA double-strand breaks elicit the DNA damage response (DDR) in eukaryotic cells. This response involves the Mre11/Rad50/NBS1 (MRN) complex and activation of DNA damage kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) that phosphorylate proteins involved in cell cycle arrest, DNA repair, and apoptosis. Adenovirus (Ad) is a linear, double-stranded DNA virus, which presents the cell with a genome that resembles a DNA double-strand break. Wild type Ad5 (Ad5) produces proteins that prevent a DDR while Ad early region 4 (E4) mutants lack this ability and therefore induce DDRs in infected cells. This work is aimed at studying the DDR during infection with a particular E4 mutant (H5dl1014) (1014), and analyzing the severe DNA replication defect that this mutant exhibits. From this work I have demonstrated that Ad E4 mutant 1014 infection induces a DDR and that 1014 DNA replication may be regulated through the MRN complex.
Advisors/Committee Members: Bridge, Eileen.
Subjects: Biology; Cellular biology; Microbiology; Molecular biology; Virology
Keywords: DNA damage response; ATM; MDC1; E4 mutant; adenovirus
More Like This
3.
Clark, Melinda Erin.
Physiological Analysis of Desulfovibrio vulgaris Hildenborough Under Conditions Relevant to the Subsurface Environment: Carbon and Energy Limitation and Biofilm Formation.
Degree: PhD, Microbiology, 2008, Miami University
► Desulfovibrio vulgaris Hildenborough is a member of the sulfate-reducing bacteria (SRB) and…
(more)
▼ Desulfovibrio vulgaris Hildenborough is a member of the sulfate-reducing bacteria (SRB) and is considered a model microorganism. SRB are beneficial because of their potential for bioremediation applications and detrimental due to their ability to corrode metals. Therefore, not only is it important to understand the metabolic capacity of SRB but also understand the mechanisms of survival and the physiology of SRB as they thrive in the subsurface. In order to elucidate the metabolic capacity and survival of SRB, one organism was analyzed under conditions relevant to the subsurface environment. In this study, D. vulgaris was analyzed under two environmentally relevant conditions, carbon and energy limitation and biofilm growth. D. vulgaris was studied throughout growth and into stasis by transcriptomic analysis which provided the responses this organism has during electron and carbon depletion. This resulted in increased expression of iron acquisition systems, phage genes, carbon starvation genes, and transcripts involved with cell division and growth. The expression of genes involved with translation, ATP synthesis, and substrate transport was reduced. Since fluxes in carbon and energy sources are common within the subsurface, responses such as these should be considered when examining ways to stimulate or hinder D. vulgaris growth. Biofilms developed by D. vulgaris were analyzed in terms of content as well as physiology. Biofilms formed upon glass slides within a defined medium by D. vulgaris were dependent upon proteins for biofilm formation and stability and did not have an extensive exopolysaccharide matrix. Flagella also appear to play an important role in biofilm formation and stabilization and a knock-out mutant made in a particular flagellin was biofilm deficient even though the cells remained motile. Physiologically, the biofilm appeared to utilize carbon and energy sources differently, had increased iron sequestration systems and stress responses, and may be nitrogen-limited. Cells within the biofilm were active, with metabolic rates similar to those of exponential phase cells. These results indicate that biofilm cells are responding to their environment and actively maintaining cell integrity. Overall, our results contribute to the understanding of D. vulgaris survival within the subsurface and provide a basis for other physiological studies.
Advisors/Committee Members: Fields, Matthew.
Subjects: Microbiology
Keywords: Desulfovibrio vulgaris; transcriptomics; proteomics; biofilm; SRB
More Like This
4.
Corbin-Lickfett, Kara.
Investigating the mechanisms used by the Adenovirus E4-34kDa protein to promote viral late gene expression.
Degree: PhD, Microbiology, 2003, Miami University
► Adenovirus (Ad) early region 4 (E4) is important for normal viral DNA…
(more)
▼ Adenovirus (Ad) early region 4 (E4) is important for normal viral DNA accumulation, late mRNA stability, late protein synthesis and virus particle assembly. The 34kDa protein encoded by E4 open reading frame 6 (E4-34kDa) and the 11kDa protein encoded by E4 open reading frame 3 (E4-11kDa) have redundant functions and each is sufficient to replace E4 functions during Ad infection. The E4-34kDa protein forms a complex with the 55kDa protein from early region 1b (E1b-55kDa). This complex promotes the post-transcriptional accumulation of viral late mRNA in the cytoplasm while subsequently inhibiting cellular mRNA nuclear export. E4-34kDa and E1b-55kDa have leucine-rich nuclear export signals and shuttle between the nucleus and the cytoplasm as a complex. E1b-55kDa binds RNA nonspecifically and together these observations suggested a direct role for E4-34kDa and E1b-55kDa in viral mRNA export. The importance of E4-34kDa nuclear localization signals for promoting viral late gene expression of a defective E4 mutant virus was determined. An E4-34kDa NES mutant promoted viral late gene expression as well as the wild type E4-34kDa protein. Further genetic analysis of the E4-34kDa protein revealed that central regions of the protein important for binding the E1b-55kDa protein as well as forming a complex with a cellular E3 ubiquitin ligase were critical for E4-34kDa to promote Ad late gene expression. This E4-34kDa/E1b-55kDa/E3 ubiquitin ligase complex ubiquitinates the tumor suppressor p53, and targets it for proteasome-mediated degradation during Ad infection. The ability of E4-34kDa to target proteins for proteasome-mediated degradation was required for promoting viral late gene expression, even in the absence of p53, suggesting that p53 was not the critical degradation target for expression of late genes. Viruses which expressed the E4-11kDa protein were able to express viral late proteins in the presence of proteasome inhibitors, whereas proteasome inhibitors dramatically affected the ability of an E4-11kDa virus mutant to express its late genes. Therefore, in the absence of the redundant functions of the E4-11kDa protein, E4-34kDa/E1b-55kDa promotion of viral late gene expression requires active proteasomes.
Advisors/Committee Members: Bridge, Eileen.
Subjects: Biology, Microbiology
Keywords: E4-34kDa; PROTEIN; LATE GENE EXPRESSION; E1b-55kDa; E4-11kDa
More Like This
5.
Crowgey, Erin Lynn.
Effects of protein malnourishment and corticosterone on thymocyte apoptosis.
Degree: MS, Microbiology, 2005, Miami University
► Previous research in this laboratory demonstrated that protein malnourishment causes increased levels…
(more)
▼ Previous research in this laboratory demonstrated that protein malnourishment causes increased levels of serum corticosterone and thymic atrophy in mice. The purpose of this study was to determine the effects of up-regulated serum corticosterone on thymocyte apoptosis, and the effects of a stress response on corticosterone-induced apoptosis. This study confirmed that protein malnourishment induces thymic atrophy. When mouse thymocytes were treated in vitro with corticosterone and assayed for apoptosis by quantifying phosphatidylserine externalization, mitochondrial permeabilization, andDNA fragmentation, corticosterone was shown to induce thymocyte apoptosis. When thymocytes from protein deficient mice were assayed for apoptosis, phosphatidylserine externalization and mitochondrial permeability were significantly altered, but DNA fragmentation was not. To determine how a stress response could alter thymocyteapoptosis, protein sufficient thymocytes were heat shocked and then treated with corticosterone in vitro. Heat shock decreased corticosterone-induced apoptosis and increased heat shock protein (hsp) 70 and hsp90 levels in these normal mouse thymocytes. However, when hsp70 and hsp90 were quantified in thymocytes from protein deficient mice, hsp70 or hsp90 were not significantly increased. In conclusion this study describes the significance of corticosterone-induced thymocyte apoptosis during protein malnourishment.
Advisors/Committee Members: Stevenson, John R.
Subjects: Biology, Molecular
Keywords: apoptosis; thymocytes; corticosterone; heat shock proteins; protein malnourishment
More Like This
6.
Day, James M.
Isolation of Streptomyces lividans ribosomes and initiation factors and their characterization using in vitro mRNA binding assays.
Degree: PhD, Microbiology, 2004, Miami University
► A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal…
(more)
▼ A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal subunits from Streptomyces lividans. This assay allowed the study of ribosome binding to streptomycete leaderless and leadered mRNA. Purified 30S subunits were unable to form a ternary complex on aph or cat leaderless mRNAs, whereas 70S ribosomes could form ternary complexes on both of these mRNAs. 30S subunits formed ternary complexes on aph and malE leadered mRNA. The translation initiation factors (IF1, IF2, IF3) from S. lividans were isolated and included in toeprint and filter binding assays with leadered and leaderless mRNA. Generally, the IFs reduced the toeprint signal on leadered mRNA; however, incubation of IF1 and IF2 with high-salt washed 30S subunits promoted the formation of a ternary complex on aph leaderless mRNA. Our data suggest that, as reported for Escherichia coli, initiation complexes with leaderless mRNAs might use a novel pathway involving 70S ribosomes or 30S subunits bound by IF1 and IF2 but not IF3. Some mRNA-ribosome-initiator tRNA reactions that yielded weak or no toeprint signals still formed ternary complexes in filter binding assays, suggesting the occurrence of ternary complex interactions that are not stable in the toeprint assay. Further, the streptomycete 50S subunit was observed forming a ternary complex on aph and cat leaderless mRNAs; a model is presented for an alternate translation initiation pathway involving 50S subunits binding to leaderless mRNA in the streptomycetes. This is the first comprehensive look at the interactions of the molecular components involved in translation in the streptomycetes.
Advisors/Committee Members: Janssen, Gary R.
Keywords: Streptomyces; ribosome; translation initiation; initiation factors; leaderless mRNA
More Like This
7.
Gaddy, Jennifer Angeline.
Acinetobacter baumannii Virulence Attributes: The Roles of Outer Membrane Protein A, Acinetobactin-mediated Iron Acquisition Functions, and Blue Light Sensing Protein A.
Degree: PhD, Microbiology, 2010, Miami University
► Acinetobacter baumannii is a gram-negative opportunistic pathogen that has emerged as a…
(more)
▼ Acinetobacter baumannii is a gram-negative opportunistic pathogen that has emerged as a problematic organism, causing severe infections in human hosts. This is compounded by the fact that clinical isolates of this organism are often resistant to multiple types of antimicrobial therapies. To better understand the molecular mechanisms involved in pathogenicity with the long-term goal of finding targets for novel therapies of the future, the work presented in this manuscript focuses on the factors involved in the adherence to abiotic and biotic surfaces, invasion of epithelial cells, and killing of invertebrate animal models. In addition, this work presents novel data about the regulation of those factors. Adherence to biotic surfaces is often the first step of pathogenesis. It is apparent that outer membrane protein A is required for adherence of A. baumannii ATCC 19606T cells to human respiratory epithelia. In addition, cells lacking the productionof this protein are deficient in their ability to kill eukaryotic cells such as A549 human alveolar cells and also Candida albicans tup1 filaments. Furthermore, it was proven that the death of these cells is a result of an induction of apoptosis and secreted proteins in addition to OmpA are implicated in that process. After the bacterial cell invades the A549 eukaryotic cell, it requires the function of an iron acquisition system, specifically acinetobactin biosynthesis and transport. Without these functions, bacterial cells can adhere to the surface of A549 cells, but cannot persist and replicate in the cytoplasm of the eukaryotic cell. In addition, the apoptotic induction in A549 cells is lowered when infections are performed with mutants lacking iron acquisition functions. Interestingly, A. baumannii cells lacking siderophore biosynthesis can be crossfed during infections of A549 cells via co-infection with parental strain expressing full function of siderophore biosynthesis. These results are also supported by animal model experiments using Galleria mellonella caterpillars. In addition, it was also proven that purified cell-free siderophore is capable of inducing an apoptotic response. The bacterial cell must coordinate its physiology in response to environmental stimuli to adapt to the transition from environmental reservoirs to the host niche. This work implicates blue light sensing protein A as a regulator capable of changing the proteins decorating the outer membrane in response to stimuli such as light and temperature. Two proteins which are regulated by BlsA are OmpA and the BauA siderophore receptor involved in siderophore transport. In summary, A. baumannii utilizes sensing molecules and response to mediate changes in its cellular biology in order to persist in many different environments and adapt quickly to changes in that environment. Some of these proteins are necessary for adherence, invasion, intracellular persistence and replication, as well as virulence in animal hosts.
Advisors/Committee Members: Actis, Luis.
Subjects: Microbiology
Keywords: Biofilm; bacterial pathogenesis; siderophore; virulence; Acinetobacter baumannii; blue light
More Like This
8.
Giliberti, Jacqueline.
FEATURES OF LEADERLESS mRNA AND RIBOSOMES THAT FACILITATE THEIR INTERACTION.
Degree: PhD, Microbiology, 2011, Miami University
► Translation initiation is the rate-limiting stage of translation and results in the…
(more)
▼ Translation initiation is the rate-limiting stage of translation and results in the formation of a 70S ternary initiation complex containing a ribosome, initiator tRNA, and mRNA, with the mRNA’s start codon positioned in the 70S ribosome’s P-site. Leaderless mRNAs lack a 5’ untranslated region; the 5’-terminal AUG is the start codon for translation. The translation initiation pathway of these mRNAs likely involves the 70S ribosome and differs from the pathway employed by leadered mRNAs and 30S subunits. The signals contained in leaderless mRNAs, other than the 5’AUG, and the properties of the ribosome that facilitate their interactions with leaderless mRNAs are unknown. Ribosome binding and expression assays showed that the presence of a 5’AUG on an RNA molecule generally allowed for ribosome binding and translation regardless of the downstream sequence. Crosslinking assays with ribosomes and a leaderless mRNA containing a 4-thiouridine at the +2 position (U of the AUG start codon) were used to identify contacts between the ribosome and a leaderless mRNA’s start codon. Crosslink analysis revealed that several 30S subunit proteins contact the start codon and a subset of these proteins, when used in purified form, continued to crosslink to the mRNA’s start codon, suggesting that these proteins are involved in start codon recognition. Using ribosome binding and expression assays, the mRNA’s 5’-triphosphate was shown to be important for stable ribosome binding and subsequent expression of leaderless but not leadered mRNA. Dissociation rate assays also suggested that the 5’- triphosphate functions in ternary complex stabilization. This work provides evidence that ribosomal proteins may function to recognize the 5’AUG of leaderless mRNA and facilitate its movement into the P-site. When positioned in the P-site, the leaderless mRNA’s 5’-triphosphate is important for ternary complex stabilization.
Advisors/Committee Members: Janssen, Gary.
Subjects: Microbiology
Keywords: translation initiation; leaderless mRNA; ribosome
More Like This
9.
Glen, McGillivary.
Comparative Genomic Analysis Between the Haemophilus influenzae biogroup aegyptius Brazilian Purpuric Fever Invasive Strain F3031 and the Haemophilus influenzae biogroup aegyptius Non-invasive Strain F1947.
Degree: PhD, Microbiology, 2004, Miami University
► Haemophilus influenzae biogroup aegyptius (H. aegyptius) strain F3031 is the causative agent…
(more)
▼ Haemophilus influenzae biogroup aegyptius (H. aegyptius) strain F3031 is the causative agent of Brazilian purpuric fever (BPF), a serious hemorrhagic disease of humans. The H. aegyptius strain F1947 is an isolate that was taken from a Brazilian child who acquired conjunctivitis but did not develop BPF. A genome subtraction procedure was utilized to isolate DNA specific to the F3031 strain and several subtracted clones were found to have no or poorly described homologs in GenBank, some translated products were similar to proteins in other bacteria, and some had similarity to phage-related proteins. One of the subtracted fragments had sequence similarity to IgA1 proteases and this fragment was found to localize to the region of the sequenced F3031 iga1 gene which would encode the proposed active site. The protease from the F3031 strain cleaved IgA1 with a different specificity than its cognate protease in the F1947 strain. Eight of the other F3031-specific subtracted fragments were found to be part of a genomic island that is not found in the F1947 strain. This 34,387-bp genomic island contains 44 predicted ORFs and is almost perfectly conserved in several other bacterial strains. The association of genomic island-like sequences in more pathogenic bacterial strains suggests that genomic island-like sequences have a role in virulence and the proximity of transposon, inverted repeat, and nutrient transport gene remnants in multiple organisms suggests that the island has been involved in the evolution of other species. The F3031 and F1947 strains contain similar but distinct plasmids which act as markers for defining BPF and non-BPF H. aegyptius strains, respectively. The resident plasmids pF3031 and pF1947 were sequenced and were found to encode 34 ORFs of which only three have similar sequences to other known Haemophilus sequences. A majority of the ORFs in both plasmids seem dedicated to expressing proteins involved in conjugation although the conditions for conjugation were not found. The two plasmids were shown to differ in several ways of which the most important was a truncation in a gene encoding the conjugation protein TraL in the pF1947 plasmid.
Advisors/Committee Members: Actis, Luis A.
Keywords: Haemophilus aegyptius; genome subtraction hybridization; genomic island; plasmid
More Like This
10.
Hatchel, Jennifer M.
Structure and Function of the Electron-dense Core in Mycoplasma pneumoniae and its Relatives.
Degree: PhD, Microbiology, 2009, Miami University
► Among the mycoplasmas, the human pathogen Mycoplasma pneumoniae is the best-characterized at…
(more)
▼ Among the mycoplasmas, the human pathogen Mycoplasma pneumoniae is the best-characterized at the cellular level. It has a polar structure, the attachment organelle, which mediates adherence to host cells, is the leading end during gliding motility, and plays a role during cell division. Within the attachment organelle is a detergent-insoluble electron-dense core composed of several proteins, some of which have been identified through the study of cytadherence deficient mutants of M. pneumoniae. Mutants lacking proteins in the electron-dense core are avirulent, suggesting that the core is essential for the proper formation of the attachment organelle, which in turn is essential for virulence. We used scanning electron microscopy (SEM) and time-lapse microcinematography to test the relationship between ultrastructure and gliding motility in M. pneumoniae and some of its close phylogenetic relatives, which vary in ultrastructure, gliding characteristics, host range, and pathogenic potential. Our results show that Mycoplasma amphoriforme, a novel species found in the respiratory tract that is possibly pathogenic to humans, is motile and shares morphological characteristics with its closest relatives, M. pneumoniae and the avian pathogen, Mycoplasma gallisepticum. Using SEM and time-lapse microcinematography, we find that the morphology of seven species of the M. pneumoniae cluster correlates with phylogeny rather than with gliding motility characteristics. We also find that in most species the electron-dense cores have fibers and filaments that remain attached to the base of the core after detergent treatment, but disappear after treatment with DNase, suggesting that they are DNA. It has been hypothesized that the electron-dense core plays a role during cell division, which might utilize protein-DNA interactions between the core and the chromosome. Using fluorescence in situ hybridization (FISH) coupled to immunofluorescence, we attempted to further investigate specific interactions between the oriC region of the chromosome and the electron-dense core in M. pneumoniae. The data suggest that a variable region of the chromosome associates with the base of the electron-dense core, although the FISH protocol still needs optimization. Overall, my work suggests that gliding motility has a major role in the partitioning of the chromosomes in cell division and a minor role in pathogenicity.
Advisors/Committee Members: Balish, Mitchell.
Subjects: Microbiology
Keywords: Mycoplasma; Mycoplasma pneumoniae; Gliding motility; Cell division; Attachment organelle; Morphology
More Like This
11.
Hwang, Chiachi.
Assessment of bacterial communities and an iron-reducing bacterium in relation to an engineered bioremediation system designed for the treatment of uranium-nitric acid contaminated groundwater.
Degree: PhD, Microbiology, 2009, Miami University
► The elucidation of how populations of interest interact in a given community…
(more)
▼ The elucidation of how populations of interest interact in a given community and how the community responds to stress and perturbations can provide insight into the interplay between stress pathways and gene networks that help optimize bacterial biochemistry. The goal of the present study was to characterize the responses of bacterial communities at multiple levels of resolution to understand microbial biochemical capacity at DOE waste sites. The field work at the Field Research Center of the U.S. Department of Energy, Oak Ridge, TN, used a field scale denitrifying fluidized bed reactor (FBR) for nitrate treatment of the groundwater and a series of wells to stimulate microbial growth via ethanol for in situ U(VI) immobilization. Bacterial community dynamics were investigated during the initial start-up of the FBR while those from the groundwater of the wells were studied over a 1.5-yr period. In addition, the physiology and the genome of the isolate, Anaeromyxobacter Fw109-5, from the site were studied to examine its potential role in U(VI) remediation. The subsurface environment was altered via engineering controls during successive phases to better understand strategies that would improve the remediation process. Within this framework, the interrelationship of bacterial communities and geochemistry was studied at different spatial and temporal scales to characterize the ecosystem ecology of an engineered system. Bacterial communities from both FBR and groundwater samples were analyzed via clone libraries of partial SSU rRNA genes. Multivariate analyses were applied to correlate the changes in the bacterial communities to the measured physicochemical parameters. Our results from the field experiments indicated that there was an important interaction between the engineering controls that altered the subsurface geochemistry over time that influenced bacterial population responses. Growth study experiments and genomic analysis also revealed insights to the physiological potential of an iron-reducing isolate, Anaeromyxobacter Fw109-5. The strong associations between particular environmental variables and certain population distributions will provide insights into establishing practical and successful remediation strategies in radionuclide-contaminated environments with respect to engineering controls.
Advisors/Committee Members: Fields, Matthew.
Subjects: Microbiology
Keywords: bacterial ecology; uranium; bioremediation
More Like This
12.
Jaffri, Sarah.
Characterization of the photosynthetic apparatus of Chlorella BI sp., an Antarctica mat alga under varying trophic growth states.
Degree: MS, Microbiology, 2011, Miami University
► The psychrophilic green alga, Chlorella BI sp. was isolated from a transient…
(more)
▼ The psychrophilic green alga, Chlorella BI sp. was isolated from a transient Antarctic pond as part of a mat consortium. Previous research on Chlorella BI sp. showed that the organism was able to utilize inorganic and organic forms of carbon and alter its photosynthetic apparatus in response to varying trophic growth states. Based on these early results, the goals of this thesis project were to: (1) characterize the photosynthetic apparatus of Chlorella BI sp. under different trophic states in comparison to the mesophilic species, Chlorella vulgaris; and, (2) determine the effect on the photosynthetic apparatus of Chlorella BI sp, when it is shifted from dark to light conditions. Chlorella BI sp. grew exponentially under the three tested trophic states. The photosynthetic apparatus exhibited functional and structural alteration. It is concluded Chlorella BI sp. has retained the ability to alter its photosynthetic apparatus in response to adaptation to a variable habitat.
Advisors/Committee Members: Morgan-Kiss, Rachael.
Subjects: Microbiology
Keywords: extreme environments; Antarctica; Antarctic microbial mat ponds; phototrophic primary producers; green algae; light environment; Chlorella BI sp.; different trophic regimes; organic carbon; modulation of photosynthetic apparatus; chlorophyll fluorescenc
More Like This
13.
Jayaram, Sumithra.
INVESTIGATING ADENOVIRUS INTERACTIONS WITH HOST DOUBLE-STRAND BREAK REPAIR DEFENSES.
Degree: PhD, Microbiology, 2005, Miami University
► The goal of this study was to investigate the role of host…
(more)
▼ The goal of this study was to investigate the role of host double-strand break repair (DSBR) on the life cycle of Adenovirus (Ad). Ad mutants that lack the entire E4 region activate a cellular DNA damage response accompanied by phosphorylation of several host DSBR and DNA damage response proteins. We find that aspects of the E4 mutant induced DNA damage response occurs at the onset of viral DNA replication and may be activated by physical replication of viral genomes. Genetic analysis of the E4 mutants revealed that the E4-34kDa protein was required to prevent the activation of DNA damage response. Redistribution of MRN complex proteins away from viral replication centers by the E4-11kDa protein was not sufficient to prevent the DNA damage response. Activation of the DNA damage response does not interfere with viral DNA replication in the presence of E4-11kDa protein. E4 mutants are severely defective for late gene expression following concatenation of their genomes by host DSBR proteins. We find that E4 mutant late gene expression improves in MO59J cells that fail to form genome concatemers. DSBR kinase inhibitors interfere with genome concatenation and also stimulate late gene expression. Concatenation of E4 mutant genomes interferes with cytoplasmic accumulation of viral late messages and leads to reduced late protein levels and poor viral yields following high multiplicity infection. However, failure to concatenate viral genomes did not rescue either the DNA replication defect or virus yield following low multiplicity E4 mutant infection. Our results indicate that if the E4 mutant DNA replication defect is overcome by high multiplicity infection, concatenation of the replicated genomes by host DSBR interferes with viral late gene expression. These studies provide insight into the role of host DSBR as an obstacle to a productive Ad infection, and how the virus dismantles this barrier.
Advisors/Committee Members: Bridge, Eileen K.
Keywords: Adenovirus; late gene expression; concatemers; DNA damage response; DSBR
More Like This
14.
Jung, Joo-Yong.
INTERLEUKIN-10 AS A NEGATIVE REGULATOR OF INTERFERON-MEDIATED IMMUNITY IN CHLAMYDIAL INFECTIONS.
Degree: PhD, Microbiology, 2007, Miami University
► IFN-induced indoleamine 2,3-dioxygenase (IDO) inhibits intracellular chlamydiae by restricting the availability of…
(more)
▼ IFN-induced indoleamine 2,3-dioxygenase (IDO) inhibits intracellular chlamydiae by restricting the availability of tryptophan required for chlamydial growth. The pro-inflammatory cytokines that synergistically enhance IDO activity, IL-1β and TNF-α, are produced by macrophages during in vitro chlamydial infections. These cytokines may be involved in the intensive inflammatory responses at sites of infection. However, asymptomatic infection occurs in 70-90% of infected patients, suggesting that antagonistic cytokines might be generated during the immune response in vivo. Interleukin-10 is an anti-inflammatory cytokine with potent immunomodulatory effects, including the inhibition of cytokine production. If IL-10 is produced during a chlamydial infection, it might inhibit the production of pro-inflammatory cytokines capable of up-regulating anti-chlamydial IDO activity. The purpose of this study was to evaluate the effectiveness of IL-10 production as a mechanism by which chlamydiae evade the effects of IFN-γ. It was found that IL-10 treatment of macrophage cultures inhibited the production of IFN-α, IL-1β and TNF α as well as the induction of IDO activity in response to chlamydial infection in a concentration-related manner. IL-1β and TNF-α were detected by 24 h after exposure to UV-inactivated Chlamydophila psittaci and live Chlamydia trachomatis, whereas IL-10 was detected at approximately 48 h after exposure to chlamydiae. The mechanism of IL-10 production in response to chlamydial infection was explored. It was found that IL-10 production from macrophages exposed to chlamydiae was dependent upon endogenous production of IL-1β and TNF-α. Since UV-inactivated C. psittaci and C. trachomatis were unable to replicate in macrophages, involvement of chlamydiae-Toll like receptor 2 (TLR2) and 4 (TLR4) interaction on IL-1β and TNF-α production was explored. Endogenous production of IL-1β and TNF-α were initiated following the interaction between Chlamydia and TLR2 and TLR4 on the surface of macrophages. These results indicate that Chlamydia-TLR interaction stimulates a cascade of cytokine production capable of either up-regulating or down-regulating IFN γ-induced IDO activity. Overall this study suggests that IL-10 produced following Chlamydia-TLR interaction may inhibit the induction of effective immunological responses by inhibiting pro-inflammatory cytokine production by macrophages.
Advisors/Committee Members: Carlin, Joseph M.
Subjects: Biology, Microbiology
Keywords: Chlamydia, IL-1β, TNF-α, macrophages, IL-10, IFN-γ,indoleamine 2,3-dioxygenase (IDO)
More Like This
15.
Jurkovic, Dominika Angelika.
Structure, Organization, and Function of the Terminal Organelle in Mycoplasma penetrans.
Degree: PhD, Microbiology, 2012, Miami University
► Bacteria utilize cytoskeletal elements to confer both morphological and functional polarity, which…
(more)
▼ Bacteria utilize cytoskeletal elements to confer both morphological and functional polarity, which are necessary for growth and survival in their native environments. Some bacteria utilize polar appendages for attachment and motility to and within a host. Additionally, some bacteria utilize functional polarity for the subcellular localization of certain molecules to a particular area of bacterium. In most bacteria the cell wall plays a critical role in generating polarity, which poses a problem for the members of the Mycoplasma genus, whose members lack cell walls. Despite reductive evolution from gram-positive bacteria, mycoplasmas harbor a wide variety of morphological and functional complexity. Polarized species of mycoplasmas utilize polar organelles conferred by unique cytoskeletal elements utilized in attachment and motility, two processes associated with pathogenic species. One of these, Mycoplasma penetrans, is a potential opportunist usually isolated from human immunodeficiency (HIV)-infected individuals, proposed to play a role in AIDS progression. Previously, it has been demonstrated that M. penetrans attach to epithelial cells by a polar tip structure, yet nothing is known about the components involved in the architecture and functioning of the tip structure. We used time-lapse microcinematography to characterize gliding motility in both M. penetrans and its relative, Mycoplasma iowae, a poultry pathogen. Gliding speeds observed among strains in both species correlated positively with cytadherence differences by hemadsorption assay, suggesting that attachment and motility are both mediated by the same cellular components. During cell division, attachment to a surface and motility were critical for cytokinesis. We used scanning electron microscopy (SEM) and electron cyro-tomography to characterize the internal cellular organization and of the Triton X-100 (TX)-insoluble, cytoskeletal structure underlying the terminal organelle. The results from the different microscopy techniques are consistent with the cytoskeletal structure being artifactually dehydrated during processing for SEM, suggesting the cytoskeletal structure to be a proteinaceous gel found in varying amounts at both cell poles. Based on the importance motility plays in proper cell division, and the internal composition and organization of M. penetrans, we propose a cell cycle model in which a pole develops by accumulating enough TX-insoluble material in order to function in attachment and/or motility, and therefore power cytokinesis. To further identify the components of the TX-insoluble structure, we sought to identify proteins enriched in the TX-insoluble fraction compared to whole cells. We identified two proteins, MYPE1560 and MYPE1570, two of six proteins with similar features found in a putative transcriptional unit. Our observations of morphology and organization of M. penetrans and the TX-insoluble material demonstrate a novel cellular organization for generating polarity. We observed continued motility in the presence of an ATP depletion agent, a proton motive force inhibitor, and a sodium motive force inhibitor, raising the possibility that M. penetrans does not use chemically-derived energy source for motility. However, analysis of gliding under different temperature and pH conditions led us to conclude that thermal energy plays an important role in gliding motility. We propose a model for M. penetrans gliding motility wherein thermal energy is converted to unidirectional motility by means of a Brownian ratchet. Additionally, we provide evidence supporting the hypothesis that terminal organelles in distantly related mycoplasma species have evolved independently of each other, rather than diverging from a common ancestor.
Advisors/Committee Members: Balish, Mitchell.
Subjects: Microbiology
Keywords: Mycoplasma penetrans, Mycoplasma iowae, terminal organelle, gliding motility, cytadherence, cytoskeleton
More Like This
16.
Klemann, Tiffany Anne.
Influence of Secretory Immunoglobulin A upon Germination Frequency and Adhesion of Candida albicans.
Degree: MS, Microbiology, 2003, Miami University
► Pathogenicity of Candida albicans, the most virulent Candida species, is attributed to…
(more)
▼ Pathogenicity of Candida albicans, the most virulent Candida species, is attributed to a diverse array of virulence factors, including the abilities to germinate and to adhere to host epithelial cells. Frequently, portals of entry for C. albicans are mucous membranes protected by secretory immunoglobulin A (SIgA). However, the role of SIgA in local host defense against C. albicans is currently unclear. This study’s objectives were to investigate effects of SIgA upon C. albicans’ germination and adherence to HEp-2 cells, using a germination assay with phase contrast microscopy and flow cytometry, respectively. Eighty percent (8/10) of C. albicans strains exposed to a 3-fold dilution series of pooled human SIgA, ranging from 1667 to 62 µg/ml, experienced a decrease in germination, whereas two showed either no change or an increase. Removal of Candida-specific SIgA negated the reduction in germination. SIgA did not affect germ tube length. To investigate the influence of SIgA upon adherence of C. albicans, eight strains were exposed to 10-fold dilutions of SIgA, ranging from 500 to 0.5 µg/ml, fluorescently stained, and incubated with HEp-2 cells prior to analysis. Two of eight strains exhibited increased adherence after SIgA exposure. In contrast, six strains demonstrated no significant change in adherence. In conclusion, germ tube formation is inhibited by Candidaspecific SIgA. However, adherence of C. albicans is largely unaffected by SIgA. These findings suggest SIgA has a novel role of controlling germination and subsequent invasion of the host by C. albicans.
Advisors/Committee Members: Lee, Marcia.
Subjects: Biology, Microbiology
Keywords: Candida albicans; Secretory Immunoglobulin A; SIgA; Germination; Adherence
More Like This
17.
Krishnan, Karthik M.
RIBOSOME - mRNA INTERACTIONS THAT CONTRIBUTE TO RECOGNITION AND BINDING OF A 5’-TERMINAL AUG START CODON.
Degree: PhD, Microbiology, 2010, Miami University
► Initiation is the rate-limiting step of translation and is an important regulatory…
(more)
▼ Initiation is the rate-limiting step of translation and is an important regulatory step in gene expression. Translation initiation requires ribosome recognition of the mRNA’s start codon followed by formation of a stable ternary complex involving initiator tRNA. Several mRNA features are recognized and contacted by the ribosome, suggesting that the ribosome may possess multiple mRNA recognition features. Studies with leaderless mRNA have shown specific contacts between a leaderless mRNA’s 5’-AUG and several ribosomal proteins (r-proteins). We employed short-leadered mRNAs in order to further characterize the path of a leaderless mRNA’s 5’-AUG from initial association with the ribosome to its final placement at the ribosome’s decoding site (P-site). Our results suggest that Escherichia coli ribosomes can bind and inspect the 5’-terminus of all mRNAs for an AUG triplet, with close proximity of AUG to the 5’-terminus positively influencing ribosome binding and ternary initiation complex formation. Crosslinking studies showed that while 5’-AUG interactions with r-proteins S1, S3, S4, S7, S10 and S18 can occur in the absence of tRNAfMet, movement of a 5’-AUG into the P-site requires tRNAfMet. Addition of a nucleotide triplet upstream (i.e., a -1 triplet) to a leaderless mRNA’s start codon resulted in interference with 5’-AUG interactions with the r-proteins S3, S4, S7, S10 and S18 in a sequence-specific manner while allowing retention of these mRNAs on the ribosome. Investigation of Streptomyces lividans high salt-washed (HS) ribosomes and the features of leaderless aph mRNA that are important for ribosome binding further suggest that the recognition, binding and movement of an mRNA’s start codon into the ribosome’s P-site involves multiple contacts and interactions between r-proteins and the mRNA.
Advisors/Committee Members: Janssen, Gary.
Subjects: Molecular biology
Keywords: translation initiation, leaderless mRNA, -1 triplet, streptomyces, E. coli, ribosome binding, toeprint, crosslinking
More Like This
18.
Mathew, Shomita S.
Investigating the role of DNA damage signaling events in the cellular interference with Adenovirus replication.
Degree: PhD, Microbiology, 2007, Miami University
► Eukaryotic cells possess mechanisms that monitor breaks in genomic DNA and repair…
(more)
▼ Eukaryotic cells possess mechanisms that monitor breaks in genomic DNA and repair them. The effectors of double strand break repair (DSBR) comprise a variety of proteins, including the Mre11/Rad50/Nbs1 (MRN) complex and the mediator of DNA damage checkpoint protein 1 (Mdc1). The Adenovirus (Ad) genome is linear, double stranded DNA that can be a substrate for “repair” by host DSBR proteins that link genomes end-to-end forming concatemers. The virus defends itself against this repair by producing regulatory proteins that interfere with DSBR. Early region 4 (E4) orf3-11kDa protein relocalizes the cellular MRN complex to nuclear track-like structures. The E4-orf6 34kDa/E1b-55kDa complex targets MRN for proteasome mediated degradation. Mutants that lack the E4-11kDa and 34kDa proteins activate the cellular damage response and are severely defective for DNA replication. We have investigated roles for the MRN complex and Mdc1 as sensors and effectors of damage signaling in an Ad-E4 mutant infection, particularly related to the onset of an efficient viral DNA replication. Briefly, we find that the MRN complex regulates the re-localization of Mdc1 early in the infection with an E4 mutant virus, consistent with its role as a sensor of DNA damage. Mdc1 is re-localized in response to viral infection and binds E4 mutant viral DNA, but does not appear to have a role in the regulation of viral DNA replication. The MRN complex, however, is relocalized to E4 mutant viral DNA replication centers in an Nbs1 dependent manner and binds viral DNA. The Nbs1 dependent binding of the complex to E4 mutant viral DNA inhibits the efficient onset of viral DNA replication consistent with the role of the MRN complex as an effector in the damage response and repair pathway. Investigating the ability of host DSBR proteins to interfere with E4 mutant DNA synthesis provides a model for understanding the mechanism by which these proteins sense and respond to the presence of aberrant or damaged DNA.
Advisors/Committee Members: Bridge, Eileen.
Keywords: Adenovirus, DNA replication, DNA damage response, foci, MRN complex, Mdc1, DNA binding
More Like This
19.
McQueary, Christin Nicole.
Variations in Biofilm Formation and Motility Displayed by Isolates of Acinetobacter baumannii.
Degree: PhD, Microbiology, 2010, Miami University
► Acinetobacter baumannii is an opportunistic nosocomial pathogen of substantial concern due to…
(more)
▼ Acinetobacter baumannii is an opportunistic nosocomial pathogen of substantial concern due to its increasing antibiotic resistance and ability to survive unfavorable conditions in the hospital environment, likely in the form of a biofilm, which potentiates its ability to spread. However, the information about its virulence factors and the regulatory networks that could control their expression is insufficient. This lack of data is compounded by large variations in genotypic and phenotypic traits among clinical isolates as illustrated by the differences in biofilm structures they form on plastic or glass. These biofilm variations did not correlate with properties of abiotic surfaces or other cell properties, such as surface hydrophobicity, pellicle formation, surface-associated motility, or somatic appendages. It is apparent that environmental signals, such as the concentration of extracellular free iron, could affect the aforementioned interactions, as measured by changes in cell motility on semisolid media and biofilm formation on plastic. These responses are independent of pilT and pilU orthologs, which impact motility and biofilm functions in other pathogens. However, these two genes play a role in the interaction of A. baumannii ATCC 17978 cells with human alveolar epithelial cells which results in the apoptotic death of these epithelial cells. The A. baumannii responses to multiple environmental cues may be attributable to the sensing and response function of regulators such as that coded by the bfmL locus present in the ATCC 19606T strain. Interestingly, this LysR-type transcriptional regulator (LTTR) is needed for the post-transcriptional expression of the CsuA/BABCDE chaperone-usher pilus assembly system needed for cell attachment and biofilm formation on plastics. Equally interesting is that inactivation of bfmL results in the production of pili different from those produced when the aforementioned assembly system is expressed. Taken together, these results demonstrate that the interaction of A. baumannii with medically relevant surfaces is affected by a multitude of cellular and environmental factors, most of which remain to be characterized. This missing basic knowledge could be used to develop much needed alternative and more efficient therapies to treat the infections this pathogen causes.
Advisors/Committee Members: Actis, Luis.
Subjects: Microbiology
Keywords: Biofilm formation; Motility; LTTR; Acinetobacter baumannii
More Like This
20.
Mumy, Karen Lynn.
Determination of degradative gene frequencies: Applications in polycyclic aromatic hydrocarbon contaminated sediments.
Degree: PhD, Microbiology, 2004, Miami University
► Aerobic polycyclic aromatic hydrocarbon (PAH) degradative pathways have been described in a…
(more)
▼ Aerobic polycyclic aromatic hydrocarbon (PAH) degradative pathways have been described in a wide variety of bacteria, fungi and algae. The occurrence and role of these pathways and their corresponding genes in natural attenuation in environmental systems remain poorly understood. The primary goal of this dissertation research was to determine the concentration and frequency of five degradative genes within natural microbial communities exposed to ambient, low and high levels of PAHs using a quantitive-competitive PCR approach adapted for microbial ecology use. PAH concentrations, total microbial biomass and microbial community structure for sediment samples were also determined using a coupled biochemical approach. Community structure analysis indicated that microeukaryotes may be indirectly responding to the contamination, suggesting the development of a PAH-based food web within the microbial communities. Gene frequencies and concentrations reflected increases with elevated levels of contamination. However, the measured frequencies for most genes indicated that only 0.1% or less of the microbial community had the genetic capacity to degrade the contaminants using described aerobic pathways and their corresponding genes and suggested these sequences are rare in these environmental samples. Metabolic assays indicated that the detected genetic capacity was sufficient to account for the overall biodegradative potential observed within the entire sediment microbial community. These data support that, although the reported frequencies were lower than those others have described, the determined frequencies are indicative of the genetic capacity of natural populations in these sediments. While it is likely that additional unidentified degradative pathways and genes exist and are operating in the environment, the data presented in this dissertation support that many of the well understood enzymes and pathways, while primarily studied and defined within laboratory settings, catalyze the majority of the metabolic activity observed in this environmental setting.
Advisors/Committee Members: Findlay, Robert H.
Keywords: Degradative genes; Microbial communities; Sediments; PAHs; QC-PCR; Aerobic degradative pathways
More Like This
21.
Mykrantz, Hallie B.
Investigation of Burkholderia cepacia Virulence.
Degree: MS, Microbiology, 2005, Miami University
► Burkholderia cepacia is an opportunistic pathogen that causes a variety of infections.…
(more)
▼ Burkholderia cepacia is an opportunistic pathogen that causes a variety of infections. Individuals with cystic fibrosis, a lethal autosomal-recessive disease, contract nosocomial infections of B. cepacia that are challenging to treat due to the organism’s resistance to multiple antibiotics. The mechanisms used by the bacterium to cause these infections are currently unknown and, therefore, alternative methods of treatment or prevention are difficult to establish. This project examined several B. cepacia strains and avirulent B. cepacia transconjugants to investigate potential virulence mechanisms. The data suggest that the extracellular enzymes protease, lipase, and phospholipase C do not significantly contribute to the pathogenicity of the organism, at least in the Caenorhabditis elegans model we used. Infection, not intoxication, by B. cepacia of C. elegans was responsible, but not necessarily sufficient for the nematocidal activities of all the strains studied. The results of these experiments also support the suitability of C. elegans as an in vivo model for studying the virulence factors of B. cepacia.
Advisors/Committee Members: Morris-Hooke, Anne.
Subjects: Biology, Microbiology
Keywords: Caenorhabditis elegans; Burkholderia cepacia; cystic fibrosis; virulence mechanisms; transposon mutagenesis; lipase; protease; phospholipase C
More Like This
22.
O'Dee, Dawn M.
EFFECT OF CORTICOSTERONE ON SELECTED ASPECTS OF MACROPHAGE AND T-CELL ACTIVITY.
Degree: MS, Microbiology, 2005, Miami University
► Previous research in this laboratory demonstrated that protein deficient (PD) mice have…
(more)
▼ Previous research in this laboratory demonstrated that protein deficient (PD) mice have elevated serum corticosterone, decreased macrophage function, and increased susceptibility to infection. The goal of this project was to determine how corticosterone affects selected aspects of macrophage activity. Peritoneal exudate cells from mice were stimulated with IFN-gamma and LPS, then treated with corticosterone. At the concentration found in PD mice, corticosterone greatly inhibited macrophage MHCII expression, IL-12 secretion and TNF-alpha secretion, but only slightly inhibited NO production. Corticosterone interferes with IL-12 upregulation at the transcriptional level, as determined using semi-quantitative RT-PCR. IL-12 secreted by macrophages cultured with corticosterone stimulated T-lymphocyte secretion of IFN-gamma in proportion to its concentration as determined by ELISA, whereas T-lymphocytes treated with corticosterone did not respond to IL-12. These findings indicate that elevated corticosterone may be a primary cause of increased susceptibility to infection in PD mice.
Advisors/Committee Members: Stevenson, John R.
Subjects: Biology, Microbiology
Keywords: Corticosterone; Macrophage; T-cell
More Like This
23.
Relich, Ryan F.
Gliding Motility Mechanisms in Divergent Mycoplasma Species.
Degree: PhD, Microbiology, 2011, Miami University
► Bacteria belonging to the Mycoplasma pneumoniae phylogenetic cluster possess polarity that is…
(more)
▼ Bacteria belonging to the Mycoplasma pneumoniae phylogenetic cluster possess polarity that is conferred by a differentiated tip structure called the attachment organelle. Among the species comprising this cluster, all but one have been experimentally demonstrated to exhibit a contact-dependent form of motility categorized as gliding, a process that is mediated by the attachment organelle. The subcelluar structures within the attachment organelle are conserved in all of these species; however, the morphology and gliding speed of each are distinct. The reasons for these phenotypic disparities are unknown, but we propose that an adhesin common to all of these species, called P30 in M. pneumoniae, contributes many of the species-specific differences, and the concentration of this protein at the attachment organelle tip dictates gliding speed. To test our hypotheses, we examined several phenotypes of an M. pneumoniae P30 null mutant, II-3, expressing a P30 ortholog, P32, from the closely related species Mycoplasma genitalium, which is phenotypically distinct from M. pneumoniae. Although these experiments did not identify a role for P30 in species-specific phenotypes, P32 was demonstrated to be a functional surrogate for P30 in M. pneumoniae. These data also comprise the first report of successful orthologous gene replacement in mycoplasmas, a technique that is potentially amenable for the study of other aspects of mycoplasma biology. We next examined phenotypes of M. pneumoniae II-3 cells expressing native P30 under the control of the M. pneumoniae ldh promoter, which gave rise to several transformant strains expressing variable low levels of P30. These data indicated a positive correlation between the concentration of P30 and the speed at which cells glide. We also used techniques for the analysis of M. pneumoniae and its relatives to examine the rod-shaped mycoplasma, Mycoplasma insons. We were able to characterize a novel cytoskeleton and gliding motility in this species, although, we were not able to define the bases for polarity or motility generation. Overall, the work described herein provides insight into the biology of mycoplasmas and their motility, as well as description of novel experimental approaches for studying these unique microorganisms.
Advisors/Committee Members: Balish, Mitchell.
Subjects: Microbiology
Keywords: Mycoplasma pneumoniae; Mycoplasma genitalium; gliding motility; P30 adhesin; orthologous gene replacement
More Like This
24.
Rhodes, Eric Robert.
Genetic and Molecular Characterization of the Iron Acquisition Systems of Actinobacillus actinomycetemcomitans.
Degree: PhD, Microbiology, 2006, Miami University
► Actinobacillus actinomycetemcomitans has been associated with a variety of human infections but…
(more)
▼ Actinobacillus actinomycetemcomitans has been associated with a variety of human infections but mainly with Localized Aggressive Periodontitis. This pathogen grows under iron-limiting conditions imposed by synthetic and natural iron chelators. Utilization and binding assays showed that while all strains tested can bind lactoferrin, hemoglobin, and hemin but not transferrin, they can only use iron chloride and hemin as iron sources. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was reduced when the cells were treated with proteinase K. Scanning electron microscopy showed that smooth strains form less complex biofilms when compared with rough isolates. The iron sources also affected biofilm formation in both rough and smooth isolates. The A. actinomycetemcomitans afeABCD iron transport system, expression of which is controlled by iron and Fur, as well as the hitABC iron transport system were identified. Transformation of the E. coli 1017 iron acquisition mutant with a plasmid harboring either afeABCD or hitABC promoted cell growth under iron-chelated conditions, which shows that the role of these systems are in iron acquisition. Insertions in each open reading frame showed that each one is needed to complement the growth defect of E. coli 1017 imposed by the iron-chelated conditions. A. actinomycetemcomitans isogenic mutants with interruptions in either afeA, afeB, afeC, afeD, hitA, hitB or hitC were all unable to grow under iron-chelated conditions in which the parental strain could grow. The afeABCD system mutants were more restricted in growth by iron-chelated conditions than the hitABC mutants. This suggests that the AfeABCD system has a bigger role in iron acquisition under the conditions tested. Mutations in exbB, exbD, tonB , components of the energy transducing system, and hasR , an outer membrane heme binding protein, did not affect the ability of this strain to use heme as the sole source of iron. This suggests the expression of alternative functions that play a role in heme acquisition. We also describe the implementation of 2-D PAGE and DNA microarray analysis to investigate global gene expression in response to different iron conditions. Preliminary results indicate differential gene expression in response to changes in the iron conditions.
Advisors/Committee Members: Actis, Luis A.
Subjects: Biology, Microbiology
Keywords: Actinobacillus actinomycetemcomitans; Iron Acquisition; Biofilms; Iron Regulation
More Like This
25.
Robinson, Cory Michael.
MOLECULAR MECHANISMS OF SYNERGISTIC TRANSCRIPTIONAL REGULATION OF INDOLEAMINE 2,3-DIOXYGENASE.
Degree: PhD, Microbiology, 2004, Miami University
► Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity inhibits the growth of some intracellular…
(more)
▼ Interferon (IFN)-gamma-induced indoleamine 2,3-dioxygenase (IDO) activity inhibits the growth of some intracellular pathogens by catalyzing the oxidative cleavage of the indole ring of L-tryptophan and depleting pools of this essential amino acid. Tumor necrosis factor (TNF)-alpha synergistically enhances the IDO activity induced by IFN at the level of transcription in human epithelial cells. The purpose of this study was to characterize the molecular mechanisms responsible for synergistic gene expression in response to IFN and TNF. It was found that IFN-induced binding of STAT-1 to gamma activation sequences (GAS) and IFN responsive factor (IRF)-1 to IFN-stimulated response elements (ISRE), is more highly activated following treatment with interferon and TNF. This enhanced signal transduction is due to the increase in IFN receptor expression following combined cytokine stimulation. CCAAT enhancer binding protein (C/EBP)-beta binds to one of three consensus C/EBP sites in the IDO regulatory region in response to TNF alone or in combination with IFN. A transcriptional reporter construct consisting of green fluorescent protein expressed from the IDO regulatory region was utilized to understand which enhancer regions are responsible for synergistic IDO gene expression in response to IFN and TNF. Mutation of other individual enhancers and large deletions within the regulatory region showed that increased binding of IFN-specific factors to GAS and ISRE sites is alone responsible for synergistic transcriptional activation. An indirect requirement for NF-KB in synergistic IDO expression by regulating IRF-1 expression was also explored. A gamma activated sequence and a KB site, respectively, reside in the IRF-1 regulatory region. It was important to identify whether increased translocation of NF-KB to the nucleus and binding to the kB site upstream of the IRF-1 gene in response to IFN and TNF, is rate-limiting in enhanced IRF-1 expression. Limiting NF-KB translocation to the nucleus with a proteasome inhibitor reduced synergistic IRF-1 expression comparable to that achieved in response to IFN alone, and subsequently blocked synergistic IDO transcription. This defines an indirect requirement for NF-KB in synergistic IDO expression in response to IFN and TNF by regulating IRF-1 expression.
Advisors/Committee Members: Carlin, Joseph M.
Keywords: transcriptional regulation; interferon; tumor necrosis factor; indoleamine 2,3-dioxygenase; STAT-1; IRF-1; C/EBP-beta; NF-KB
More Like This
26.
Rovito, Holly A.
The rate of formation and stability of translation initiation complexes with leaderless mRNA in Escherichia coli.
Degree: MS, Microbiology, 2003, Miami University
► The translation initiation region for leaderless mRNA includes the nucleotide sequence and…
(more)
▼ The translation initiation region for leaderless mRNA includes the nucleotide sequence and structures near the 5’end of mRNA; translation signals located upstream to the coding sequence of leadered mRNAs are missing. In primer extension inhibition assays, 30S ternary complexes with leaderless mRNA are unstable and disassociate quickly in the presence of excess competitor mRNA, suggesting leaderless mRNA might be readily replaced in 30S ternary complexes by mRNA with stronger ribosome binding signals. Leaderless mRNA are more stable within 50S or 70S ternary complexes than 30S ternary complexes. Kinetic analyses revealed that 70S ribosomes and 50S subunits form ternary complexes 55 and 3.5 times faster, respectively, than 30S subunits, suggesting that leaderless mRNA might initiate translation through a faster and more stable 50S or 70S ternary complex.
Advisors/Committee Members: Janssen, Gary R.
Subjects: Biology, Microbiology
Keywords: translation initiation; Escherichia coli; ribosomes
More Like This
27.
Sethman, Chad Robert.
Attachment of Streptococcus pyogenes to Host Epithelial Cells.
Degree: PhD, Microbiology, 2003, Miami University
► For most pathogens, the first step in colonization of host surfaces is…
(more)
▼ For most pathogens, the first step in colonization of host surfaces is the initial approach and attachment to host epithelium. Surface molecules on both the bacterial cell and host cell are mediators of this interaction. For S. pyogenes, at least 12 different surface molecules have been implicated in mediating the attachment to host cells. Using a newly developed flow cytometry assay, we have shown that the adhesion of S. pyogenes to host cells occurs as a two-affinity stage process. During this process, the initial interactions between bacterial cell and host cell are relatively weak and mediated primarily by ionic and hydrophobic forces. As the interaction persists, particular surface molecules are able to interact specifically with host molecules, leading to a stronger affinity binding. We have shown that the hyaluronic acid capsule of S. pyogenes can mediate an early (specific) stage of binding by interacting with the host membrane glycoprotein CD44. We have also shown that, in addition to its role as a surface hydrophobin, lipoteichoic acid also mediates an early (specific) stage binding by interacting with the host membrane Toll-like receptor-2 molecule. Using fluorescent labeling of surface HA and LTA we have showed that, depending on the growth phase of the bacterium, (with respect to the extent of encapsulation) either HA or LTA may occupy the outermost layer of the bacterium and, therefore, may independently mediate the first attractive interactions between the bacterium and host.
Advisors/Committee Members: Cowan, Marjorie M.
Subjects: Biology, Microbiology
Keywords: Streptococcus Pyogenes; flow cytometry; adhesion; hyaluronic acid; lipoteichoic acid
More Like This
28.
Shirey, Kari Ann.
Modulation of Interferon-gamma Receptor Expression During Infection with Chlamydia psittaci 6BC and Its Influence on Indoleamine 2,3-dioxygenase.
Degree: PhD, Microbiology, 2006, Miami University
► Interferon-gamma (IFNγ) induces indoleamine dioxygenase (IDO), which effectively inhibits the growth of…
(more)
▼ Interferon-gamma (IFNγ) induces indoleamine dioxygenase (IDO), which effectively inhibits the growth of intracellular Chlamydia in vitro. Furthermore, tumor necrosis factor-alpha (TNFα) and interleukin-1 (IL-1) synergistically increase IFN-induced, anti-chlamydial IDO activity. The mechanism of synergistic IDO activity is multifactorial. While one mechanism is the nuclear factor-κB (NF-κB)-dependent increase in interferon regulatory factor-1 (IRF-1) activation, increased expression of IFNγ receptors (IFNγR), could also enhance IDO activation. It was found that IFNγR expression was up-regulated in epithelial cells upon stimulation with IL-1, also through the transactivation of NF-κB. This increase in receptor expression was shown to enhance IDO activity by increasing activation of the transcription factor signal transducer and activator of transcription-1 (STAT-1). Moreover, Chlamydia was found to significantly increase IFNγR expression in HeLa cells, even when inactivated, suggesting that chlamydial antigens and not infection up-regulate cytokine receptor expression. Cytokine receptor increase was found to be independent of cytokine secretion as supernatants from infected cells failed to increase IFNγR expression. The component of Chlamydia responsible for stimulating the cell to up-regulate cytokine receptor expression is heat stable; receptors increased occurred upon stimulation with Chlamydia inactivated at 100°C. The mechanism by which Chlamydia increases receptor expression requires stimulation of the Toll-like receptors (TLR). While cells either TLR2 or TLR4+MD2 increased IFNγ receptor expression, cells not possessing TLRs were unresponsive. Similar to IL-1, Chlamydia required TLR-mediated NF-κB activation to enhance IFNγR expression. However, unlike stimulation with cytokine, no increase in IDO induction was observed. This effect is not due to the inability of IFNγ to bind to the newly expressed receptors, but rather the impairment in signaling of these receptors. No increase in phosphorylated STAT-1 could be detected in infected cells suggesting that the JAK/STAT pathway was affected.
Advisors/Committee Members: Carlin, Joseph M.
More Like This
29.
Tomaras, Andrew P.
Genetic Determinants Required for Biofilm Formation by Acinetobacter baumannii.
Degree: PhD, Microbiology, 2004, Miami University
► Acinetobacter baumannii is an important human pathogen that causes severe respiratory diseases…
(more)
▼ Acinetobacter baumannii is an important human pathogen that causes severe respiratory diseases in compromised patients. This bacterium is capable of surviving on nutrient-limited abiotic surfaces, such as bed linens, hospital equipment, and medical devices. This survival property suggested the possibility that this pathogen could form biofilms to survive such unfavorable conditions. Biofilms are aggregates of bacterial cells that are metabolically and physiologically distinct from their planktonic counterparts. Cells composing these structures work together to acquire nutrients and survive harsh environmental conditions. This hypothesis was proven by observing biofilm formation by A. baumannii on abiotic surfaces such as plastics. This formation seems to occur independently of cell motility, as we have been unable to detect such a capability in this organism. Random insertion mutagenesis revealed the presence of a chaperone-usher secretion system, which was found to be involved in the assembly of pili on the bacterial cell surface. These pili are essential for attachment and subsequent biofilm formation on abiotic surfaces, and are assembled using a cellular machinery similar to that described previously in other gram-negative pathogenic organisms. The same mutagenesis approach allowed for the discovery of a putative two-component regulatory system, which is also involved in this attachment process. Interestingly, this regulatory circuit controls pili production by activating the transcription of the chaperone-usher system described above. Additionally, this regulatory system controls cellular morphology in a nutrient-dependent fashion. The ability of A. baumannii to form biofilms on biotic surfaces was also tested in this work. It was found that this organism could attach to two distinct biotic surfaces, Candida albicans and HeLa cells, and that the presence of pili on the bacterial cell surface was not as essential to attachment as it was for abiotic surface colonization. In addition, the successful binding of A. baumannii cells to either biotic surface led to the death of either eukaryotic cell type. Therefore, our results demonstrate that the abiotic and biotic systems described in this work can be used to identify the virulence factors produced by A. baumannii to cause serious human infections.
Advisors/Committee Members: Actis, Luis A.
Subjects: Biology, Microbiology
Keywords: Biofilm Formation; Acinetobacter baumannii; Pili Biogenesis; Two-Component Regulatory Systems
More Like This
30.
Xue, Jianli.
Comparison of ascovirus and baculovirus genomes and their replication and gene expression strategies.
Degree: PhD, Microbiology, 2011, Miami University
► A new member of the newly discovered insect virus family Ascoviridae, Spodoptera…
(more)
▼ A new member of the newly discovered insect virus family Ascoviridae, Spodoptera frugiperda ascovirus-1d (SfAV-1d) was identified from South Carolina, USA. The genome size of SfAV-1d is estimated to be about 100 kb which makes SfAV-1d the smallest ascovirus genome so far. SfAV-1d is closely related to the previously reported SfAV-1a with 99% DNA sequence identity to SfAV-1a. A deletion of 14 kb was found in the SfAV-1d genome that corresponds to the inverted repeat region in SfAV-1a. Cloning and sequencing revealed that the deleted region is highly variable in the SfAV-1d genome with different lengths deleted in individual isolates. SfAV-1d has a narrower host-range than SfAV-1a and it can only develop full infections in S. frugiperda but not in S. exigua in which SfAV-1a is highly infectious. In order to understand better ascoviruses, ascoviruses gene transcription and expression strategies were studied and compared with baculoviruses. The DNA polymerase gene of SfAV-1d was demonstrated to be an early gene while the major capsid protein gene was confirmed to be a late gene. Three RNA polymerase homologues were found in the SfAV-1d genome. In vitro transcription assays showed that an early gene of SfAV-1d was transcribed by the nuclear extract from Sf21 insect cells while a late gene of SfAV-1d was not, suggesting that host RNA polymerase transcribes early genes of SfAV-1d while late gene transcription needs viral factors. Therefore, SfAV-1d follows the same transcription patterns as baculoviruses: early genes are transcribed by the host RNA polymerase while late genes transcription needs viral factors. We further characterized specifically a baculovirus late gene gp37 expression to understand the gene expression strategies. The 3’ untranslated region (UTR) of gp37 gene from the well-studied baculovirus Autographa californica Multicapsid NPV (AcMNPV) was studied. It has multiple polyadenylation sites and can reduce polyhedron production at the polyhedrin locus without changing the total amount of protein expressed.
Advisors/Committee Members: Cheng, Xiao-Wen.
Subjects: Molecular Biology; Virology
Keywords: ascovirus, baculovirus, genomes comparison, replication and gene expression strategies
More Like This
