Browsing by Author "Jonathan Olson, Committee Member"

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Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5
(2009-04-20) House, Alan; James Brown, Committee Member; Jonathan Olson, Committee Member; Steven Peretti, Committee Member; Michael Hyman, Committee Chair
Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5. (Under the direction of Michael R. Hyman.) Cometabolic transformations are unable to support cell growth. This process is often catalyzed by, and superimposed upon, enzyme systems expressed to catalyze carbon- or energy-yielding reactions. Biodegradation of the gasoline additive methyl tertiary butyl ether (MTBE) is known to be superimposed upon a propane-oxidizing system in the aerobic bacterium Mycobacterium austroafricanum (vaccae) JOB5. Taking a whole-cell approach, we investigated the physiology of propane metabolism and MTBE cometabolism in this strain. Multiple major gasoline components are frequent co-contaminants with MTBE in the environment, and we determined the impacts of these hydrocarbons on the cometabolism of both MTBE and its commonly encountered metabolite, tertiary butyl alcohol (TBA). Most of the hydrocarbons tested supported cell growth and concurrent MTBE and TBA oxidation occurred without affecting final culture optical density. Results suggest hydrocarbon-grown cells simultaneously expressed more than one alkane-oxidizing enzyme system. Nuclear magnetic resonance spectroscopy (NMR) was used to study the pathway of MTBE oxidation in propane-grown cells of strain JOB5. We confirmed the existence of predicted intermediates, including a hemiacetal, formate and formaldehyde. Hydroxyisobutyraldehyde, a predicted intermediate in MTBE oxidation by some bacterial strains was not detected, despite attempts to promote its accumulation. As the pathway of MTBE oxidation progressed, the rate of daughter product oxidation decreased, which may be preventing MTBE-dependent cell growth in strain JOB5. Propane metabolism was examined using a series of growth experiments and substrate oxidation assays. We observed the simultaneous production, and later consumption of both 1- and 2-propanol during cell growth. This divergent oxidation of propane was apparently followed by the divergent oxidation of propionate and the divergent oxidation of acetone. Our results suggest at least two CO2-fixation steps are involved in propane metabolism in strain JOB5. Finally, we used NMR to contribute to several studies that characterized the pathway of (i) MTBE oxidation by Nitrosomonas europaea or (ii) bacterial oxidation of a fluorinated analog of TBA. The later study identified a compound that may serve as a tracer for TBA degradation in situ.
The Genomic Basis of Bile Tolerance in Lactobacillus acidophilus
(2009-09-25) Pfeiler, Erika Ann; Todd Klaenhammer, Committee Chair; Robert Kelly, Committee Member; Dahlia Nielsen, Committee Member; Jonathan Olson, Committee Member
Global Effects of the Transcriptional Regulators ArcA and FNR in Anaerobically Grown Salmonella enterica sv. Typhimurium 14028s.
(2008-11-25) Evans, Matthew Richard; Hosni M. Hassan, Committee Chair; Craig Altier, Committee Member; Matthew D. Koci, Committee Member; Eric S. Miller, Committee Member; Jonathan Olson, Committee Member
EVANS, MATTHEW RICHARD. Global Effects of the Transcriptional Regulators ArcA and FNR in Anaerobically Grown Salmonella enterica sv. Typhimurium 14028s. (Under the direction of Dr. Hosni M. Hassan.) The purpose of this research was to assess and compare the genome-wide transcriptional profiles and virulence in mice of the global regulators, FNR (Fumarate Nitrate Reductase) and ArcA (Aerobic Respiratory Control) in anaerobically grown Salmonella enterica serovar Typhimurium 14028s. FNR controls the expression of target genes by sensing and responding to the presence or absence of dioxygen via assembly-disassembly of oxygen-liable iron-sulfur clusters, while ArcA is a two-component (ArcA/ArcB), cytosolic redox response regulator. This work demonstrates that FNR is a positive regulator of motility, flagellar biosynthesis, and pathogenesis. An fnr mutant was non-motile, lacked flagella, attenuated for virulence in mice, and did not survive inside macrophages. In S. Typhimurium, as in Escherichia coli, the FNR modulon encompassed the core metabolic and energy functions as well as motility. Salmonella-specific genes/operons regulated by FNR included those required for ethanolamine utilization, newly identified flagellar genes (mcpAC, cheV), several virulence genes in Salmonella pathogenicity island 1 (SPI-1), and the srfABC operon. ArcA serves as a transcriptional repressor/activator coordinating cellular metabolism and motility. An arcA mutant was non-motile, lacked flagella, and was as virulent as the wild-type strain via intraperitoneal challenge in mice. In S. Typhimurium, as in E. coli, the ArcA modulon encompassed the core metabolic and energy functions as well as motility. Salmonella-specific genes/operons regulated by ArcA included those for propanediol utilization, newly identified flagellar genes (mcpAC, cheV), Gifsy-1 prophage genes, and a few virulence genes located in SPI-3 (mgtBC, slsA, STM3784). Regulation by either ArcA or FNR in S. Typhimurium is similar, but distinct from that in E. coli. Genes/operons involved in the succinyl-CoA pathway, fatty acid degradation, flagellar biosynthesis, motility, chemotaxis, cytochrome oxidase complexes are regulated similarly in the two organisms by ArcA. Genes/operons involved in aerobic metabolism, NOâ€¢ detoxification, flagellar biosynthesis, motility and chemotaxis, and anaerobic carbon utilization are regulated similarly in the two organisms by FNR. Herein, we present the first report on the global role of these two redox regulators in S. Typhimurium. According this study, we hypothesize that FNR plays a more heirarchical role than ArcA in pathogenesis and during the transition between aero- and anaerobiosis in the host. Furthermore, when comparing the motility and virulence results from our work on FNR to those on ArcA, we show that the lack of motility does not necessarily correspond to the lack of virulence in S. Typhimurium.
Pathway and Enzymology of Cyclic Ether Biodegradation by Mycobacterium vaccae JOB5
(2007-03-22) Lan, Renny Shang-Lun; Jonathan Olson, Committee Member; Francis L. de los Reyes III, Committee Member; Michael R. Hyman, Committee Chair; Amy Grunden, Committee Member
In this study we investigated the cometabolic oxidation of tetrahydrofruran (THF), 1,4-dioxane (14D) and other cyclic ethers by alkane-grown Mycobacterium vaccae JOB5. An initial screening demonstrated this strain oxidized all six ethers tested after growth on propane, n-butane, n-pentane, isobutane and isopentane. Ether-degrading activity was limited in dextrose-grown cells or in alkane-grown cells incubated with ethers and acetylene. Oxidation of THF and 14D was further characterized using propane-grown cells. Propane competitively inhibited THF oxidation and γ-butyrolactone (γBL) accumulated and was also consumed during THF oxidation. In contrast, no products were detected during 14D oxidation. Oxidation of γBL and the 3-hydroxy homolog of its unstable but presumed precursor, 2-hydroxytetrahydrofuran, were largely unaffected by acetylene. This suggested other enzymes co-expressed with the acetylene-sensitive, propane-oxidizing monooxygenase contributed to THF oxidation. Although strain JOB5 did not grow on any cyclic ethers tested, slight growth was observed with several lactones while vigorous growth was supported by 4-hydroxybutyrate, the expected immediate product of γBL degradation. The ability of strain JOB5 to assimilate THF metabolites during growth on n-alkanes was examined in carbon-limited batch cultures. Relative to growth on n-pentane alone, the final culture density and protein concentration were doubled in the presence of THF while no stimulation was observed with 14D. Our results are discussed in terms of the overlap between the enzymes and pathway involved in alkane and ether oxidation, their significance to our understanding of cometabolism and their potential impact on approaches for cyclic ether biodegradation in the environment.
Prevalence and Dissemination of Antimicrobial Resistance among Campylobacter jejuni and Campylobacter coli from Meat-Animals
(2009-12-02) Islam, Mohammed Shahidul; Jonathan Olson, Committee Member; Jonathan C. Allen, Committee Member; Sophia Kathariou, Committee Chair
Campylobacter jejuni and C. coli from conventionally grown turkeys have been frequently reported to be resistant to nalidixic acid and ciprofloxacin. To reduce possible contributions of fluoroquinolone use in poultry production to fluoroquinolone resistance in poultry-derived Campylobacter, use of the fluoroquinolone enrofloxacin (trade name, Baytril) was banned in July 2005. However, the impact of this ban on fluoroquinolone resistance in thermophilic campylobacters from turkeys has not been rigorously evaluated yet. In this study, we investigated prevalence of quinolone/fluoroquinolone resistance and multidrug resistance among 1552 Campylobacter isolates (81% C. coli and 19% C. jejuni) derived from 2371 cecal samples of young turkeys (10 days to six weeks of age). The isolates were derived from different flocks and farms, representing three different integrators, from 2002 to 2008. Resistance to ciprofloxacin and nalidixic acid was highly prevalent (585/722, 81%) among isolates from 2002 to August 2005, and prevalence was even higher (779/830, 94%) among isolates from 2006 to 2008, the three surveyed years subsequent to the ban (p<0.0001). Multidrug resistant C. coli (resistant to tetracycline, streptomycin, kanamycin, erythromycin, nalidixic acid and ciprofloxacin) and C. jejuni (resistant to all of the above except erythromycin) represented 46% and 66% of the campylobacters isolated in the periods prior to and subsequent to the enrofloxacin ban, respectively. Multidrug resistance prevalence increased significantly post-ban (p<0.0001) for both C. coli and C. jejuni. Ciprofloxacin MIC determinations suggested that there was no obvious difference in MIC distribution before and after the enrofloxacin ban. Our results suggest that there was no detectable decrease in prevalence of fluoroquinolone resistance among turkey-derived campylobacters within the surveyed three-year period following the enrofloxacin ban. Instead, and for reasons that remain unknown, prevalence values for fluoroquinolone resistance and for multidrug resistance were higher post-ban than pre-ban (p<0.0001). Continued surveys are needed to further evaluate the potential impact of the ban on resistance of campylobacters from turkeys to fluoroquinolones and other antibiotics. Even though fluorioquinolones are no longer permitted for use in poultry, several antimicrobials, for instance tetracycline, continue to be used extensively in conventional production. Naturally competent C. coli C. jejuni are able to acquire foreign DNA from different sources through transformation but the role of transformation in the dissemination of tetracycline resistance in Campylobacter has not been investigated. We therefore investigated transfer of tetracycline resistance from tetracycline resistant C. coli and C. jejuni to tetracycline-susceptible (TS) C. coli and C. jejuni derived from meat animals. Tetracycline-resistant (TR) but kanamycin susceptible (KS) C. coli both from turkey and swine could serve as donors in transformation-mediated transfer of tetracycline resistance to TS C. coli from turkeys. TR and kanamycin-resistant (KR) C. coli from turkeys were unable to serve as donors of tetracycline resistance whereas certain TR KR swine-derived strains successfully transformed tetracycline resistance. None of the TR KR C. jejuni strains could serve as donors and overall C. coli proved to be better donors in transforming tetracycline resistance than C. jejuni. However, a bovine TR KS C. jejuni donors strain was successful in transforming turkey-derived as well as bovine C. jejuni to tetracycline resistance. Further studies are needed to evaluate the impact of transformation in dissemination of tetracycline resistance among C. jejuni and C. coli from animal production systems.
Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii as a Microbial Catalyst.
(2010-11-17) Tirado Acevedo, Oscar; Amy Grunden, Committee Chair; Eric Miller, Committee Member; Jonathan Olson, Committee Member; Mari Chinn, Committee Member