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- Title
- AN EFFORT TO DISCOVER NOVEL BACTERIAL DSZ GENES TO HELP IMPROVE THE BIODESULFURIZATION OF CRUDE OIL
- Creator
- Salazar, Joelle Krieger
- Date
- 2011-04-25, 2011-05
- Description
-
In this thesis study, soil samples were procured from various locations thought to contain high petroleum content, such as gas stations. Those...
Show moreIn this thesis study, soil samples were procured from various locations thought to contain high petroleum content, such as gas stations. Those samples were enriched for those that contained bacteria that were able to metabolize sulfur-containing compounds, such as DBT. The bacteria were then subjected to various experiments to determine if they possess any or all of the genes (dsz) encoding the three enzymes of the dibenzothiophene desulfurization pathway, as compared with R. erythropolis IGTS8. This feat was accomplished by using both growth experiments on medium containing dibenzothiophene as the sole sulfur source and genomic DNA extraction followed by PCR with dszABC universal primers. The resultant amplified DNA was then ligated to pGEM®-T Easy vector and transformed into electrocompetent E. coli cells. The transformants were subsequently screened and sent for sequencing. Using Blast, the sequences were analyzed. No obvious dsz homologies were identified although homologues in some cases to enzymes involved in sulfur metabolism were found. Several isolates were found to have dsz-like activity via the growth experiments as well as the Gibbs assay, which measures accumulation of the end product of the dsz-encoded pathway, 2-HBP Soil sample #32 showed promise in growth experiments in that the culture grew well in M9 with DBT as the sole sulfur source at temperatures of 30 °C, 37 °C, 45 °C, and 55 °C. Streaking the culture discovered two colonies with distinct morphology that were designated 32-W and 32-Y, for white and yellow colonies. 32-W and 32-Y also grew independently in M9 with DBT. Positive Gibbs Assays were obtained from the cultures 32-mixed, 32-W, and 32-Y.
M.S. in Biology, May 2011
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- Title
- PRFA-LIKE TRANSCRIPTION FACTOR, LMO0753, CONTRIBUTES TO VIRULENCE, L-RHAMNOSE UTILIZATION, AND PERSISTENCE OF LISTERIA MONOCYTOGENES HUMAN FOODBORNE OUTBREAK LINEAGES
- Creator
- Salazar, Joelle Krieger
- Date
- 2013, 2013-12
- Description
-
Listeria monocytogenes is a foodborne bacterial pathogen and the causative agent of a human and animal disease, listeriosis. Among the three...
Show moreListeria monocytogenes is a foodborne bacterial pathogen and the causative agent of a human and animal disease, listeriosis. Among the three major genetic lineages of L. monocytogenes (i.e. LI, LII, and LIII), LI and LII are predominantly associated with foodborne listeriosis outbreaks, whereas LIII is rarely implicated in human infections. In a previous study, we identified a Crp/Fnr family transcription factor lmo0753 that was highly specific to outbreak-associated LI and LII but absent from LIII. Lmo0753 shares two conserved functional domains including a DNA-binding domain with the well-characterized master virulence regulator PrfA in L. monocytogenes. In this study, lmo0753 deletion and complementation mutants were constructed in two fully sequenced L. monocytogenes LII strains 10403S and EGDe, and compared virulence-associated mechanisms of flagellar motility, phospholipase C production, hemolysis, and intracellular growth of the mutants and their respective wild types. Persistence-associated mechanisms of growth, biofilm production, attachment and soil survival were also assayed. Results suggested that lmo0753 plays a role in some virulence- and persistence-associated mechanisms in both EGDe and 10403S. More importantly, it was found that deletion of lmo0753 led to the loss of L-rhamnose utilization in EGDe but not in 10403S. Transcriptomic comparison of the EGDe lmo0753 deletion mutant and the wild type incubated in Phenol-red medium containing L-rhamnose as the sole carbon source revealed 126 (4.5%) and 546 (19.5%) out of 2,798 genes in the EGDe genome that were up- and down-regulated for more than 2-fold, respectively. Genes involved in biotin biosynthesis, general stress response and rhamnose metabolism were shown to be differentially regulated by Lmo0753. Findings from this study may partially explain why xvi LIII of L. monocytogenes is underrepresented in the environment and rarely associated with human listeriosis outbreaks due to the inability of rhamnose utilization.
PH.D in Biology, December 2013
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