ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.

Background: The incidence of Pseudomonas aeruginosa bacteremia has not been defined in a population-based investigation. Methods: We performed a retrospective, population-based incidence study using resources of the Rochester Epidemiology Project of Olmsted County, Minnesota. We identified all Olmsted County residents with P. aeruginosa bacteremia between January 1, 1997, and December 31, 2006, by microbiology records in the only 2 laboratories in the county. Medical records were reviewed to confirm diagnosis, residency status, and clinical characteristics. Results: Age-adjusted incidence per 100,000 person-years was 10.8 (95% confidence interval [CI], 7.5-14.0) in men and 3.7 (95% CI, 2.2-5.2) in women for total P. aeruginosa bacteremia, and 8.4 (95% CI, 5.5-11.2) in men and 2.5 (95% CI, 1.3-3.8) in women for monomicrobial P. aeruginosa bacteremia. There was no significant change in incidence of total P. aeruginosa bacteremia during the past decade (P = .418). Incidence increased exponentially with age, with a greater magnitude of increase in men compared with women for total and monomicrobial P. aeruginosa bacteremia (P = .007 and P = .015, respectively). In patients with monomicrobial P. aeruginosa bacteremia, the median age was 69 years, and 78.4% of cases were either nosocomial or health care associated. Most patients had multiple comorbid conditions. The urinary tract was the most common primary source of infection. The 28-day all-cause mortality of monomicrobial P. aeruginosa bacteremia was 25.5%. In vitro susceptibility to ciprofloxacin was 95.3%. Conclusion: To our knowledge, this is the first population-based incidence study of P. aeruginosa bacteremia. The incidence of P. aeruginosa bacteremia has remained stable during the past decade. Fluoroquinolone susceptibility is high among local P. aeruginosa bacteremia isolates.

The identification of antimicrobial compounds that inhibit multidrug-resistant (MDR) bacteria continues to be a significant area of research to combat the public health threat posed by MDRs. Pseudomonas aeruginosa (PA) is a Gram-negative, MDR bacterium found within both the environment and healthcare settings. Our laboratory has observed another Gram-negative bacterium, Proteus species, exerts an interesting polymicrobial interaction with Pseudomonas aeruginosa by compromising the quorum sensing (QS) factor, pyocyanin. Production of pyocyanin by Pseudomonas is a main method the bacterium uses for communication and coordination of virulence. In this study, we examined Proteus mirabilis (PM) and Proteus vulgaris (PV13) effectiveness to compromise pyocyanin production in Pseudomonas aeruginosa. Through the use of pyocyanin isolation and extraction techniques, data was gathered for Pseudomonas aeruginosa’ s molecular interaction with the two Proteus bacteria described. Further observations were made on microbial interaction between Pseudomonas aeruginosa and Proteus mirabilis through measuring the rate of metabolic activity, twitching motility rate, and observing differences in biofilm formation. Using the data obtained from this research, we hope to identify new methods of controlling Pseudomonas virulence and infection by inhibiting its ability to communicate and coordinate in polymicrobial infections.

East Fork Poplar Creek (EFPC) in Oak Ridge, TN was highly contaminated with elemental mercury in the 1950 and 1960. The area is still experiencing the effects of mercury contamination, and researchers are searching for ways to remediate the EFPC. One possible mechanism for bioremediation is the use of biogenic Mn oxides to remove heavy metals from water systems. Native Pseudomonas bacteria species were isolated from EFPC in order to examine biogenic Mn oxides production and bioremediation of Oak Ridge slurries. Pseudomonas isolates did produce Mn oxides which bound to mercury, and mercury bound to organic matter significantly decreased. However, after a significant decrease of dissolved mercury, dissolved mercury was cycled back into the water system on day 10. Given a longer experimental timeline, biogenic Mn oxides have the potential to decrease mercury cycling.

No description available.

Plants rely on innate immunity to perceive and respond to pathogenic microbes. Pathogenic microbes suppress and evade plant immune responses to obtain nutrients and multiply resulting in plant diseases and death. One battleground for the arms race between plants and microbial invaders is located in the leaf intercellular space, specifically between Pseudomonas bacteria and Arabidopsis. This thesis seeks to understand the virulence mechanisms that allow Pseudomonas bacteria to grow within the leaves of Arabidopsis and how the plant immune response reduces pathogen growth and reproduction. Some plant pathogens produce specific extracellular polysaccharides to potentially enhance pathogenicity during infection of plants. The objective of this thesis is to understand the importance of biofilms for Pseudomonas success and determine if Arabidopsis suppresses biofilm formation as part of the plant immune response. It was hypothesized that biofilm formation contributes to Pseudomonas success in planta and Arabidopsis suppresses biofilm formation during PAMP-Triggered Immunity (PTI) to reduce bacterial growth. Wild-type plants and defense mutants were infiltrated with flg22 or mock (water) treatments to induce or mock-induce PTI in plants, followed by observing GFP-expressing Pseudomonas via florescence microscopy to determine if biofilm-like aggregate formation was occurring. In vivo studies in this thesis indicate that biofilm-like aggregate formation contributes to bacterial success during Arabidopsis infection. Additionally, the phytohormone, salicylic acid (SA), accumulates in leaf intercellular spaces of resistant plants during PTI that suppresses biofilm formation, suggesting that SA acts as an anti-microbial and anti-biofilm agent that contributes to the suppression of pathogen growth during plant defense. / Thesis / Master of Science (MSc)

Biofertilizer (or microbial soil inoculants) may be used to reduce current fertilizer inputs (organic or conventional methods), while maintaining or improving crop plant growth and yield. Pseudomonas putida is a plant-growth promoting rhizobacterium (PGPR) that solubilizes inorganic phosphorous or mineralizes organic phosphates, produces siderophores (enhancing Fe availability for plants, reducing Fe access to pathogenic fungi), and is known to improve plant growth by assisting with nutrient availability, synthesis of plant hormones (indole acetic acid regulation production and/or degradation, or ethylene regulation through aminocyclopropane carboxylate deaminase activity), and acts as a biological control of several plant pathogens and pests (Rhizoctonia solani, Bemisia tabaci). Recommendations for inoculum population density, application timing, and species of PGPR, vary mainly based on geographic and weather conditions, and their relationship to each horticultural crop, which needs to be better understood. Two studies were conducted in 2014 and 2015 at the Southern Illinois Horticulture Research Center to determine the optimum application timing and dosage of Pseudomonas putida strains 17-29 and G11-32 to improve plant growth and yield on two important horticultural crops: field grown ‘Revolution’ bell peppers and greenhouse grown ‘Rocky Top’ determinate tomatoes. Field pepper results indicated that the inoculum population density increased early-season vigor, plant height and stem caliper (P < 0.05), as well as late-season plant vigor, height (cm), leaf chlorophyll index (SPAD), and stem caliper (mm) (P < 0.05) for inoculum population density. Although early harvests (first two harvests) fruit yield increased with rhizobacteria inoculum population density (P < 0.05) for total fruit weight (2014, 2015) and number (2014), late-season fruit yields (last three harvests) were not affected. Combined fruit yield total weight (all five harvests) also increased (P < 0.05) by inoculum population density in the order: 10^0 < 10^3 < 10^5 < 10^7 < 10^9. It appears that higher early-season and combined harvest yields were higher resulting from increasing inoculum population densities were greater due to increased early season growth from the bacterial treatments during plug growth phase. Tomato results indicated that inoculum population density increased early growth and late growth vigor, height (cm), leaf chlorophyll index (SPAD), leaf number, flower number, fresh leaf weight (g), fresh stem weight, and dry leaf weight (P < 0.05). Tomato growth was effected by inoculum population density treatment however, yields differences were not observed.

No description available.

Life on Earth is incredibly diverse. The process of diversification that gives rise to this diversity is not the same for all lineages. Diversification is often driven by ecological opportunity. Pseudomonas aeruginosa is an opportunistic pathogen present in a variety of environments that causes chronic lung infections in cystic fibrosis (CF) patients. It diversifies rapidly within the CF lung and CF lung-like environments. Here we aim to assess both ecological and genetic factors in diversification of several strains of P. aeruginosa. We evolved 12 replicate populations of 8 different strains of P. aeruginosa in a nutritionally complex (LB) and simple environment (MIN) for 750 generations. We then measured diversity over time by observing the number of colony morphologies in each population every 250 generations. We also measured competitive fitness relative to the ancestor for endpoint populations. To provide a more complete analysis, phylogeny was factored into our statistical models. First, we found no significant differences in diversification between populations evolved in LB versus MIN media. Ancestor population size had no significant effect on diversification. We found that in both selection environments, CF strains diversified less than environmental strains, but this difference was marginally significant and only present when comparing these two niches directly and excluding acute strains. Finally, we found no correlation between gains in fitness and endpoint diversity. Our results suggest that diversification is limited by niche specialization (domestication) of P. aeruginosa to the CF lung.