Browsing by Subject "Pseudomonas aeruginosa"

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    Comparison Of Oxygen Sensitivities Of Nitrous Oxide Reductase Of Pseudomonas aeruginosa PAO1 and Pseudomonas stutzeri ZoBell
    (2019-12) Vishwanathan, Nisha
    Nitrous oxide, a potent greenhouse gas and a key agent in stratospheric ozone depletion, can be produced as an end or intermediate product of denitrification or as a byproduct of nitrification. Some denitrifiers can reduce exogenous N2O to N2 by using nitrous oxide reductase (N2OR) encoded by nosZ gene. The activity of N2OR is greatly decreased in the presence of oxygen (O2) gas, although several strains were reported capable of reducing N2O under aerobic conditions. Based on our studies, we hypothesize that O2 is an un-competitive inhibitor for N2OR. To better understand this mechanism, Clark-type microsensors and H-shaped chambers were used to calculate N2O and O2 reduction rates of various bacterial strains. From microsensor studies, we identified Pseudomonas stutzeri strain Zobell and Pseudomonas aeruginosa strain PAO1 having O2 tolerant and O2-sensitive N2OR, respectively. Gene knock out of PAO1 and ZoBell nosZ genes ensured null N2OR activity by microsensor assay. The gene nosZ originated from the same strain or different strain was re-introduced via plasmid to the knockout mutants to clarify the role of NosZ on the N2OR activities. The phenotypes of the wildtype and mutants were analyzed by monitoring N2O and O2 consumption in real time using microsensors. PAO1ΔnosZ_p.Z.nosZ and ZoBellΔnosZ_p.P.nosZ were both unexpectedly found to be N2OR - oxygen sensitive which rejects our hypothesis that the difference in nosZ genes is responsible for the difference in oxygen sensitivities of N2OR. However, nosZ genes were re-introduced into original positions and switched on the genome to further understand ZoBell N2OR’s oxygen tolerance mechanism. PAO1ΔnosZ_g.P.nosZ and ZoBellΔnosZ_g.Z.nosZ showed the N2O and O2 consumption behaviors exactly as the wildtype as expected: i.e., their N2OR were oxygen-sensitive and -tolerant, respectively. nosZ switch on the genome, between PAO1 and ZoBell unexpectedly did not show any N2OR activity based on microsensor studies. This implies the need for further exploration and optimization of genetic manipulation of all nos genes in the cluster to fully understand the oxygen tolerance mechanism of ZoBell.
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    Effects of deletion mutations in aguA on fitness and survival of Pseudomonas aeruginosa cystic fibrosis isolates
    (2018-08) McCurtain, Jennifer
    Pseudomonas aeruginosa is a pathogen commonly associated with the disease cystic fibrosis (CF) due to the bacterium’s ability to adapt to numerous environments. These adaptations include mutations that benefit the pathogen such as antibiotic resistance and host immune evasion. Five P. aeruginosa isolates from CF sputum were found to have deletion mutations in aguA that encodes the agmatine deiminase for this species. These mutations cause the cationic molecule agmatine to accumulate both within and outside the cell. The purpose of this thesis was to determine the effects of agmatine accumulation on fitness and survival of P. aeruginosa in the context of CF. Compared to the isogenic strains with a native aguA, the mutations led to decreased susceptibility to cationic antibiotics (aminoglycosides and polymyxins). At sub-minimal inhibitory concentrations (MIC) of these antibiotics, growth of the aguA+ strains was delayed and stunted more than the aguA mutants. This growth phenotype at these sub-MICs was also seen when Mg2+ and Ca2+ concentrations in the growth medium were reduced. In an acute pneumonia the aguA mutant recruited fewer neutrophils to the murine lungs relative to the aguA+ strain. When testing the ex vivo bronchial epithelial cell response to LPS, agmatine caused reduced IL-8 production in response to LPS in a dose-dependent manner, with the higher concentration of agmatine (100 μM) eliciting a response similar to the negative control without LPS. Together, these data show that the mutations in aguA that prevent the bacteria from breaking down agmatine may be beneficial to the P. aeruginosa isolates while colonizing the CF airways.