Date of Award

2010

Document Type

Thesis

Degree Name

Master of Science

School

College of Arts and Sciences

Program

Biology MS

First Advisor

Robert Zdor

Abstract

Pseudomonas putida ATH2-1RI/9, a bacterium that colonizes plant roots, can suppress weed seedling growth of velvetleaf by producing hydrogen cyanide in the rhizosphere. The operon hcnABC constitutively expresses HCN synthase, an enzyme which catalyzes glycine to form carbon dioxide and hydrogen cyanide, a secondary metabolite. Cyanogenesis is influenced by several environmental factors including iron, phosphate, and oxygen-limiting conditions. This study used a hcnABC::luxAB gene fusion in Pseudomonas putida ATH2-1RI/9 involving the insertion of a promoterless luxAB gene into the hcnABC operon, which results in bacterial luciferase expression instead of HCN synthase. Bioluminescence by this reporter strain was compared to cyanide production by the wild-type P. putida ATH2-1RI/9. The reporter strain, Pseudomonas putida ATH2-1RI/9, was grown in four different cultures as well as rhizosphere conditions: aerobic +FeCl3, microaerobic +FeCl3, aerobic –FeCl3, and microaerobic –FeCl3. In two of three experiments, aerobic+FeCl3 stationary phase cultures had statistically higher levels of HCN in comparison to log phase cultures. In two of three of the culture experiments, microaerobic+FeCl3 log phase cultures had statistically higher levels of gene expression in comparison to the other three treatment groups. A possible explanation for this result is that the anaerobic regulator ANR, which acts as an iron sensor in the hcnABC operon, converts to its active form under low oxygen supply. The effect of iron and oxygen levels on bioluminescence and cyanide production in the rhizosphere was less clear. Further research in the rhizosphere environment will clarify some of the unknown variables that could have been involved in triggering the hcnABC gene expression of this soil microbe Pseudomonas putida ATH2-1RI/9.

Subject Area

Pseudomonas., Gene expression.

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