Masters Thesis

Development of a Genetic System for the Mn(ii)-oxidizing Bacterium Erythrobacter Sp. Sd21 for Transposon Mutagenesis and Inducible Protein Expression

Manganese (Mn) oxides play an important role in the biogeochemical cycling of carbon, sulfur, and iron, and can also act as a sorbent or oxidant for various toxic trace metals. the formation of Mn oxides is primarily driven by Mn-oxidizing microbes, as abiotic Mn oxidation is slow. Erythrobacter sp. SD21 is unique among Mn-oxidizing bacteria, since it carries a novel Mn(II)-oxidizing protein (MopA). To better study MopA and Mn oxidation in this strain, a genetic system was developed in Erythrobacter sp. SD21. Transformation efficiencies were tested for three broad host-range plasmids and a transposon carrying plasmid (pUT/mini-Tn5Km ) through both chemical transformation and electroporation. Following uptake of both broad host-range and transposon carrying plasmids, two applications of the genetic systems were explored: transposon mutagenesis and inducible protein expression. Transposon-insertion mutants were produced and analyzed through inverse PCR and sequencing to identify genes associated with a change in Mn oxidation. A mutant with reduced Mn(II)-oxidation had a transposon insertion in a chemotaxis receptor-related gene. The potential for inducible protein expression was developed by cloning the sequence coding the catalytic domain of MopA into pJN105, using the Polymerase Incomplete Primer Extension (PIPE) cloning method.

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