Protein transplant: Cloning genes for two NtaA proteins from Gordonia sp. NB4-1Y

Some telomerisation-based aqueous film-forming foams (AFFF) used to fight forest fires contain 6:2 fluorotelomer sulfonate (6:2 FTS), or precursors that degrade to 6:2 FTS, which has been found to accumulate in groundwater systems and appear in surface waters and in the livers of fish following AFFF deployment. By examining the microbial metabolism of organosulfur compounds such as 6:2 FTS, therein lies potential for development of bioremediation tools. To date, the metabolism of 6:2 FTS has been observed in the soil bacterium Gordonia sp. NB4-1Y, an organism that was isolated in Kamloops, British Columbia Canada. When NB4-1Y was supplied 6:2 FTS as the sole sulfur source, the differential production of two nitrilotriacetate (NtaA) monooxygenases was observed when compared to a MgSO4 supplemented culture. This led to the hypothesis that these enzymes initiate the desulfurization reaction used by NB4-1Y for 6:2 FTS biodegradation. To examine this hypothesis, DNA was isolated from NB4-1Y using a modified Streptomyces genomic DNA isolation protocol, and this DNA was used as a template to amplify the protein-coding region for each NtaA enzyme by polymerase chain reaction (PCR). PCR primers were designed to introduce restriction enzyme sites to the 5' and 3' ends of the genes to allow for subsequent directional cloning. These genes were then transferred into DH5α competent E. coli using a pBluescript cloning vector for sequencing. Once the gene sequence data has been obtained, the gene inserts will be moved into a pMAL-c2 expression vector that will allow for the production and purification of large quantities of the NtaA enzymes for characterization. The enzymes will be purified from an E. coli expression host using affinity chromatography by taking advantage of a maltose binding protein introduced to the N-terminal region of the target proteins using the pMAL-c2 expression vector, a binding protein that has an affinity for amylose. Once purified, spectrophotometric assays monitoring the rate of NADH oxidation will be performed to definitively determine if these enzymes are involved in 6:2 FTS desulfurization in Gordonia sp. NB4-1Y, helping us further understand the potential value of Gordonia as a bioremediation tool.