Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform
Transcriptional fusions with gfp driven by the promoter region of mbla (NE2571) in pPRO/mbla4 and clpB (NE2402) in pPRO/clpb7 were used to transform the ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718). The two genes were chosen because their transcript levels were found at much higher levels in N. europaea in response to oxidation of chloroform and chloromethane. In N. europaea transformed with pPRO/mbla4, green fluorescent protein (GFP)-dependent fluorescence increased from 3- to 18-fold above control levels in response to increasing chloroform concentrations (7 to 28 μM), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.5–7.5 mM). The GFP-dependent fluorescence of N. europaea transformed with pPRO/clpb7 also showed an increase of 6- to 10-fold in response to chloroform (28–100 μM) but did not respond to H2O2. Our data provide proof of concept that biosensors can be fabricated in ammonia-oxidizing bacteria using “sentinel” genes that up-regulate in response to stress caused either by co-oxidation of chlorinated solvents or by the presence of H2O2. The fabricated biosensors had a consistent concentration-dependent response to chloroform; however, these did not respond to other chlorinated compounds that cause similar cellular stress.
KeywordsNitrosomonas europaea Biosensor Green fluorescent protein Chloroform Hydrogen peroxide
This research was funded through an NSF biocomplexity grant number 0412711. The authors thank the laboratory of L.W. Lindow for the pPROBE-NT vector used in this work. We wish to also thank C. Bayne for the use of his laboratory facilities and R. Bender for his help. We thank S. Long for her technical assistance funded through an undergraduate internship from the Subsurface Biosphere Initiative at OSU.
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