Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in response to co-oxidation of chloroform

Appl Microbiol Biotechnol (2009) 82:11791185 DOI 10.1007/s00253-009-1914-y

ENVIRONMENTAL BIOTECHNOLOGY

Construction of recombinant Nitrosomonas europaea expressing green fluorescent protein in responseto co-oxidation of chloroform

Barbara O. Gvakharia & Peter J. Bottomley &

Daniel J. Arp & Luis A. Sayavedra-Soto

Received: 13 January 2009 /Revised: 5 February 2009 /Accepted: 8 February 2009 /Published online: 27 February 2009 # Springer-Verlag 2009

Abstract 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 M), and from 8- to 10-fold in response to increasing hydrogen peroxide concentrations (2.57.5 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 (28100 M) 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.

Keywords Nitrosomonas europaea . Biosensor .

Green fluorescent protein . Chloroform . Hydrogen peroxide

Introduction

Pollution that results from increases in population, urbanization, and industrialization is a pressing problem that can affect ecologically important microbes. The search for solutions to this problem demands relatively simple, specific, sensitive, and reliable methods to detect a potential pollutant at levels that influence microbial communities. Bacteria containing sensitive promoterreporter gene fusions responding to a pollutant can serve as specific biosensors for that particular compound. A promising method is to use biosensors as indicators so that steps can be taken to prevent damage to microbial communities in engineered processes such as in bio-pharmaceutics production, in bio-treatments, or in the reclamation of wastewater (Ron 2007). Whole-cell biosensors ideally contain an environmentally sensitive promoter...