Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA′::lux fusions (original) (raw)
Related papers
2002
A set of genetically engineered Escherichia coli strains was constructed, in which the promoter of the fabA gene is fused to Vibrio fischeri luxCDABE either in a multi-copy plasmid or as a single copy chromosomal integration. The fabA gene codes for b-hydroxydecanoyl-ACP dehydrase, a key enzyme in the synthesis of unsaturated fatty acids, and is induced when fatty acid biosynthesis pathways are interrupted. A dose-dependent and highly sensitive bioluminescent response to a variety of chemicals was controlled by the fadR gene. A tolC mutant E. coli host displayed generally lower detection threshold for toxicants. A chromosomal integration of a single copy of the fabA%::lux fusion led to a markedly lower background luminescence, but did not yield an improvement in overall performance. It is proposed that these or similarly constructed reporters of fatty acid biosynthesis inhibition may serve as novel microbial toxicity biosensors.
Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains
Sensors and Actuators B: Chemical, 2003
Genetically modified bacteria, engineered to generate a quantifiable signal in response to pre-determined sets of environmental conditions, may serve as combined sensing/reporting elements in whole-cell biosensors. We have compared two of the several available reporter genes in such cells: green fluorescent proteins (GFPs) (Aquorea victoria gfp) and bioluminescence (Vibrio fischeri luxCDABE) genes, fused to either SOS (recA) or heat shock (grpE) promoters. In both cases, bacterial bioluminescence allowed faster and more sensitive detection of the model toxicants; the fluorescent reporter proteins were much more stable, and following long-term exposure allowed detection at levels similar to that of the bioluminescent sensors. From the two green fluorescent proteins tested, enhanced GFP (EGFP) displayed a more rapid response and higher signal intensity than GFPuv. To combine the advantages of both reporter functions, representatives of both types were jointly encapsulated in a sol-gel matrix and immobilized onto a glass surface, to generate a bioluminescent toxicity and a fluorescent genotoxicity sensor. The dual-function sensor detected both toxic and genotoxic model compounds with no interference from the co-immobilized member. #
International Workshop on Monitoring and Sensor for Water Pollution Control, 2007
The increasing of poisons discharged in water body causes more and more serious harm to the environment. In order to evaluate the whole toxicity of poisonous pollutants in water, it is necessary to use rapid and sensitive analytical methods. Bioassay based on bioluminescent bacteria test is a good and promising method to evaluate toxicity. Comparing to natural bioluminescent bacteria, recombinant bioluminescent bacteria test reveals its advantages in more convenient test condition and higher sensitivity. Constructing different kinds of recombinant bioluminescent bacteria, we can also perform diverse evaluations for various purposes, including specifically identifying type of pollutants and toxicity. The RecA protein of E. coli is involved in important cellular functions such as cell division, recombination-repair, mutagenesis, and phage induction, and recA promoter is among the "strongest" E. coli promoters. In this study, a recombinant plasmid pUCD-recA for genetic toxicity evaluation of environmental pollutants was constructed by fusing the DNA damage-inducible promoter recA to the Vibrio fischeri luxCDABE operon. The PCR analysis and DNA sequencing indicated that the recA promoter had been fused into the multiple cloning site of the promoterless luxCDABE plasmid, pUCD615 and the recombinant for genetic toxicity evaluation of environmental pollutants was constructed successfully. The recombinant bioluminescent strain was named THZH07.
Monitoring subtoxic environmental hazards by stress-responsive luminous bacteria
Environmental Toxicology and Water Quality, 1996
A novel approach to toxicant detection is described, based on monitoring bacterial reactions to environmental threats. In response to such stress, various defense mechanisms are turned on by initiating gene transcription at specific DNA sites known as promoters. To follow this transcription sensitively, such promoters were genetically fused in fscherichia coli to the lux (luminescence) genes from the bacterium Vibrio fischeri. The bacteria thus engineered now produce light in response to different environmental insults; this light is easy to measure and quantify.
Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions
Applied and environmental microbiology, 1994
Heat shock gene expression is induced by a variety of environmental stresses, including the presence of many chemicals. To address the utility of this response for pollutant detection, two Escherichia coli heat shock promoters, dnaK and grpE, were fused to the lux genes of Vibrio fischeri. Metals, solvents, crop protection chemicals, and other organic molecules rapidly induced light production from E. coli strains containing these plasmid-borne fusions. Introduction of an outer membrane mutation, tolC, enhanced detection of a hydrophobic molecule, pentachlorophenol. The maximal response to pentachlorophenol in the tolC+ strain was at 38 ppm, while the maximal response in an otherwise isogenic tolC mutant was at 1.2 ppm. Stress responses were observed in both batch and chemostat cultures. It is suggested that biosensors constructed in this manner may have potential for environmental monitoring.
A Dual-Color Bacterial Reporter Strain for the Detection of Toxic and Genotoxic Effects
Engineering in Life Sciences, 2006
To genetically engineer a bacterial whole cell dual-function toxicity/genotoxicity bioreporter system, a plasmid was constructed containing two independent fusions of stress-responsive promoters (of the recA and grpE genes) to green and red fluorescent protein reporter genes, respectively. An Escherichia coli strain harboring this plasmid exhibited distinct green fluorescence in response to the presence of the SOS inducing agent nalidixic acid, and red fluorescence in reaction to ethanol. The different fluorescent responses, which exhibited little or no overlap, were quantified by microtiter plate fluorometry, confocal microscopy, and fluorescence emission spectroscopy. Mutations in lexA and rpoH, which affected the E. coli SOS and heat shock systems, respectively, abolished the green and red fluorescence. Similar constructs may serve as biological entities in future wholecell toxicity/genotoxicity biosensor systems.
Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation
Microbial Biotechnology, 2010
Bioluminescent bacterial sensors are based upon the fusion of bacterial bioluminescence (lux) genes, acting as a reporter element, to selected bacterial stress-response gene promoters. Depending upon the nature of the promoter, the resulting constructs react to diverse types of environmental stress, including the presence of toxic chemicals, by dosedependant light emission. Two bacterial sensors, harbouring sulA::luxCDABE and grpE::luxCDABE fusions, activated by the model chemicals nalidixic acid (NA) and ethanol, respectively, were subjected to molecular manipulations of the promoter region, in order to enhance the intensity and speed of their response and lower their detection thresholds. By manipulating the length of the promoter-containing segment (both promoters), by introducing random or specific mutations in the promoter sequence or by duplicating the promoter sequence (sulA only), major improvements in sensor performance were obtained. Improvements included significantly enhanced sensitivity, earlier response times and an increase in signal intensity. The general approaches described herein may be of general applicability for optimizing bacterial sensor performance, regardless of the sensing or reporting elements employed.
A panel of stress-responsive luminous bacteria for the detection of selected classes of toxicants
Water Research, 1997
A panel of bacteria, each genetically engineered to respond by increased luminescence to a different tyl:¢ of environmental stress, is presented. Members of the panel were shown to be sensitive to several groups of chemicals including phenols, halomethanes and several oxidants. The increase in light emission depended upon toxicant concentration and could, thus, be used to calculate a characteristic value, EC~, designating the sample concentration causing a two-fold luminescence induction. In almost all cases, EC200 values, were lower than the corresponding Microtox TM ECs0 values, indicating a generally higher sensitivity. One of the panel members, DPD2794, a designated DNA-damage sensor, responded within 2 h to the presence of genotoxicants, including metabolically activated nitropropane. It is suggested that these or similarly constructed strains can be used for the rapid and sensitive detection of potentially toxic and genotoxic pollutants and that the concept of genetically engineering a panel of microbial toxicity sensors can readily be implemented for environmental monitoring. © 1997 Elsevier Science Ltd