Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 μM, 1.1 μM, and 141 μM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.     

Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.     

Chromosomally located gene fusions constructed in Acinetobacter sp. ADP1 for the detection of salicylate

Acinetobacter sp. ADP1 is a common soil-associated bacterium with high natural competency, allowing it to efficiently integrate foreign DNA fragments into its chromosome. This property was exploited to engineer salicylate-inducible luxCDABE and green fluorescent protein (GFP) variants of Acinetobacter sp. ADP1. Specifically, Acinetobacter sp. ADPWH_lux displayed the higher sensitivity when comparing the two variants (minimum detection c. 0.5-1 microM salicylate) and a faster turnover of the lux marker gene, making it suitable for whole-cell luminescence assays of salicylate concentration. In contrast, the longer maturation and turnover times of the GFP protein make the Acinetobacter sp. ADPWH_gfp variant more suited to applications involving whole-cell imaging of the presence of salicylate. The sensitivity of the luxCDABE variant was demonstrated by assaying salicylate production in naphthalene-degrading cultures. Assays using ADPWH_lux specifically mapped the kinetics of salicylate production from naphthalene and were similar to that observed by high-performance liquid chromatography (HPLC) data. However, ADPWH_lux exhibited the higher sensitivity, when compared with HPLC, for detecting salicylate production during the first 24 h of naphthalene metabolism. These data demonstrate that the engineered Acinetobacter variants have significant potential for salicylate detection strategies in laboratory and field studies, especially in scenarios where genetic stability of the construct is required for in situ monitoring.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins

Here we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsive cda promoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).     

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.     

Two novel bacterial biosensors for detection of nitrate availability in the rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5alpha and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 microM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 microM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenase-peroxidase coupling reactions

We have developed a simple, whole-cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)-coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 microM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX-related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10-week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.     

Construction of a whole-cell gene reporter for the fluorescent bioassay of nitrate

The development of a whole-cell fluorescence-based biosensor for nitrate is reported. The sensor is Escherichia coli transformed with a plasmid (pPNARGFP) in which the promoter and regulatory regions of the membrane-bound nitrate reductase narGHJI operon (Pnar) are fused to a gfp gene encoding green fluorescent protein (GFP). Pnar-gfp activity was measured at a range of nitrate concentrations using whole-cell GFP fluorescence. The bioassay conditions have been optimized so that the fluorescence intensity is proportional to the extracellular nitrate concentration. The developed bioassay has established that E. coli (pPNARGFP) can be used for the quantitative determination of nitrate in environmental waters without interference from other electron acceptors, e.g., nitrite, dimethyl sulfoxide, trimethylamine-N-oxide and fumerate, and azide, an inhibitor of redox-active proteins.     

Radiation-induced recovery processes in cultured marsupial cells

The ultraviolet sensitivity of Potorous tridactylus male kidney (PtK-2) cells is markedly increased by post irradiation treatment for 24 h with 5 microM emetine (which inhibits protein synthesis by acting on the 40S ribosomal subunit), or with 5 microM cycloheximide (which inhibits by interaction with the 60S subunit), or with the RNA polymerase II inhibitor 5,6-dichloro-1-beta-ribofuranosylbenzimidazole at 50 microM. All 3 treatments give the same sensitivity, while unirradiated cells are little affected. Shortening the time of treatment, or delaying application of the drugs decreases their effect on the same time schedule. Preiirradiation of cells, with no drug treatment in the following 8 h, diminishes the sensitivity to a subsequent irradiation with protein synthesis blocked afterwards. Photoreactivation immediately following such preirradiation eliminates its desensitizing effect. Inhibiting protein synthesis after irradiation also markedly reduces the frequency of UV-induced mutants in the surviving population. These facts suggest that gene expression in the period following irradiation facilitates recovery from radiation damage, with an increased probability of mutation, reminiscent of the "SOS response" in Escherichia coli.     

The cda GenoTox assay: a new and sensitive method for detection of environmental genotoxins, including nitroarenes and aromatic amines

A new bacterial test system for detection of genotoxic compounds was developed, based on two new Salmonella typhimurium tester strains, TGO1 and TGO2. Both strains contain a gene fusion between a strong SOS-promotor, P(cda), and the gfp gene, which allows detection of genotoxic compounds that induce the SOS response. SOS induction was detected by means of flow cytometry. TGO1 showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine compared with a previously developed strain, which had an Escherichia coli strain as host instead of S. typhimurium. S9 mix was introduced into the assay, making the test system suitable for detection of indirect mutagens. Furthermore, the genes for bacterial nitro-reductase (NR) and o-acetyl transferase (o-AT) were inserted into TGO2, making it an NR- and o-AT-over-expressing strain. This resulted in an assay that was able to detect the nitroarene 1-nitropyrene and the aromatic amine 2-aminoanthracene with high sensitivity.     

Characterization of stress-responsive behavior in Pseudomonas aeruginosa PAO: isolation of Tn3-lacZYA fusions with novel damage-inducible (din) promoters.

Although the pervasive soil and water microorganism Pseudomonas aeruginosa demonstrates heightened sensitivity to UV radiation, this species possesses a recA gene that, based on structural and functional properties, could mediate a DNA damage-responsive regulon similar to the SOS regulon of Escherichia coli. To determine whether P. aeruginosa encodes such stress-inducible genes, the response of P. aeruginosa to DNA-damaging agents including far-UV radiation (UVC) and the quinolone antimicrobial agent norfloxacin was investigated by monitoring the expression of fusions linking P. aeruginosa promoters to a beta-galactosidase reporter gene. These fusions were obtained by Tn3-HoHoI insertional mutagenesis of a P. aeruginosa genomic library. Eight different damage-inducible (din) gene fusions were isolated which lack homology to the P. aeruginosa recA gene. Expression of the three gene fusions studied, dinA::lacZYA, dinB::lacZYA, and dinC::lacZYA, increased following UVC and quinolone exposure but not following heat shock. Similar to E. coli SOS genes, the din genes were induced to different extents and with dissimilar kinetics following UVC irradiation.