Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

Response of bioluminescent bacteria to sixteen azo dyes

Recombinant bioluminescent bacteria were used to monitor and classify the toxicity of azo dyes. Two constitutive bioluminescent bacteria,Photobacterium phosphoreum andEscherichia coli, E. coli GC2 (lac::luxCDABE), were used to detect the cellular toxicity of the azo dyes. In addition, four stress-inducible bioluminescentE. coli, DPD2794 (recA::luxCDABE), a DNA damage sensitive strain; DPD2540 (fabA::luxCDABE), a membrane damage sensitive strain; DPD2511 (katG::luxCDABE), an oxidative damage sensitive strain; and TV1061 (grpE::luxCDABE), a protein damage sensitive strain, were used to provide information about the type of toxicity caused by crystal violet, the most toxic dye of the 16 azo dyes tested. These results suggest that azo dyes result in serious cellular toxicity in bacteria, and that toxicity monitoring and classification of some azo dyes, in the field, may be possible using these recombinant bioluminescent bacteria.     

Bioluminescent assay for sphingolipid ceramide N-deacylase using <Emphasis Type="Italic">Vibrio harveyi</Emphasis> dark mutant M-17

A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 μU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.     

In vitro antagonism of bioluminescent fungi by Trichoderma harzianum

Two species of bioluminescent fungi, Panellus stypticus and Omphalotus olearius were placed in contact with three different strains of interfungal pathogenic Trichoderma harzianum. Subsequent light emission by the luminous fungi and advance of the interfungal pathogens were compared. Relative differences among the pathogens were reflected in their rate of mycelial advance, the total area over which they produced spores upon the host fungi, and decreases in host bioluminescence. After ten days differences in the total surface areas of spore production varied from 1 to 53 per cent. Differences in the reduction of bioluminescence of the same material ranged over 2 orders of magnitude. Final reduction in luminescence ranged over 6 orders of magnitude. A marked reduction in bioluminescence was observed to precede the advance of spore production. The greatest reduction in luminescence was correlated with the presence of T. harzianum hyphae. Two strains of T. harzianum, NRRL 1698 and ATCC 58674, were effective against both bioluminescent fungi within the study period while a third strain, NRRL 13019, was only effective against Omphalotus olearius.     

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02–850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.     

Characterization of superoxide-stress sensing recombinant Escherichia coli constructed using promoters for genes zwf and fpr fused to lux operon

To measure the toxicity experienced by superoxide-generating compounds, two plasmids were constructed in which the superoxide-inducible fpr and zwf promoters from Escherichia coli were fused to promoterless Vibrio fischeri luxCDABE operon present in plasmid pUCD615. The bioluminescent response of E. coli harboring these constructs was studied as a function of the toxicity and was shown to be specific for superoxide generating chemicals. The two promoters employed, fpr and zwf, responded differentially to the redox-chemicals tested. Furthermore, a ΔmarA strain bearing the fpr::luxCDABE fusion had a weaker response to paraquat (methyl viologen) than its isogenic parent strain, whereas zwf induction was not inhibited in ΔmarA or Δrob strains. The fpr and zwf promoters were also induced by alkylating agents but were unresponsive in ΔmarA or Δrob strains. Using optimized assay conditions, the abilities of these strains to differentially respond to superoxide stress and alkylating agents that may be present in contaminants proves them to be good biosensor candidates for monitoring toxicity.     

Conjugates of the Сa2+-Regulated Photoprotein Obelin with Immunoglobulins: Synthesis and Use as Labels in Bioluminescent Immunoassay

An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 IU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).     

Electrophysiological membrane characteristics of the salt tolerant Plantago maritima and the salt sensitive Plantago media

Plasmid pUCD607 was mobilized into the biocontrol agent Enterobacter cloacae strain E6 by conjugation and the resultant strain, E6(pUCD607), was bioluminescent. Biocontrol of Pythium ultimum by E6(pUCD607) was similar to that of the parent strain, E6. The location of E6(pUCD607) in the soil and in the rhizosphere of lettuce was readily determined by pressing agar medium against plant roots in a root box, allowing the bacteria to grow overnight on the medium, and detecting the presence of bioluminescence by autophotography. There was a positive, linear correlation between population sizes determined by dilution plating and the quantity of light emitted due to bioluminescence. However, both the intercept and slope of this line varied among experiments possibly due to the differing physiological states of cells recovered from soils. The amount of light emitted by the bioluminescent strain E6(pUCD607) was not quantitative. This technique is useful for qualitative determinations of populations and for photographically locating bacteria.     

Fabrication of a bio-MEMS based cell-chip for toxicity monitoring

A bio-MEMS based cell-chip that can detect a specific toxicity was fabricated by patterning and immobilizing bioluminescent bacteria in a microfluidic chip. Since the emitted light intensity of bioluminescent bacteria changed in response to the presence of chemicals, the bacteria were used as the toxicity indicator in this study. A pattern of immobilized cells was successfully generated by photolithography, utilizing a water-soluble and negatively photosensitive polymer, PVA-SbQ (polyvinyl alcohol-styrylpyridinium) as an immobilization material. Using the recombinant Escherichia coli (E. coli) strain, GC2, which is sensitive to general toxicity, the following were investigated for the immobilization: an acceptable dose of long-wavelength UV light, the biocompatibility of the polymer, and the effect of the chip-environment. We found that 10 min of UV light exposure, the toxicity of polymer (SPP-H-13-bio), and the other chip-environment did not inhibit cell metabolism significantly for making a micro-cell-chip. Detection of a specific toxicity was demonstrated by simply immobilizing the bioluminescent bacteria, DK1, which increased bioluminescence in the presence of oxidative damage in the cells. An injection of hydrogen peroxide of 0.88 mM induced 10-fold increase in bioluminescent intensity confirming the capability of the chip for toxicity monitoring.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a bioindicator for estimating pentachlorophenol toxicity and concentration

Pentachlorophenol (PCP) and its sodium salt (Na-PCP) are extremely toxic chemicals responsible for important soil and groundwater pollution, mainly caused by wastes from wood-treatment plants, because chlorinated phenols are widely used as wood preservatives. The methods most commonly used for routine analysis of pesticides such as PCP and Na-PCP are high-performance liquid chromatography (HPLC) and gas chromatography–mass spectroscopy (GC–MS). A variety of rapid biological screening tests using marine organisms, bioluminescent bacteria, and enzymes have also been reported. In this study, rapid biological screening analysis using Bacillus subtilis was developed, to assess the biodegradation of PCP and its by-products in liquid samples. An empirical model is proposed for spectrophotometric analysis of Na-PCP concentration after growth of Bacillus subtilis.     

Multi-gene analysis reveals previously unrecognized phylogenetic diversity in Aliivibrio

The “Vibrio fischeri species group” recently was reclassified as a new genus, Aliivibrio, comprising four species, Aliivibrio fischeri, Aliivibrio logei, Aliivibrio salmonicida, and Aliivibrio wodanis. Only limited phylogenetic analysis of strains within Aliivibrio has been carried out, however, and taxonomic ambiguity is evident within this group, especially for phenotypically unusual strains and certain strains isolated from bioluminescent symbioses. Therefore, to examine in depth the evolutionary relationships within Aliivibrio and redefine the host affiliations of symbiotic species, we examined several previously identified and newly isolated strains using phylogenetic analysis based on multiple independent loci, gapA, gyrB, pyrH, recA, rpoA, the luxABE region, and the 16S rRNA gene. The analysis resolved Aliivibrio as distinct from Vibrio, Photobacterium, and other genera of Vibrionaceae, and resolved A. fischeri, A. salmonicida, A. logei, and A. wodanis as distinct, well-supported clades. However, it also revealed that several previously reported strains are incorrectly identified and that substantial unrecognized diversity exists in this genus. Specifically, strain ATCC 33715 (Y-1) and several other strains having a yellow-shifted luminescence were not members of A. fischeri. Furthermore, no strain previously identified as A. logei grouped with the type strain (ATCC 29985^T), and no bona-fide strain of A. logei was identified as a bioluminescent symbiont. Several additional strains identified previously as A. logei group instead with the type strain of A. wodanis (ATCC BAA-104^T), or are members of a new clade. Two strongly supported clades were evident within A. fischeri, a phylogenetic structure that might reflect differences in the host species or differences in the ecological incidence of strains. The results of this study highlight the importance of basing taxonomic conclusions on examination of type strains.     

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.     

Enhancement in the viability and biosensing activity of freeze-dried recombinant bioluminescent bacteria

The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biosensor field was determined.     

Evaluation and application of aquatic toxicity tests: use of the Microtox test for the prediction of toxicity based upon concentrations of contaminants in soil

The quality control for the reuse of cleaned soil from a contaminated site consisted in the determination of the main contaminants by analytical chemical methods such as GC and HPLC. Since it is not possible to analyze for all contaminants a toxicity test should be used to detect large concentrations of not routinely analyzed chemicals. The aim of the study was to develop a system for toxicity testing, which should be able to predict the toxicity of soil samples based on the concentration of chemicals in the soil and to detect toxic chemicals not analyzed by the routinely conducted soil analysis.Based upon the relative sensitivity to various contaminants as well as practical aspects such as test duration and costs the Microtox® test was favoured over the bioassays with Daphnia magna and Scenedesmus subspicatus. The Microtox® test was used to measure the toxicity of various pesticides and their major metabolites. The toxicity data of the pure compounds were used to predict the toxicity (EC₅₀ and % inhibition of the bioluminescence reaction) of defined mixtures of chemicals in water by applying two different mathematical appriaches which are based on the additivity of the effects of the single chemicals. The predicted values were compared with the experimental data and showed good agreement.In order to be able to predict the toxicity of soil samples using the Microtox® test the soil/water partition coefficient (K _d) was measured for the main contaminants. The toxicity of soil samples was predicted by calculating the concentration of the contaminants in the leachate by using the corresponding concentration in the soil and applying the K _d values determined. From the calculated composition of the leachate the expected toxicity was estimated. This value was compared with the toxicity experimentally determined in the Microtox® test.     

Biodegradation of diphenyl ether and transformation of selected brominated congeners by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. PH-07

Polybrominated diphenyl ethers (PBDEs) are common flame-retardant chemicals that are used in diverse commercial products such as textiles, circuit boards, and plastics. Because of the widespread production and improper disposal of materials that contain PBDEs, there has been an increasing accumulation of these compounds in the environment. The toxicity and bioavailability of PBDEs are variable for different congeners, with some congeners showing dioxin-like activities and estrogenicity. The diphenyl ether-utilizing bacterium Sphingomonas sp. PH-07 was enriched from activated sludge of a wastewater treatment plant. In liquid cultures, this strain mineralized 1 g of diphenyl ether per liter completely within 6 days. The metabolites detected and identified by gas chromatography/mass spectrometry (MS) and electrospray ionization/MS analysis corresponded with a feasible degradative pathway. However, the strain PH-07 even catabolized several brominated congeners such as mono-, di-, and tribrominated diphenyl ethers thereby producing the corresponding metabolites.     

群体感应动态调控促进大肠杆菌合成酪醇

酪醇是一种多酚类天然产物,广泛应用于化工、医药和食品等领域。目前大肠杆菌(Escherichia coli)从头合成酪醇存在发酵菌体密度低和产量低等问题。为此,本研究将前期获得苯丙酮酸脱羧酶突变体ARO10F138L/D218G与不同来源的醇脱氢酶融合表达,最优组合ARO10F138L/D218G-L-YahK酪醇产量达到1.09 g/L。为进一步提高酪醇产量,敲除了4-羟基苯乙酸竞争途径关键基因feaB,使酪醇产量提高了21.15%,达到1.26g/L。针对酪醇发酵菌体密度低的问题,通过群体感应系统动态调控酪醇合成途径,减轻酪醇对底盘细胞的毒性作用,缓解生长抑制,使其产量提高了33.82%,达到1.74 g/L。在2 L发酵罐中,群体感应动态调控工程菌TRFQ5的酪醇产量达到4.22g/L,OD600值达到42.88,分别较静态诱导表达工程菌TRF5提高了38.58%和43.62%。本研究应用基因敲除技术,阻断了酪醇合成竞争途径;同时结合群体感应动态调控策略,减轻了酪醇毒性对底盘细胞的生长抑制,从而有效地提高了酪醇产量。本研究对其他高毒性化学品的生物合成具有良好的借鉴和应用价值。     

Efficient screening and breeding of Bacillus thuringiensis subsp. kurstaki for high toxicity against Spodoptera exigua and Heliothis armigera

Spodoptera exigua is one of the most renowned agricultural pest insects and relatively insensitive to Bacillus thuringiensis subsp. kurstaki strains which are widely used commercial products to control lepidopterans such as Heliothis armigera. In the current study, we have developed a new and efficient approach to screen and breed a B. thuringiensis subsp. kurstaki strain exhibiting high toxicity against S. exigua while retaining its high toxicity against H. armigera. UV and diethyl sulfate methods were used for mutagenesis, followed by an agar plug plate diffusion assay for preliminary screening of Zwittermicin A over-producing mutants, from which we obtained a mutant strain, designated here as B. thuringiensis subsp. kurstaki D1-23, with high toxicity against S. exigua. The toxicity of D1-23 against S. exigua and H. armigera was improved by 115.4 and 25.9%, respectively, compared to its parental commercial strain BMB005.     

Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli

AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA. A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study. METHODS AND RESULTS: The recombinant bioluminescent E. coli strain DPD2794, containing a recA promoter fused to luxCDABE from V. fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage. DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity. CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2. Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter. SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E. coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E. coli.