International round-robin study on the Ames fluctuation test.

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Optical and electrical interfacing technologies for living cell bio-chips.

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by micro-system-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such whole-cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro-electronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Water toxicity detection by a panel of stress-responsive luminescent bacteria.

A panel of Escherichia coli strains harbouring different stress-responsive promoters fused to a lux reporter system was used to assess the potential toxicity of 17 unknown model water samples. Using liquid cultures, nine out of 14 toxic samples were properly identified as toxic, whereas five were false negatives. All three non-toxic controls were identified correctly (no false positives). Two strains containing promoter-lux fusions were also tested when immobilized onto fibre-optic tips. One genotoxic sample and six toxic samples were correctly identified in this manner. The potential advantages and limitations in the use of genetically engineered bacteria as biosensors for water toxicity are discussed in view of these results.

Biological laser printing of genetically modified Escherichia coli for biosensor applications.

One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed a technique, biological laser printing or BioLP, which satisfies these requirements and has advantages over current technologies. BioLP is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates. The BioLP technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.

Survival of enteric bacteria in seawater.

Enteric bacteria exposed to the marine environment simultaneously encounter a variety of abiotic and biotic challenges. Among the former, light appears to be critical in affecting seawater survival; previous growth history plays a major part in preadaptation of the cells, and stationary phase cells are generally more resistant than exponentially growing ones. Predation, mostly by protozoa, is probably the most significant biotic factor. Using Escherichia coli as a model, a surprisingly small number of genes was found that, when mutated, significantly affect seawater sensitivity of this bacterium. Most prominent among those is rpoS, which was also dominant among genes induced upon transfer to seawater.

Monitoring of phosphorus bioavailability in water by an immobilized luminescent cyanobacterial reporter strain.

Massive growth of cyanobacteria, known as "algal blooms", has become a major concern for water monitoring. It has been observed that environmental factors like temperature, light, and certain patterns of availability of nutrients such as P, N, Fe influence cyanobacterial proliferation and toxin production. In order to monitor nutrients in aquatic ecosystems, an assay for monitoring phosphorus bioavailability to cyanobacteria was developed. The test consists of an immobilized luminescent reporter strain of Synechococcus PCC 7942, designated APL. The reporter strain harbours the gene coding the reporter protein luciferase from Vibrio harveyi under control of the inducible alkaline phosphatase promoter from Synechococcus PCC 7942, and can be induced under phosphorus limitation. The resultant CyanoSensor detects PO(3-)(4)-P in a concentration range of 0.3-8 microM after a sample incubation time of 8 h under continuous illumination (50 microE m(-2) s(-1)). The sensor also responded to a variety of organic phosphorus sources and was storable for 3 weeks at 4 degrees C. It could be demonstrated that the CyanoSensor for bioavailability monitoring is an improvement to conventional phosphorus detection methods.

Seawater Activation of Escherichia coli Gene Promoter Elements: Dominance of rpoS Control.

The release of non-disinfected wastewater into the marine environment is a common practice in many countries; nevertheless, the molecular mechanisms involved in determining the survival of enteric bacteria in seawater are poorly understood, in spite of the obvious public health implications. In a methodological attempt to address this issue, a plasmid-based collection of 687 Escherichia coli distinct promoter::luxCDABE fusions was screened to identify promoters that are induced upon exposure to seawater. The luminescence driven by 22 out of these promoters reproducibly increased at least two-fold in an artificial seawater medium; only 9 of the corresponding genes have previously been assigned a function. The most prominent characteristic of the induced genes was that most (18 out of 22) were under rpoS control. The induction of these seawater-responsive promoters was evaluated in different media to identify the cause of the increased transcription. Salinity or osmolarity was instrumental in only four cases, and in three promoters, increased pH also seemed to play a role; however, the most significant environmental effector in inducing the majority of the seawater-induced promoters appeared to be nutrient limitation.

Whole-cell biodetection of halogenated organic acids.

A whole-cell bacterial sensor system for short-chain halo-organic acids was constructed, using 2-chloropropionic acid (2-CPA) as a model pollutant. An Escherichia coli host was transformed with a moderate-copy plasmid containing a fusion of two foreign genetic elements: (a) a promoter-containing segment of the Pseudomonas DL-DEX (DL-2-haloacid dehalogenase) encoding gene and (b) bioluminescence (luxCDABE) genes of Photorhabdus luminescens. The resulting construct, named MT1, responded to the presence of 2-CPA by dose-dependent light emission, in a highly specific albeit a very insensitive manner. Thus, while the desired concept was successfully demonstrated, further genetic work is needed in order to make such a construct practical for environmental monitoring purposes.

Antibody-based immobilization of bioluminescent bacterial sensor cells.

Whole-cell luminescent bioreporter sensors based on immobilized recombinant Escherichia coli are described and evaluated. The sensors were prepared by glutaraldehyde-anchoring of nonspecific anti-E. coli antibodies on aminosylilated gold or silica glass surfaces with subsequent attachment of the probe bacteria. We demonstrate the generality of the concept by attachment of several E. coli strains that express luciferase in response to different physiological stress conditions including heat shock, DNA damage (SOS), fatty acid availability, peroxide and oxidative stress. The sensors can be used either as single- or multiple-use disposable sensing elements or for continuous operation. We show compatibility with optical fiber technology. Storage stability of the sensors exceeded 5 months with no measurable deterioration of the signal. Repeatability on exposure in successive days was <15%, as was sensor to sensor reproducibility. Sensitivity and detection limits of the immobilized cells were comparable to that of non-immobilized bacteria.

Cationic peptide antimicrobials induce selective transcription of micF and osmY in Escherichia coli.

Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.

Improved bacterial SOS promoter∷lux fusions for genotoxicity detection.

Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants. In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E. coli host. A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed. Generally, fastest responses were exhibited by Sal94, a S. typhimurium strain harboring a plasmid-borne fusion of V. fischeri lux to the E. coli recA promoter. Highest sensitivity, however, was demonstrated by DPD3063, an E. coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant. Overall, the two latter strains appeared to perform better and seemed preferable over the others. The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

Reporter gene bioassays in environmental analysis.

In parallel to the continuous development of increasingly more sophisticated physical and chemical analytical technologies for the detection of environmental pollutants, there is a progressively more urgent need also for bioassays which report not only on the presence of a chemical but also on its bioavailability and its biological effects. As a partial fulfillment of that need, there has been a rapid development of biosensors based on genetically engineered bacteria. Such microorganisms typically combine a promoter-operator, which acts as the sensing element, with reporter gene(s) coding for easily detectable proteins. These sensors have the ability to detect global parameters such as stress conditions, toxicity or DNA-damaging agents as well as specific organic and inorganic compounds. The systems described in this review, designed to detect different groups of target chemicals, vary greatly in their detection limits, specificity, response times and more. These variations reflect on their potential applicability which, for most of the constructs described, is presently rather limited. Nevertheless, present trends promise that additional improvements will make microbial biosensors an important tool for future environmental analysis.

Microbial sensors of ultraviolet radiation based on recA'::lux fusions.

Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA'::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA'::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37 degrees C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.

Reactive oxygen species are partially involved in the bacteriocidal action of hypochlorous acid.

Hypochlorous acid (HOCl) is probably the most widely used disinfectant worldwide and has an important role in inflammatory reaction and in human resistance to infection. However, the nature and mechanisms of its bactericidal activity are still poorly understood. Bacteria challenged aerobically with HOCl concentrations ranging from 9.5 to 76 microM exhibit higher ability to form colonies anaerobically than aerobically. Conversely, aerobic plating greatly increased lethality after an anaerobic HOCl challenge, although anaerobic survival did not depend on whether HOCl exposure was aerobic or anaerobic. Even a short transient exposure to air after anaerobic HOCl challenge reduced anaerobic survival, indicative of immediate deleterious effects of oxygen. Exposure to HOCl can cause lethal DNA damage as judged by the fact that recA sensitivity to HOCl was oxygen dependent. Antioxidant defenses such as reduced glutathione and glucose-6-phosphate dehydrogenase were depleted or inactivated at 10 microM HOCl, while other activities, such as superoxide dismutase, dropped only above 57 microM HOCl. Cumulative deficiencies in superoxide dismutase and glucose-6-phosphate dehydrogenase rendered strains hypersensitive to HOCl. This indicates that part of HOCl toxicity on Escherichia coli is mediated by reactive oxygen species during recovery.

Combinations of chlorocatechols and heavy metals cause DNA degradation in vitro but must not result in increased mutation rates in vivo.

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure-activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E. coli mutants deficient in oxidative stress defense or DNA repair. In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E. coli avoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded.

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.

Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli.

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.