Papers by Shimshon Belkin
A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detecti... more A novel micro-fluidic whole cell biosensor for toxicity analysis based on bioluminescence detection was developed. The optical part of the biochip was modelled and simulated to optimize the total light collection efficiency and the system response. The optimization elucidated some of the optical aspects of the biochip. A study evaluating the bioluminescence reaction kinetics was performed and revealed the interdependence between the detected bioluminescence and the physical parameters of the introduced toxin.
Analytica Chimica Acta, 2010
Electrochemical signal detection can be readily integrated in biosensors and is thus an attractiv... more Electrochemical signal detection can be readily integrated in biosensors and is thus an attractive alternative to optical detection methods. In the field of environmental chemistry and ecotoxicology there is a growing demand for lab-independent devices based on whole cell biosensors for the detection of genotoxic compounds. Because of the broad occurrence of pre-genotoxic compounds that need to be bio-activated, the integration of a system for metabolic activation into such a biosensor is important. The present study evaluates a chrono-amperometric detection method in which para-aminophenyl beta-D-galactopyranoside is used as substrate for a reporter gene assay based on the bacterial SOS-response in comparison to a test system for the determination of genotoxicity in water that is standardized according to the International Organization for Standardization (ISO). The evaluation was done in order to analyze the potential of the electrochemical signal detection to be used as a complementary method for the standard test system and thus to evaluate the usability of electrochemical biosensors for the assessment of genotoxicity of environmental samples. In the present study it is shown that the chrono-amperometric detection of para-aminophenol is specific even in the presence of electro-active species generated by the enzymatic system used for the external bio-activation of contaminants. Under optimized conditions electrochemistry is sufficiently sensitive with a limit of detection that is comparable to the respective ISO-standard.
< A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay res... more < A new bacterial bioassay for toxicity of particulate air pollution is presented. < Bioassay responses indicating toxicity were observed for several PM samples. < These responses suggest oxidative stress, respiration inhibition and Fe deficiency. < A metal chelating treatment of the samples relieved the bioassay's responses. < Bioavailability analysis suggested that Cr was related to these toxic responses. a b s t r a c t Numerous studies have demonstrated that elevated concentrations of suspended atmospheric particulate matter (PM) are associated with adverse health effects. In order to minimize the adverse public health effects of atmospheric PM by exposure management, there is a need for a greater understanding of the toxic mechanisms and the components that are responsible for the toxic effects. The aim of this study was to utilize bioassay techniques to investigate these aspects. For this purpose a reporter panel of 9 genetically engineered bacterial (Escherichia coli) strains was composed. Each panel member was designed to report on a different stress condition with a measurable light signal produced by the luciferase enzyme. Toxic mechanisms and components were studied using six anthropogenic PM source samples, including two vehicle combustion particles, three coal fly ash (CFA) samples and an urban dust sample. The most prominent outcome of the panel exposure results were broad panel responses observed for two of the CFA samples, indicating oxidative stress, respiration inhibition and iron deficiency. These responses were relieved when the samples were treated with EDTA, a non-specific metal chelator, suggesting the involvement of metals in the observed effects. Bioavailability analysis of the samples suggests that chromium was related to the toxic responses induced by two of the CFA samples. Oxidative stress was also observed in several samples of ambient atmospheric aerosols and excess metal toxicity in an urban dust sample collected in a parking lot. The reporter panel approach, as demonstrated in this study, has the potential of providing novel insights as to the mechanisms of atmospheric PM toxicity. Furthermore, combining the panel's results with bioavailability data can enlighten about the role of different PM components in the observed toxicity.
Cellular Origin, Life in Extreme Habitats and Astrobiology, 2010
The surfaces of aboveground parts of plants – the phyllosphere – are normally colonized by a vari... more The surfaces of aboveground parts of plants – the phyllosphere – are normally colonized by a variety of bacteria, yeasts, and fungi (Lindow and Leveau, 2002). Bacteria are the most numerous colonists of leaves, often being found in numbers averaging 106–107 cells/cm2 of leaf (Andrews and Harris, 2000; Beattie and Lindow, 1995; Hirano and Upper, 1989). In spite of their worldwide distribution (Morris and Kinkel, 2002), studies of the composition of bacterial communities on leaves have been relatively limited in scope, mostly focusing on potential pathogens of agriculturally relevant plants (Beattie and Lindow, 1994; Dik, et al., 1992; Ercolani, 1991).
Environmental Toxicology & Water Quality, 1994
... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been e... more ... Michael Stieber ... As a direct consequence, the biodegradation of these compounds has been extensively studied, and their degradative pathways are known to a large extent (Cer-niglia andHeitkamp, 1989; Morgan and Watkinson, 1989; Mueller et al., 1989, Heitkamp et al ...
NATO Science Series, 2006
At the heart of every biosensor is a biological entity, the purpose of which is to react with the... more At the heart of every biosensor is a biological entity, the purpose of which is to react with the target analyte(s) and generate a readily quantifiable signal. Traditional biosensors are based on the unique specificity of enzymes to their substrates, antibodies to antigens or that of nucleic acids to their complementary sequences.
PLANT PHYSIOLOGY, 1987
The internal pH values of two unicellular cyanobacterial strains were determined with electron sp... more The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.
FEMS Microbiology Ecology, 2000
This paper describes the microbial ecosystem found on the leaves of Atriplex halimus, a salt-excr... more This paper describes the microbial ecosystem found on the leaves of Atriplex halimus, a salt-excreting plant in the central Negev highlands of Israel. Because of the regular nightly occurrence of dew at this location, these leaves undergo a diurnal wetting so that phylloplane microorganisms experience large fluctuations in salinity and water activity, as well as tolerate repeated desiccation. During the dry season, in the late spring and summer, a significant amount of salts and organic material coats the leaf surface. During dew events the salt concentration at the leaf surface was calculated to be > 0.4 M. Direct counts of the respiring bacteria on the leaf surface ranged from 1.06× 104 to 5.06× 105 per cm 2. Using a variety of media it was shown that there was limited bacterial diversity which could be cultured, with greater than 90% of the isolates being orange colored Gram-negative rods. Viable counts ranged from 0.32 to 2.32× 104 bacteria per cm 2 of A. halimus leaf surface. No bacteria capable of nucleating ice were recovered in these studies. The dominant orange pigmented bacterium, identified as a halotolerant Pseudomonas sp., grew optimally at 30°C and at 5% NaC! and was capable of growth in media containing up to 20% NaCI. This bacterium could grow on a variety of organic compounds, including some associated with plant materials. The leaf bacteria were desiccation-tolerant when on the leaf surface or when directly washed off the leaves, but much less so when in isolated culture. A major component of the tolerance to desiccation is probably related to the compounds on the leaf surface.
Chemosphere, 1999
In this communication we report on a genetically engineered bacterium that reacts by light emissi... more In this communication we report on a genetically engineered bacterium that reacts by light emission to the presence of 4-chlorobenzoic acid. To construct this strain, DNA fragment ( 1.7 kb) upstream from the 4-chlorobenzoic acid dehalogenase (fcb) operon of Arthrobacter SU was fused to Vibriofischeri 1mCDABE
Biological Bulletin, 1986
Biodegradation, 1992
Halogenated alkanes constitute a significant group among the organic pollutants of environmental ... more Halogenated alkanes constitute a significant group among the organic pollutants of environmental concern. Their industrial and agricultural uses are extensive, but until 1978 they were considered to be nonbiodegradable. In recent years, microorganisms were described that could degrade, partially or fully, singly or in consortia, many of the compounds tested. The first step in haloalkane degradation appears to be universal: removal of the halogen atom(s). This is mediated by a group of enzymes, generally known as dehalogenases, acting in most cases either as halidohydrolases or oxygenases. Nevertheless, information is still severely lacking regarding the biochemical pathways involved in these processes, as well as their genetic control.
Archives of Microbiology, 1990
We describe a new species, Thermococcus litoralis, which is different from the type species Therm... more We describe a new species, Thermococcus litoralis, which is different from the type species Thermococcus celer in molecular, morphological and physiological characteristics.
Archives of Microbiology, 1988
A second species of the extremely thermophilic, eubacterial genus Thermotoga is described as clea... more A second species of the extremely thermophilic, eubacterial genus Thermotoga is described as clearly distinguished from the type species Thermotoga maritima by physiological and phylogenetic criteria. It is named Thermotoga neapolitana.
Archives of Microbiology, 1985
An extremely thermophilic (optimum growth at 88~ anaerobic bacterium was isolated from a shallow ... more An extremely thermophilic (optimum growth at 88~ anaerobic bacterium was isolated from a shallow submarine thermal spring. It appears to be an obligate heterotroph, capable of reducing sulfur to HzS. Oxygen sensitivity is apparent only at and above those temperatures where growth occurs, while the cultures retain their viability for long periods under air at 4 ~ C. Insensitivity to chloramphenicol, vancomycin and streptomycin, and lack ofmuramic acid in its cell wall, indicate a possible affilitation of the new isolate to the thermoacidophilic archaebacteria. However, its neutrophilic and heterotrophic nature, as well as its DNA base composition (39.1 tool % guanine plus cytosine) set it apart from the known genera of this group.
Archives of Biochemistry and Biophysics, 1987
A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemi... more A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and Oz-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by superoxide dismutase, indicating that superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible "destruction" of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carboncentered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was. o 1987 Academic press, I,,~.
Fresenius' journal of analytical chemistry
In parallel to the continuous development of increasingly more sophisticated physical and chemica... more 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 ...
Microfluidics, BioMEMS, and Medical Microsystems IV, 2006
A novel water chemical toxin sensor has been successfully developed and evaluated as a working po... more A novel water chemical toxin sensor has been successfully developed and evaluated as a working portable laboratory prototype. This sensor relies on a disposable plastic biochip prepared with a 4x4 micro-laboratory (muLab) chambers array of Escherichia coli reporter cells and micro-fluidic channels for liquids translocation. Each bacterial strain has been genetically modified into a bioluminescent reporter that responds to a
Sensors and Actuators B: Chemical, 2011
Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of haza... more Cell-based toxicity bioassays harbor the potential for efficient detection and monitoring of hazardous materials. However, their use in the field has been limited by harsh and unstable environmental conditions that shorten shelf-life, introduce significant noise, and reduce the signal and signal-to-noise ratio; such conditions may thus decrease the probability of correct decisions, increasing both false positive and false negative outcomes. Therefore, there is a need for a stable cell-on-chip integration that offers longterm storage and resilience to environmental factors. The use of intact microbial biofilms as biological elements in a whole-cell biosensor, and their integration into specialized biochips, holds promise for enhancing sensor stability as well as providing an innovative platform for biofilm research. We report here for the first time on the integration of a bacterial biofilm as the sensing element of a whole-cell biosensor, as a means to stabilize and preserve reproducibility, viability and functionality of the bacterial sensor cells. We have employed a genetically engineered Escherichia coli sensor strain, tailored to respond to the presence of genotoxic (DNA damaging) agents by the induction of a reporter enzyme, alkaline phosphatase, and tested its functionality in colorimetric and electrochemical assays. Three different bacterial integration forms were examined: planktonic cells, electronically deposited sessile cells, and biofilms. For all integration forms, a clear dose-dependent positive response to the presence of the model toxicant nalidixic acid was observed, with biofilms displaying higher current density and detection sensitivity than planktonic and sessile cells. We present the electrode apparatus and methods and biochip characterization of such chips, e.g. signal vs. time and induction factor, and discuss the advantage and potential problems of the new biofilm-biochip technology.
Research in Microbiology, 2011
The Escherichia coli yjbEFGH operon, encoding genes involved in exopolysaccharide production, has... more The Escherichia coli yjbEFGH operon, encoding genes involved in exopolysaccharide production, has previously been shown to be induced by osmotic stress and to be negatively regulated by s 38 . Promoter analysis suggested that like most E. coli genes, its transcription is driven by the housekeeping sigma factor s 70 . Indeed, manipulation of any of the other five alternative sigma factors did not affect its induction by osmotic stress. Surprisingly, when assayed in a strain expressing low levels of s 70 , yjbEFGH induction in response to osmotic stress was higher than in a strain expressing normal levels of s 70 . Similar phenomena were observed in the s 70 -driven promoters of sulA, uvrA, recA, fecI, entC and lacZ, the transcription of which is directly controlled by a repressor protein (LexA, Fur and LacI), but not in promoters of the housekeeping genes ftsA and ftsY, or in s 38 -driven treA promoter. Since transcription factors are generally present in the cell in low numbers, we hypothesize that a decrease in s 70 , that drives the expression of lexA, fur and lacI as well, further diminishes their number in the cell and thus de-represses the induction of genes which are subjected to their repression.
Uploads
Papers by Shimshon Belkin