On-line and in situ biosensors for monitoring environmental pollution

Efficient tools for on-line and in situ monitoring of environmental pollutants are required to provide early warning systems. In addition, such tools can contribute important information on the progress of various remediation treatments. One of the recently developed monitoring technologies involves the use of whole-cell biosensors. Such biosensors could be constructed to detect general toxicity or specific toxicity caused by one or more pollutants. Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, color or electric current. Electrochemical monitoring is of special interest for in situ measurements as it can be performed using simple, compact and mobile equipment and is easily adaptable for on-line measurements. Here we survey the potential application of electrochemical biosensors in monitoring of general toxicity as well as hydrocarbons and heavy metals.     

Enhanced mercury biosorption by bacterial cells with surface-displayed MerR

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg(2+) biosorption than that found in the wild-type JM109 cells. Hg(2+) binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd(2+) and Zn(2+). The Hg(2+) binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Understanding cellular responses to toxic agents: a model for mechanism-choice in bacterial metal resistance

Summary Bacterial resistances to metals are heterogeneous in both their genetic and biochemical bases. Metal resistance may be chromosomally-, plasmid- or transposonencoded, and one or more genes may be involved; at the biochemical level at least six different mechanisms are responsible for resistance. Various types of resistance mechanisms can occur singly or in combination and for a particular metal different mechanisms of resistance can occur in the same species. To understand better the diverse responses of bacteria to metal ion challenge we have constructed a qualitative model for the selection of metal resistance in bacteria. How a bacterium becomes resistant to a particular metal depends on the number and location of cellular components sensitive to the specific metal ion. Other important selective factors include the nature of the uptake systems for the metal, the role and interactions of the metal in the normal metabolism of the cell and the availability of plasmid (or transposon) encoded resistance mechanisms. The selection model presented is based on the interaction of these factors and allows predictions to be made about the evolution of metal resistance in bacterial populations. It also allows prediction of the genetic basis and of mechanisms of resistance which are in substantial agreement with those in well-documented populations. The interaction of, and selection for resistance to, toxic substances in addition to metals, such as antibiotics and toxic analogues, involve similar principles to those concerning metals. Potentially, models for selection of resistance to any substance can be derived using this approach.     

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Imaging single-channel calcium microdomains

The Ca(2+) microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca(2+) signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca(2+) imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of 'optical patch-clamp recording' is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca(2+) ions.     

Design and Application of a Biosensor for Monitoring Toxicity of Compounds to Eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Enhanced Mercury Biosorption by Bacterial Cells with Surface-Displayed MerR

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg²⁺ biosorption than that found in the wild-type JM109 cells. Hg²⁺ binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd²⁺ and Zn²⁺. The Hg²⁺ binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.     

大肠杆菌拓扑异构酶I结合重金属的特性及功能影响

【背景】大肠杆菌拓扑异构酶Ⅰ(Escherichia coli topoisomerase I,E.coli TopA)在DNA复制、转录、重组和基因表达调控等过程发挥关键作用。研究表明E.coli TopA只有结合锌离子才具有活性,然而E.coli TopA能否结合其他金属离子尤其是重金属离子,以及结合其他金属后是否具有活性,目前仍不清楚。【目的】探究大肠杆菌拓扑异构酶Ⅰ是否结合环境中常见重金属离子,研究重金属离子结合E.coli TopA蛋白后对其活性的影响。【方法】在分别添加有锌、钴、镍、镉、铁、汞、砷、铬、铅、铜离子的M9基础培养中表达、纯化出E.coli TopA蛋白,并对纯化得到的蛋白用电感耦合等离子体质谱仪进行相应金属离子含量的测定;利用表达E.coli TopA锌指结构的突变体蛋白鉴定重金属离子的结合位点;通过体外超螺旋DNA松弛实验测定不同金属结合E.coli TopA的拓扑异构酶活性;通过测定蛋白内源性荧光推测不同金属结合E.coli TopA的空间构象差异。【结果】E.coli TopA在体内除了能结合锌和铁之外,还能够结合钴、镍、镉3种离子,但是不能结合汞、砷、铬、铅、铜离子。钴、镍、镉结合形式的E.coli TopA,每个蛋白分子最多可以结合3个相应的金属离子,他们与TopA蛋白的结合位点也是位于3个锌指结构域,而且每个锌指结构域结合1个金属离子。此外,E.coli TopA结合钴、镍、镉离子后,其DNA拓扑异构酶活性并未受到影响,可能是由于钴、镍、镉离子结合形式的E.coli TopA蛋白,其空间构象与锌结合形式相比并未发生显著变化。【结论】由于DNA拓扑异构酶在维持细胞正常生理功能中发挥关键作用,研究表明E.coli TopA的功能不会受到常见重金属的干扰(不结合或者结合后活性无影响),这也有可能是大肠杆菌在进化过程中产生的对抗环境中重金属离子毒害作用的一种自我保护和耐受机制,具有重要的生理意义。     

Microbial Processes of Heavy Metal Removal from Carbon‐Deficient Effluents in Constructed Wetlands

This paper reviews the main microbial processes involved when toxic metals are removed from wastewater in constructed wetlands. Microbial activity is thought to play a key role in the detoxification of these metals. The paper concentrates on the microbial processes which affect the mobility, the toxicity and bioavailability of metals, namely biosorption, metal sulfide precipitation by sulfate reducers, redox transformations, and methylation, as well as microbe‐plant interactions. These reactions result in either the precipitation and accumulation of metals in wetland soils, or their volatilization and emission into the atmosphere. The possibilities of optimizing the microbially mediated reactions for the development of wetland technology are discussed as a long‐term metal retention strategy.     

Imaging single-channel calcium microdomains

The Ca²⁺ microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca²⁺ signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca²⁺ imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of ‘optical patch-clamp recording’ is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca²⁺ ions.     

Heavy metals generate reactive oxygen species in terrestrial and aquatic ciliated protozoa

Reactive oxygen species (ROS) induction by exposure to heavy metals (Cd, Cu or Zn) in diverse free-living ciliated protozoa (Tetrahymena sp. and three strains of Colpoda steinii, isolated from freshwater and soils with different level of metal pollution) has been evaluated. Using specific fluorophores, such as 2',7'-dichlorofluorescein diacetate, hydroethidine and dihydrorhodamine 123, and a fluorescence microscope with the program MetaMorph Imaging System 4.0, we have analyzed both the average fluorescence emission and the heterogeneous distribution of fluorescence in control and treated cells. This is the first time that these fluorophores are used to detect ROS production in ciliated protozoa. All metals generate ROS, mainly superoxide and peroxides, showing a remarkable inter- and intra-specific variations. Likewise, resistance against each metal was also very diverse. Cu and specially Cd, the most toxic heavy metal for these ciliates, are the best oxidative stress inducers. However, a correlation between fluorescence emission intensity and cellular metal sensitivity for each strain cannot be established. Results are discussed and compared with similar findings previously published in other unicellular and pluricellular organisms.     

Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA'::lux fusions.

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 beta-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.     

Metal imaging in non-denaturating 2D electrophoresis gels by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for the detection of metalloproteins

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed as a powerful analytical technique for metal imaging of 2D gels for the detection of metalloproteins in rat kidney after electrophoretic separation. Protein complexes, extracted with water, were separated in their native state in the first and second dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, manganese and lead, were monitored by LA-ICP-MS after gel ablation by a focused laser beam in a way that the total surface of a selected fragment of the gel was totally ablated. The metal distribution of this part of the gel was then constructed by plotting the metal (isotope) signal intensity as a function of the x,y (isoelectric point, molecular mass) coordinates of the gel. The proteins at locations rich in metals were cut out, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).     

Toxicity evaluation of complex metal mixtures using reduced metal concentrations: application to iron oxidation by Acidithiobacillus ferrooxidans

In this study, we investigated the inhibition effects of single and mixed heavy metal ions (Zn2+, Ni2+, Cu2+, and Cd2+) on iron oxidation by Acidithiobacillus ferrooxidans. Effects of metals on the iron oxidation activity of A. ferrooxidans are categorized into four types of patterns according to its oxidation behavior. The results indicated that the inhibition effects of the metals on the iron oxidation activity were noncompetitive inhibitions. We proposed a reduced inhibition model, along with the reduced inhibition constant (alphai), which was derived from the inhibition constant (KI) of individual metals and represented the tolerance of a given inhibitor relative to that of a reference inhibitor. This model was used to evaluate the toxicity effect (inhibition effect) of metals on the iron oxidation activity of A. ferrooxidans. The model revealed that the iron oxidation behavior of the metals, regardless of metal systems (single, binary, ternary, or quaternary), is closely matched to that of any reference inhibitor at the same reduced inhibition concentration, [I]reduced, which defines the ratio of the inhibitor concentration to the reduced inhibition constant. The model demonstrated that single metal systems and mixed metal systems with the same reduced inhibitor concentrations have similar toxic effects on microbial activity.     

Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34

In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments. Ralstonia sp. CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions. These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export. Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment. Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell. Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment. Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics. Received: July 11, 1999 / Accepted: December 27, 1999     

Microbial biosensors.

A microbial biosensor consists of a transducer in conjunction with immobilised viable or non-viable microbial cells. Non-viable cells obtained after permeabilisation or whole cells containing periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells make use of the respiratory and metabolic functions of the cell, the analyte to be monitored being either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors have also been developed using genetically engineered microorganisms constructed by fusing the lux gene with an inducible gene promoter for toxicity and bioavailability testing. In this review, some of the recent trends in microbial biosensors with reference to the advantages and limitations are been discussed. Some of the recent applications of microbial biosensors in environmental monitoring and for use in food, fermentation and allied fields have been reviewed. Prospective future microbial biosensor designs have also been identified.     

Bioactive proteinaceous hydrogels from designed bifunctional building blocks

Stimulus-responsive, or "smart" protein-based hydrogels are of interest for many bioengineering applications, but have yet to include biological activity independent of structural functionality. We have genetically engineered bifunctional building blocks incorporating fluorescent proteins that self-assemble into robust and active hydrogels. Gelation occurs when protein building blocks are cross-linked through native protein-protein interactions and the aggregation of alpha-helical hydrogel-forming appendages. Building blocks constructed from different fluorescent proteins can be mixed to enable tuning of fluorescence loading and hydrogel strength with a high degree of independence. FRET experiments suggest a macro-homogeneous structure and that intragel and interprotein reactions can be engineered. This design approach will enable the facile construction of complex hydrogels with broad applicability.     

核酸切割酶在病原微生物检测中的研究进展

DNA是遗传信息的重要载体,其空间构象折叠性质使其具有很多的功能。利用核酸切割酶(cleaving DNAzyme)识别特定单链DNA分子并能够切割其中某条单链的性质来构建传感器,将特异性识别过程转化为凝胶电泳表征、释放荧光、比色现象的信号输出,同时能很好的和扩增反应结合来实现信号放大。核酸切割酶通过体外筛选技术获得,可以与靶物质(小分子、蛋白质,甚至整个细胞)特异性结合。由于具有制备简单,易于修饰和良好稳定性等优点,核酸切割酶被用于构建生物传感器以检测病原微生物,已应用到现场检测甚至医疗中的体内检测,结合已经成熟的检测设备血糖仪、横流层析试纸条带进行微生物检测,并广泛地应用到生物传感、食品安全、医疗在内的重要领域中。综述了近年来核酸切割酶在微生物检测中的应用,讨论了核酸切割酶在微生物检测中的切割机理和产物、靶标以及表征手段,探索核酸切割酶在微生物实际检测中的意义。对该技术的发展前景及其面临的问题进行展望,以期核酸切割酶在微生物检测领域能够更好的发展。