PHYSIOLOGICAL CHARACTERIZATION OF A SYNECHOCOCCUS SP. (CYANOPHYCEAE) STRAIN PCC 7942 IRON‐DEPENDENT BIOREPORTER FOR FRESHWATER ENVIRONMENTS1

The complex chemical speciation of Fe in aquatic systems and the uncertainties associated with biological assimilation of Fe species make it difficult to assess the bioavailability of Fe to phytoplankton in relation to total dissolved Fe concentrations in natural waters. We developed a cyanobacterial Fe‐responsive bioreporter constructed in Synechococcus sp. strain PCC 7942 by fusing the Fe‐responsive isiAB promoter to Vibrio harveyi luxAB reporter genes. A comprehensive physiological characterization of the bioreporter has been made in defined Fraquil medium at free ferric ion concentrations ranging from pFe 21.6 to pFe 19.5. Whereas growth and physiological parameters are largely constrained over this range of Fe bioavailability, the bioreporter elicits a luminescent signal that varies in response to Fe deficiency. A dose‐response characterization of bioreporter luminescence made over this range of Fe^{3 +} bioavailability demonstrates a sigmoidal response with a dynamic linear range extending between pFe 21.1 and pFe 20.6. The applicability of using this Fe bioreporter to assess Fe availability in the natural environment has been tested using water samples from Lake Huron (Laurentian Great Lakes). Parallel assessment of dissolved Fe and bioreporter response from these samples reinforces the idea that measures of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton communities.     

Luminescent whole-cell cyanobacterial bioreporter for measuring Fe availability in diverse marine environments

A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.     

利用蓝藻生物报告体检测水体中可利用铁的研究

鱼腥藻PCC 7120 中的alr2581 基因编码的蛋白质在缺铁胁迫时显著上调。将该基因的启动子Palr2581和费氏弧菌的luxAB 基因融合, 通过同源单交换, 整合到鱼腥藻PCC 7120 的基因组上, 构建了可以感知环境中铁的生物报告体Palr2581-luxAB。该藻株在不含铁的BG11 中培养时, 启动子Palr2581 的转录活性增强, LuxAB酶活显著升高。通过测定Palr2581-luxAB 藻株在不同铁浓度Fraquil 培养基中的LuxAB 酶活, 得到了铁浓度pFe(-lgFe3+)与 LuxAB 酶活的剂量反应曲线。结果显示, 12h 时, LuxAB 酶活随培养基中Fe3+浓度增加呈S形递减关系, 其中在pFe=20.7—21.2 范围内有很好的线性关系。根据这一特性, 我们利用Palr2581-luxAB 作为铁的生物报告体, 测定了武汉市东湖水体中可利用的铁浓度为10-20.56 mol/L。研究显示, 通过这一方法可以较方便地监测各种淡水中可利用的铁浓度。       

A high sensitivity iron-dependent bioreporter used to measure iron bioavailability in freshwaters

A Nostoc sp. PCC 7120 iron bioreporter containing iron-regulated schizokinen transporter gene alr0397 promoter fused to the luxAB genes was examined to optimize its response to bioavailable iron. Dose-response relationships between luciferase activity and free ferric ion (Fe(3+) ) concentrations pFe (-lg [Fe(3+) ]) were generated by measuring luciferase activities of the bioreporter in trace metal-buffered Fraquil medium with various incubation times. The results were best demonstrated by sigmoidal curves (pFe 18.8-21.7, Fe(3+) =?10(-18.8) -10(-21.7) M) with the linear range extending from pFe 19.6-21.5 (Fe(3+) =?10(-19.6) -10(-21.5) M) after a 12-h incubation time. Optimal conditions for the use of this bioreporter to sense the iron bioavailability were determined to be: a 12-h exposure time, initial cell density of OD(730?nm) =?0.06, high nitrate (100?μM), high phosphate (10?μM), moderate Co(2+) (0.1-22.5?nM), Zn(2+) (0.16-12?nM), Cu(2+) (0.04-50?nM), and wide range of Mn(2+) concentration (0.92-2300?nM). The applicability of using this iron bioreporter to assess iron availability in the natural environment has been tested using water samples from eutrophic Taihu, Donghu, and Chaohu lakes. It is indicated that the bioreporter is a useful tool to assess bioavailable iron in various water quality samples, especially in eutrophic lakes with high bioavailable iron.     

Fluorescence-Based Bacterial Bioreporter for Specific Detection of Methyl Halide Emissions in the Environment

Methyl halides are volatile one-carbon compounds responsible for substantial depletion of stratospheric ozone. Among them, chloromethane (CH₃Cl) is the most abundant halogenated hydrocarbon in the atmosphere. Global budgets of methyl halides in the environment are still poorly understood due to uncertainties in their natural sources, mainly from vegetation, and their sinks, which include chloromethane-degrading bacteria. A bacterial bioreporter for the detection of methyl halides was developed on the basis of detailed knowledge of the physiology and genetics of Methylobacterium extorquens CM4, an aerobic alphaproteobacterium which utilizes chloromethane as the sole source of carbon and energy. A plasmid construct with the promoter region of the chloromethane dehalogenase gene cmuA fused to a promotorless yellow fluorescent protein gene cassette resulted in specific methyl halide-dependent fluorescence when introduced into M. extorquens CM4. The bacterial whole-cell bioreporter allowed detection of methyl halides at femtomolar levels and quantification at concentrations above 10 pM (approximately 240 ppt). As shown for the model chloromethane-producing plant Arabidopsis thaliana in particular, the bioreporter may provide an attractive alternative to analytical chemical methods to screen for natural sources of methyl halide emissions.     

Bioluminescent bioreporter for assessment of arsenic contamination in water samples of India

In the present study the most efficient R-factor controlling the ars operon was selected after screening of 39 Escherichia coli isolates by minimum inhibitory concentration test (MIC) studies from water samples of different geographical locations of India. Among all, strain isolated from Hooghly River (West Bengal) was found to have maximum tolerance towards arsenic and was further used for the development of bioreporter bacteria. Cloning of the ars regulatory element along with operator-promotor and luxCDABE from Photobacteria into expression vector has been accomplished by following recombinant DNA protocols. The bioreporter sensor system developed in this study can measure the estimated range of 0.74–60 μg of As/L and is both specific and selective for sensing bioavailable As. The constructed bacterial biosensor was further used for the determination of arsenic ion concentration in different environmental samples of India.     

Construction and application of an Escherichia coli bioreporter for aniline and chloroaniline detection

Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P _todX promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP–pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9?h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose–response relationship, from which the K _1/2 value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57’sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.     

NITRATE UTILIZATION BY PHYTOPLANKTON IN LAKE SUPERIOR IS IMPAIRED BY LOW NUTRIENT (P,Fe) AVAILABILITY AND SEASONAL LIGHT LIMITATION — A CYANOBACTERIAL BIOREPORTER STUDY1

We previously developed a luminescent Synechococystis sp. strain PCC 6803 cyanobacterial bioreporter that is used as a real‐time whole‐cell sensor to assess nitrate assimilatory capacity in freshwaters. Applying the bioreporter assay to Lake Superior, a system whose nitrate levels have increased 6‐fold since 1900, we investigated factors that constrain nitrate utilization in this oligotrophic system. Clean sampling methods were used to collect water from Lake Superior during spring and summer 2004, and nitrate utilization was measured by monitoring bioreporter luminescence. Bioreporter response was monitored during experiments in which the lake water was amended with nutrients and incubated under light regimes simulating integrated spring and summer mixing depths. These studies demonstrated that nitrate utilization was enhanced at most stations following addition of phosphorus (P). Moreover, at many stations, addition of iron (Fe) enhanced the P effect. Strength‐of‐effect statistical analysis provided the individual contribution of P and Fe toward stimulating bioreporter response. In general, distance from shore and season were not good predictors of nitrate assimilatory capacity. Manipulation of light flux during bioreporter experiments also showed that light intensities experienced during spring mixing are likely insufficient to saturate the rate of nitrate utilization. Overall, these data suggest that P‐limited algae are deficient in their ability to assimilate nitrate in Lake Superior. Furthermore, we suggest that a secondary limitation for Fe may occur that further constrains nitrate drawdown. Lastly, during spring, light fluxes are sufficiently low to prevent maximal nitrate utilization, even in the absence of nutrient limitation.     

Assimilation of Fe and carbon by marine copepods from Fe-limited and Fe-replete diatom prey

Previous studies have demonstrated that growth of marine phytoplankton, bacteria and protozoa can be limited by the availability of Fe. We report evidence that the amount of Fe assimilated by crustacean grazers is affected by the Fe status of their pry. ⁵⁹Fe and ¹⁴C radiotracers were used to follow the fate of Fe and carbon during trophic transfer from diatoms to copepods. Fe assimilation efficiency was higher for copepods ingesting Fe-limited Thalassiosira weissflogii (17%) compared to diatoms that were not limited by Fe (10%), but assimilated Fe was lost more rapidly by copepods ingesting Fe-limited prey. Fe:C assimilation ratios were lower in copepods (5-12 mol Fe:mol C) than the cellular ratios of the phytoplankton prey (17-35 mol Fe:mol C), suggesting that copepods do not accumulate Fe relative to C during grazing. The largest single fate for Fe and C after grazing was regeneration to dissolved pools. Fe:C ratios in dissolved pools were approximately equal to the ratios in the original prey, but Fe:C ratios were higher in particular pools (largely fecal pellets), which should facilitate the export of Fe from the euphotic zone relative to C. Although copepod grazing does recycle cellular Fe and C, our results indicate that grazing may also tend to enhance Fe stress for lower trophic levels by removing Fe from the euphotic zone faster than C.      

Genetic indicators of iron limitation in wild populations of Thalassiosira oceanica from the northeast Pacific Ocean

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe- and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from the northeast Pacific, demonstrating the sensitivity of this method. Overall, FLDA1 and ISIP3 expression was inversely related to Fe concentrations and offered insight into the Fe nutritional health of T. oceanica in the field. As T. oceanica is a species tolerant to low Fe, indications of Fe limitation in T. oceanica populations may serve as a proxy for severe Fe stress in the overall diatom community. At two shallow coastal locations, FLD1A and ISIP3 expression revealed Fe stress in areas where dissolved Fe concentrations were high, demonstrating that this approach may be powerful for identifying regions where Fe supply may not be biologically available.     

OPTIMIZATION OF IRON‐DEPENDENT CYANOBACTERIAL (SYNECHOCOCCUS,CYANOPHYCEAE) BIOREPORTERS TO MEASURE IRON BIOAVAILABILITY1

Complex chemistry and biological uptake pathways render iron bioavailability particularly difficult to assess in natural waters. Bioreporters are genetically modified organisms that are useful tools to directly sense the bioavailable fractions of solutes. In this study, three cyanobacterial bioreporters derived from Synechococcus PCC 7942 were examined for the purpose of optimizing the response to bioavailable Fe. Each bioreporter uses a Fe‐regulated promoter (isiAB, irpA and mapA), modulated by distinct mechanisms under Fe deficiency, fused to a bacterial luciferase (luxAB). In order to provide a better understanding of the way natural conditions may affect the ability of the bioreporter to sense iron bioavailability, the effect of relevant environmental parameters on the response to iron was assessed. Optimal conditions (and limits of applicability) for the use of these bioreporters on the field were determined to be: a 12 h (12–24 h) exposure time, temperature of 15°C (15°C–22°C), photon flux density of 100 μmol photons·m^?2·s^?1 (37–200 lmol photons·m^?2·s^?1), initial biomass of 0.6–0.8 lg chlorophyll a (chl a)·L^?1 (0.3–1.5 lg chl a·L^?1) or approximately 10⁵ bioreporter cells·mL^?1, high phosphate (10 lM), and low micronutrients (absent). The measured luminescence was optimal with an exogenous addition of 60 lM aqueous decanal substrate allowing a 5 min reaction time in the dark before analysis. This study provides important considerations relating to the optimization in the use of bioreporters under field conditions that can be used for method development of other algal and cyanobacterial bioreporters in aquatic systems.     

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Bioavailable iron in oligotrophic Lake Superior assessed using biological reporters

Iron bioavailability in Lake Superior was assessed during fieldsurveys conducted in 2001–2002. Dissolved iron (Fe_d) rangedbetween 1 and 4 nM at offshore stations and >10 nM at mostnearshore sites. Iron availability was assessed using a luminescentSynechococcus bioreporter comprising a luciferase reporter controlledby an iron-responsive promoter isiAB. Bioreporter luminescencewas negatively correlated to Fe_d measured in the samples. Distancefrom shore was a better predictor of iron bioavailability thanwas season. Water collected from most offshore stations sampledduring spring and summer elicited higher bioreporter luminescencethan did nearshore sites. Iron availability did not vary withdepth during summer, despite the presence of elevated levelsof Fe_d in the hypolimnion at most stations. Ultrafiltration(0.02 µm) of Fe_d at two offshore sites demonstrated Fe_dto be present mainly in a colloidal phase, yet the bioreporterresponded solely to iron contained in the soluble phase. Duringspring, a parallel immunochemical assay of diatoms resultedin the detection of ferredoxin (Fd) but not flavodoxin (Flvd)at five stations indicating the presence of an iron sufficientassemblage of diatoms at these sites. Whereas neither bioreporternor immunochemical approaches conducted during spring supportedphysiological iron deficiency among Lake Superior phytoplankton,the results did point to differences in the availability ofiron to prokaryotes and eukaryotes.     

Effect of Inorganic and Organic Ligands on the Bioavailability of Methylmercury as Determined by Using a mer-lux Bioreporter

A mer-lux bioreporter was constructed to assess the bioavailability of methylmercury [CH₃Hg(II)] in Escherichia coli. The bioreporter was shown to be sensitive, with a detection limit of 2.5 nM CH₃Hg(II), and was used to investigate the effects of chlorides, humic acids, and thiols on the bioavailability of CH₃Hg(II) in E. coli. It was found that increasing the concentration of chlorides resulted in an increase in CH₃Hg(II) bioavailability, suggesting that there was passive diffusion of the neutral complex (CH₃HgCl⁰). Humic acids were found to reduce the bioavailability of CH₃Hg(II) in varying degrees. Complexation with cysteine resulted in increased bioavailability of CH₃Hg(II), while assays with equivalent concentrations of methionine and leucine had little or no effect on bioavailability. The mechanism of uptake of the mercurial-cysteine complexes is likely not passive diffusion but could result from the activities of a cysteine transport system. The bioavailability of CH₃Hg(II) decreased with increasing glutathione concentrations.     

Whole‐cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills

Acinetobacter baylyi ADP1 was found to tolerate seawater and have a special ability of adhering to an oil–water interface of 10–80 µm emulsified mineral and crude oil droplets. These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to actively search and sense oil spill in water and soils. Acinetobacter baylyi bioreporter ADPWH_alk was developed and applied to the detection of alkanes and alkenes in water, seawater and soils. Bioreporter ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. So far, ADPWH_alk is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. This bioreporter responded to the alkanes in about 30 min and it was independent to the cell growth phase because of two point mutations in alkM promoter recognized by alkane regulatory protein ALKR. ADPWH_alk was applied to detect mineral oil, Brent, Chestnut and Sirri crude oils in water and seawater in the range 0.1–100 mg l^?1, showing that the bioreporter oil detection was semi‐quantitative. This study demonstrates that ADPWH_alk is a rapid, sensitive and semi‐quantitative bioreporter that can be useful for environmental monitoring and assessment of oil spills in seawater and soils.