Detection of Cadmium in Human Biospecimens by a Cadmium-Selective Whole-Cell Biosensor Based on Deoxyviolacein.

Cadmium poses a severe health risk, impacting various bodily systems. Monitoring human exposure is vital. Urine and blood cadmium serve as critical biomarkers. However, current urine and blood cadmium detection methods are expensive and complex. Being cost-effective, user-friendly, and efficient, visual biosensing offers a promising complement to existing techniques. Therefore, we constructed a cadmium whole-cell biosensor using CadR10 and deoxyviolacein pigment in this study. We assessed the sensor for time-dose response, specific response to cadmium, sensitivity response to cadmium, and stability response to cadmium. The results showed that (1) the sensor had a preferred signal-to-noise ratio when the incubation time was 4 h; (2) the sensor showed excellent specificity for cadmium compared to the group 12 metals and lead; (3) the sensor was responsive to cadmium down to 1.53 nM under experimental conditions and had good linearity over a wide range from 1.53 nM to 100 µM with good linearity (R2 = 0.979); and (4) the sensor had good stability. Based on the excellent results of the performance tests, we developed a cost-effective, high-throughput method for detecting urinary and blood cadmium. Specifically, this was realized by adding the blood or urine samples into the culture system in a particular proportion. Then, the whole-cell biosensor was subjected to culture, n-butanol extraction, and microplate reading. The results showed that (1) at 20% urine addition ratio, the sensor had an excellent curvilinear relationship (R2 = 0.986) in the range of 3.05 nM to 100 µM, and the detection limit could reach 3.05 nM. (2) At a 10% blood addition ratio, the sensor had an excellent nonlinear relationship (R2 = 0.978) in the range of 0.097-50 µM, and the detection limit reached 0.195 µM. Overall, we developed a sensitive and wide-range method based on a whole-cell biosensor for the detection of cadmium in blood and urine, which has the advantages of being cost-effective, ease of operation, fast response, and low dependence on instrumentation and has the potential to be applied in the monitoring of cadmium exposure in humans as a complementary to the mainstream detection techniques.

High-throughput screening of human mercury exposure based on a low-cost naked eye-recognized biosensing platform.

Whole-cell biosensors could be helpful for in situ disease diagnosis. However, their use in analyzing biological samples has been hindered by unstable responses, low signal enhancement, and growth inhibition in complex media. Here, we offered a solution by building a visual whole-cell biosensor for urinary mercury determination. With deoxyviolacein as the preferred signal for the mercury biosensor for the first time, it enabled the quantitative detection of urinary mercury with a favorable linear range from 1.57 to 100 nM. The biosensor can accurately diagnose urine mercury levels exceeding the biological exposure index with 95.8% accuracy. Thus, our study provided a biosensing platform with great potential to serve as a stable, user-friendly, and high-throughput alternative for the daily monitoring or estimating of urinary mercury.

Pb(II)-inducible proviolacein biosynthesis enables a dual-color biosensor toward environmental lead.

With the rapid development of synthetic biology, various whole-cell biosensors have been designed as valuable biological devices for the selective and sensitive detection of toxic heavy metals in environmental water. However, most proposed biosensors are based on fluorescent and bioluminescent signals invisible to the naked eye. The development of visible pigment-based biosensors can address this issue. The pbr operon from Klebsiella pneumoniae is selectively induced by bioavailable Pb(II). In the present study, the proviolacein biosynthetic gene cluster was transcriptionally fused to the pbr Pb(II) responsive element and introduced into Escherichia coli. The resultant biosensor responded to Pb(II) in a time- and dose-dependent manner. After a 5-h incubation with Pb(II), the brown pigment was produced, which could be extracted into n-butanol. Extra hydrogen peroxide treatment during n-butanol extract resulted in the generation of a stable green pigment. An increased brown signal was observed upon exposure to lead concentrations above 2.93 nM, and a linear regression was fitted from 2.93 to 3,000 nM. Extra oxidation significantly decreased the difference between parallel groups. The green signal responded to as low as 0.183 nM Pb(II), and a non-linear regression was fitted in a wide concentration range from 0.183 to 3,000 nM. The specific response toward Pb(II) was not interfered with by various metals except for Cd(II) and Hg(II). The PV-based biosensor was validated in monitoring bioaccessible Pb(II) spiked into environmental water. The complex matrices did not influence the regression relationship between spiked Pb(II) and the dual-color signals. Direct reading with the naked eye and colorimetric quantification enable the PV-based biosensor to be a dual-color and low-cost bioindicator for pollutant heavy metal.

Metabolic engineering of the carotenoid biosynthetic pathway toward a specific and sensitive inorganic mercury biosensor.

The toxicity of mercury (Hg) mainly depends on its form. Whole-cell biosensors respond selectively to toxic Hg(ii), efficiently transformed by environmental microbes into methylmercury, a highly toxic form that builds up in aquatic animals. Metabolically engineered Escherichia coli (E. coli) have successfully produced rainbow colorants. By de novo reconstruction of the carotenoid synthetic pathway, the Hg(ii)-responsive production of lycopene and ß-carotene enabled programmed E. coli to potentially become an optical biosensor for the qualitative and quantitative detection of ecotoxic Hg(ii). The red color of the lycopene-based biosensor cell pellet was visible upon exposure to 49 nM Hg(ii) and above. The orange ß-carotene-based biosensor responded to a simple colorimetric assay as low as 12 nM Hg(ii). A linear response was observed at Hg(ii) concentrations ranging from 12 to 195 nM. Importantly, high specificity and good anti-interference capability suggested that metabolic engineering of the carotenoid biosynthesis was an alternative to developing a visual platform for the rapid analysis of the concentration and toxicity of Hg(ii) in environmentally polluted water.

Metabolic engineering of the violacein biosynthetic pathway toward a low-cost, minimal-equipment lead biosensor.

Metabolic engineered bacteria have been successfully employed to produce various natural colorants, which are expected to be used as the visually recognizable signals to develop mini-equipment biological devices for monitoring toxic heavy metals. The violacein biosynthetic pathway has been reconstructed in Escherichia coli (E. coli). Here the successful production of four violacein derivatives was achieved by integrating metabolic engineering and synthetic biology. Lead binding to the metalloregulator enables whole-cell colorimetric biosensors capable of assessing bioavailable lead. Deoxyviolacein-derived signal showed the most satisfied biosensing properties among prodeoxyviolacein (green), proviolacein (blue), deoxyviolacein (purple), and violacein (navy). The limit of detection (LOD) of pigment-based biosensors was 2.93 nM Pb(II), which is lower than that of graphite furnace atomic absorption spectrometry. Importantly, a good linear dose-response model in a wide dose range (2.93-6000 nM) was obtained in a non-cytotoxic deoxyviolacein-based biosensor, which was significantly better than cytotoxic violacein-based biosensor (2.93-750 nM). Among ten metal ions, only Cd(II) and Hg(II) exerted a slight influence on the response of the deoxyviolacein-based biosensor toward Pb(II). The deoxyviolacein-based biosensor was validated in detecting bioaccessible Pb(II) in environmental samples. Factors such as low cost and minimal-equipment requirement make this biosensor a suitable biological device for monitoring toxic lead in the environment.

Indigoidine biosynthesis triggered by the heavy metal-responsive transcription regulator: a visual whole-cell biosensor.

During the last few decades, whole-cell biosensors have attracted increasing attention for their enormous potential in monitoring bioavailable heavy metal contaminations in the ecosystem. Visual and measurable output signals by employing natural pigments have been demonstrated to offer another potential choice to indicate the existence of bioavailable heavy metals in recent years. The biosynthesis of the blue pigment indigoidine has been achieved in E. coli following heterologous expression of both BpsA (a single-module non-ribosomal peptide synthetase) and PcpS (a PPTase to activate apo-BpsA). Moreover, we demonstrated herein the development of the indigoidine-based whole-cell biosensors to detect bioavailable Hg(II) and Pb(II) in water samples by employing metal-responsive transcriptional regulator MerR and PbrR as the sensory elements, and the indigoidine biosynthesis gene cluster as a reporter element. The resulting indigoidine-based biosensors presented a good selectivity and high sensitivity to target metal ions. High concentration of target metal exposure could be clearly recognized by the naked eye due to the color change by the secretion of indigoidine, and quantified by measuring the absorbance of the culture supernatants at 600 nm. Dose-response relationships existed between the exposure concentrations of target heavy metals and the production of indigoidine. Although fairly good linear relationships were obtained in a relatively limited concentration range of the concentrations of heavy metal ions, these findings suggest that genetically controlled indigoidine biosynthesis triggered by the MerR family transcriptional regulator can enable a sensitive, visual, and qualitative whole-cell biosensor for bioindicating the presence of bioaccessible heavy metal in environmental water samples. KEY POINTS: • Biosynthesis pathway of indigoidine reconstructed in a high copy number plasmid in E. coli. • Visual and colorimetric detection of Hg(II) and Pb(II) by manipulation of indigoidine biosynthesis through MerR family metalloregulator. •Enhanced detection sensitivity toward Hg(II) and Pb(II) achieved using novel pigment-based whole-cell biosensors.

Versatile artificial mer operons in Escherichia coli towards whole cell biosensing and adsorption of mercury.

Mercury exists naturally and mainly as a man-made pollutant in the environment, where it exerts adverse effects on local ecosystems and living organisms. It is important to develop an appropriate synthetic biological device that recognizes, detects and removes the bioavailable fraction of environmental mercury. Both single-signal and double-signal output mercury biosensors were assembled using a natural mer operon as a template. Selectivity and sensitivity of whole-cell biosensors based on artificial mer operons were determined. Three whole-cell biosensors were highly stable at very high concentrations of mercuric chloride, and could detect bioavailable Hg(II) in the concentration range of 6.25-200 µM HgCl2. A novel Hg(II) bioadsorption coupled with biosensing artificial mer operon was assembled. This would allow Hg(II)-induced Hg(II) binding protein cell surface display and green fluorescence emission to be achieved simultaneously while retaining the linear relationship between fluorescent signal and Hg(II) exposure concentration. The present study provides an innovative way to simultaneously detect, quantify, and remove bioavailable heavy metal ions using an artificially reconstructed heavy metal resistance operon.

[Response of Bacteriohopanepolyols to Hypoxic Conditions in the Surface Sediments of the Yangtze Estuary and Its Adjacent Areas].

Bacteriohopanepolyols (BHPs), as a novel bacterial biomarker, show clear potential for tracking organic matter sources and environmental change. To evaluate BHPs as indicators of seasonal hypoxia in the Yangtze Estuary and its adjacent areas, the composition, distribution, and source of BHPs in surface sediments were analyzed using high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS). A total of 12 BHPs were detected with a normalized TOC concentration of 3.79-269 µg·g-1. The BHPs present in the surface sediments were dominated by bacteriohopanetetrol (BHT), 2-methyl-BHT, amino-BHPs, and adenosylhopane and its homologues, accounting for 40%, 22%, 12%, and 4% of the total BHPs, respectively. Each of these components and their corresponding indices show clear spatial trends. Specifically, BHT showed an "offshore increase" trend, which was mainly attributed to marine autochthonous inputs; and soil marker BHPs including adenosylhopane, which were dominated by terrestrial sources, showed an "offshore decrease" trend. The Rsoil index indicated a similar spatial pattern to the soil marker BHPs, with the relative contribution of terrestrial organic matter decreasing from 61.5% in coastal waters to 1.66% in the open ocean. This suggests that the organic matter in the coastal waters was mainly derived from terrestrial sources while marine sources were dominant in the open ocean. BHT-Ⅱ, the BHT stereoisomer, was derived from anaerobic ammonium oxidizing bacteria. High BHT-Ⅱ ratios were consistent with seasonal hypoxic zones in the Yangtze Estuary and, furthermore, these ratios were significantly negatively correlated with dissolved oxygen (DO) concentrations in the bottom waters. These observations indicate that hypoxic environments are beneficial to BHT-Ⅱ production, implying that BHT-Ⅱ can be used as an indicator of marine hypoxia.

Development of Cadmium Multiple-Signal Biosensing and Bioadsorption Systems Based on Artificial Cad Operons.

The development of genetic engineering, especially synthetic biology, greatly contributes to the development of novel metal biosensors. The cad operon encoding cadmium resistance was previously characterized from Pseudomonas putida. In this study, single-, dual-, and triple-signal output Cd(II) biosensors were successfully developed using artificial translationally coupled cad operons. Sensitivity, selectivity, and response toward Cd(II) and Hg(II), of three biosensors were all determined. Reporter signals of three biosensors all increased within the range 0.1-3.125 µM Cd(II). Three biosensors responded strongly to Cd(II), and weakly to Hg(II). However, the detection ranges of Cd(II) and Hg(II) do not overlap in all three biosensors. Next, novel Cd(II) biosensing coupled with bioadsorptive artificial cad operons were assembled for the first time. Cd(II)-induced fluorescence emission, enzymatic indication, and Cd(II) binding protein surface display can be achieved simultaneously. This study provides an example of one way to realize multiple signal outputs and bioadsorption based on the redesigned heavy metal resistance operons, which may be a potential strategy for biodetection and removal of toxic metal in the environment, facilitating the study of the mechanism and dynamics of bioremediation.

Genetic control of violacein biosynthesis to enable a pigment-based whole-cell lead biosensor.

Environmental risks continue to grow due to heavy metal contamination caused by anthropogenic activities. Accumulation of harmful quantities of lead poses a threat to aquatic organisms, plants, and human beings. Whole-cell biosensors, which can proliferate independently, can detect the bioavailable fraction to assess the effect of target heavy metal on the environmental ecosystem. In this study, the biosynthesis pathway of violacein was heterogeneously constructed under the control of the T7 lac promoter in E. coli. A dose-response relationship existed between the inducer and the production of violacein. The biosynthesis pathway of violacein was finally engineered under the regulation of Pb(ii)-dependent metalloregulator PbrR to assemble Pb(ii)-inducible whole-cell biosensor. It permitted specific biosensing of Pb(ii) with extraordinary selectivity, and could resist the interferences from various metal ions. Color change by the intracellular accumulation of violacein could be recognized with the naked eye directly with high concentration of lead exposure, and quantified by determining the absorbance at 490 nm after butanol extraction. A good linear range for Pb(ii) concentrations of 0.1875-1.5 µM was obtained. The novel pigment-based whole-cell biosensor could detect concentrations as low as 0.1875 µM Pb(ii) based on in vitro quantification of violacein extracted by butanol, which is significantly lower than reported fluorescent protein-based PbrR-regulated biosensors based on direct measurement of whole cell fluorescence. These results indicate that genetically controlled violacein biosynthesis can enable a sensitive, visual, and qualitative biosensor for monitoring the presence of bioavailable Pb(ii) in lead-contaminated water.

Vibrio profundi sp. nov., isolated from a deep-sea seamount.

A Gram-stain negative, rod-shaped, facultative anaerobic, motile bacterial strain, designated TP187T, was isolated from a seamount near the Yap Trench in the tropical western Pacific. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain TP187T is related to members of the genus Vibrio and has high 16S rRNA gene sequence similarity with the type strains of Vibrio chagasii (97.3%) and Vibrio gallaecicus (97.1%). Sequence similarities to all other type strains of current species of the genus Vibrio were below 97%. The polar lipids profile was found to contain diphosphatidylglycerol, phosphatidylglycerol, an aminophospholipid, two aminolipids, four phospholipids and eleven unidentified polar lipids. Ubiquinone Q-8 was detected as the predominant quinone. The genomic DNA G + C content of strain TP187T was determined to be 43.7 mol%. In addition, the maximum values of in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI) between strain TP187T with V. chagasii LMG 21353T were 22.40 and 77.50% respectively. Both values are below the proposed cutoff levels for species delineation, i.e. 70 and 95%, respectively. Combined data from phenotypic, phylogenetic, isDDH and ANI data demonstrated that the strain TP187T is representative of a novel species of the genus Vibrio, for which we propose the name Vibrio profundi sp. nov. (type strain TP187T = KACC 18555T = CGMCC 1.15395T).

[Heavy metals in the surface sediment of the dumping ground outside Jiaozhou Bay and their potential ecological risk].

Based on the monitoring data of heavy metals (Cr, Hg, Cd, Pb, Zn, Cu) in the surface sediment of the dumping ground outside Jiaozhou Bay from 2003 to 2008, the distribution patterns, factors controlling the distribution, and the potential ecological risks of heavy metals were studied with the data in 2007-08, and the fluctuation trends of heavy metals in the surface sediment over the 6 years were also discussed. The average concentrations of heavy metals Cr, Hg, Cd, Pb, Zn, Cu in the surface sediment were 29.47, 0.065, 0.105, 1.145, 9.63, 3.355 microg/g, respectively. Except for Cr, the concentration of heavy metals was high in the central dumping area while low outside the dumping ground, suggesting that the dredged material dumped was the main source of heavy metals. Organic carbon content in the surface sediment had a significant positive correlation with heavy metals except for Cr. Based on the results of ecological risk assessment, Hg had a medium potential ecological risk, while the other heavy metals had low potential ecological risk. The overall risk index (RI) of the heavy metals was 100.50, which was considered as a level of low potential ecological risk. The average concentration of heavy metals showed a decreasing trend over the 6 years, except Hg. In conclusion, the quality of surface sediment in term of heavy metals in the dumping ground outside Jiaozhou Bay is relatively good.

[Marine inorganic carbon system responses to macro-DIN supply coupled with Ulva pertusa in simulated experiments].

Effects of macronutrient (NO3(-) -N and NH4(+) -N) on inorganic carbon system of water with Ulva pertusa existed were studied in laboratory simulation experiments. The results demonstrated that nutrient enrichment induced DIC, HCO3- and p(CO2) decreased while pH and CO3(2-) increased. The seawater changed from carbon source to carbon sink. During the experiments, the concentration of DIC, HCO3- and p(CO2) decreased with increasing concentration of nutrient when the NO3(-) -N and N4(+) -N were less than critical concentration. The concentration of DIC changed most at the NO3(-3) and NH4(-)3 groups, which decreased 151 micromol x L(-1) and 232 micromol x L(-1) compared with the control groups in the end of experiment. The increased dry weight of Ulva pertusa (deltam) of nutrient addition groups showed a significant negative correlation with deltaDIC (r = - 0.91, p < 0.0001, n = 11). The main controlling factor to inorganic carbon variation is the adaptation of Ulva pertusa to different DIN. When the concentration of DIN facilitates the growth of Ulva pertusa, the concentration of DIC decreased and dry weight of Ulva pertusa increased. NH(4) -N has more influence on inorganic carbon system than NO(3-) -N.

[Effects of excessive inorganic nitrogen on inorganic carbon system in seawater simulated experiments].

The influence of excessive inorganic nitrogen (NO3-, NH4+, NO2-) on the pH, inorganic carbon system and air-sea CO2 flux was studied in laboratory seawater simulation experiments. The result indicates that the excessive inorganic nitrogen significantly has an important influence on the inorganic carbon system in all conditions. At the NO3- addition groups, HCO3- and p (CO2) increase while pH and CO3(2-) decrease when the concentration of NO3- < or = 37.60 micromol x L(-1). But high concentrations > or = 188 micromol x L(-1) is just the reverse. The seawater changes from carbon source to carbon sink of atmospheric CO2 with NO3- addition increased. At the NH4+ addition groups, HCO3- and p (CO2) decrease while pH and CO3(2-) increase when the concentration of NH4+ < or = 25.20 micromol x L(-1). However, when the concentration of NH4+ > or = 126 micromol x L(-1), the concentrations of HCO3- and p (CO2) increase with NH4+ addition and the seawater changes from carbon sink to carbon source of atmospheric CO2 and the intensity of carbon source has a positive relationship with the concentration of NH4+. At the NO2- addition groups, the concentrations of HCO3- and p (CO2) decrease while pH and CO3(2-) increase with time scale and the seawater acts as a strong carbon sink with NO2- addition of 7.90 micromol x L(-1). While at the other NO2- concentration levels, the carbon sink is weak in seawater. The Chl-a concentration of nutrient addition groups compared with control (delta Chl-a) shows a significant negative correlation with delta p (CO2) (r = -0.87, p < 0.000 1, n = 16). The main controlling factor to the variation of inorganic carbon system is the adaptation of phytoplankton to different form and concentration of nitrogen.

[Variation characteristics and controlling factors of heavy metals in the South Yellow Sea surface seawaters].

Based on the 8 cruises data of surface seawater heavy metals and other related environmental factors from 1997 to 2004 of the South Yellow Sea (SYS), distribution patterns, mechanisms controlling the distributions and pollution levels of heavy metals (As, Cd, Cu, Hg, Pb, Zn) were studied with the data of 2003-10, and 8-year-fluctuation trends of heavy metals were also discussed. The average concentrations of heavy metals in surface seawater were 2.33, 0.078, 1.41, 0.003 6, 0.37, 6.21 microg/L respectively. The average concentrations showed a relatively stable trend in 8 years, except Zn's distinct upward tendency. The distribution patterns corresponded to the distance away from the coastline,that was, the content of heavy metals (except Pb) was low in central area while high inshore. Those were responses of human activity in the marginal sea, however, in some local areas, Pb was controlled by atmospheric deposition, Cd was relative to pH and salinity, Hg was related to organic carbon, Cu and Zn were influenced by runoff and drainage, and the resuspending of sediments played important roles on the content and distribution of As. Compared to the Marine Water Quality Standard of China, heavy metals indicated that SYS was a first class sea, and Ecological Risk Index analysis showed that SYS was a low ecological risk sea. In conclusion, seawater quality of heavy metals in SYS surface seawater is relatively good in general.