Time-Dependent Biosensor Fluorescence as a Measure of Bacterial Arsenic Uptake Kinetics and Its Inhibition by Dissolved Organic Matter

Abstract

Microbe-mediated transformations of arsenic (As) often require As to be taken up into cells prior to enzymatic reaction. Despite the importance of these microbial reactions for As speciation and toxicity, understanding of how As bioavailability and uptake are regulated by aspects of extracellular water chemistry, notably dissolved organic matter (DOM), remains limited. Whole-cell biosensors utilizing fluorescent proteins are increasingly used for high-throughput quantification of the bioavailable fraction of As in water. Here, we present a mathematical framework for interpreting the time series of biosensor fluorescence as a measure of As uptake kinetics, which we used to evaluate the effects of different forms of DOM on uptake of trivalent arsenite. We found that thiol-containing organic compounds significantly inhibited uptake of arsenite into cells, possibly through the formation of aqueous complexes between arsenite and thiol ligands. While there was no evidence for competitive interactions between arsenite and low-molecular-weight neutral molecules (urea, glycine, and glyceraldehyde) for uptake through the aquaglyceroporin channel GlpF, which mediates transport of arsenite across cell membranes, there was evidence that labile DOM fractions may inhibit arsenite uptake through a catabolite repression-like mechanism. The observation of significant inhibition of arsenite uptake at DOM/As ratios commonly encountered in wetland pore waters suggests that DOM may be an important control on the microbial uptake of arsenite in the environment, with aspects of DOM quality playing an important role in the extent of inhibition. IMPORTANCE The speciation and toxicity of arsenic in environments like rice paddy soils and groundwater aquifers are controlled by microbe-mediated reactions. These reactions often require As to be taken up into cells prior to enzymatic reaction, but there is limited understanding of how microbial arsenic uptake is affected by variations in water chemistry. In this study, we explored the effect of dissolved organic matter (DOM) quantity and quality on microbial As uptake, with a focus on the role of thiol functional groups that are well known to form aqueous complexes with arsenic. We developed a quantitative framework for interpreting fluorescence time series from whole-cell biosensors and used this technique to evaluate effects of DOM on the rates of microbial arsenic uptake. We show that thiol-containing compounds significantly decrease rates of As uptake into microbial cells at environmentally relevant DOM/As ratios, revealing the importance of DOM quality in regulating arsenic uptake, and subsequent biotransformation, in the environment.

Keywords: arsenic; arsenite; bioavailability; biosensors; complexation; dissolved organic matter; kinetics; microbial uptake; modeling; thiols.

FIG 1

Graphic map of the reporter construct inserted in the pUCP19 vector. The map was obtained using ApE software (https://jorgensen.biology.utah.edu/wayned/ape/) after annotating fragments with the pLannotate web-based service (96). Intracellular As(III) binds to ArsR and induces the expression of arsR and the subsequent mCherry reporter gene. Any potential inhibitors reduce fluorescence indicating less As(III) uptake.

FIG 2

Illustration of the determination of As(III) uptake rates based on biosensor fluorescence time series data. (a) Measured fluorescence intensities (circles) and OD₆₀₀ (triangles) during a representative biosensor assay. The linear regression line (black dashed line) was fit to the measured fluorescence data. (b) dF_RFU(t)/dt (circles) and ln(N(t)) (triangles) over time. The linear regression line (red dashed line) was fit to N(t) data converted from measured OD₆₀₀ data through a calibration curve based on a serial dilution assay. The black dashed line represents the slope of the regression line (44.1 RFU h⁻¹) in panel a.

FIG 3

(a) Histograms of cell ratio (percentage of counted cells) versus fluorescence intensities measured by ImageJ software at four different As(III) concentrations. (b) Microscopic image of biosensor cells incubated with As(III) and quantification of fluorescence associated with individual cells. (c) Cross-analysis between mean fluorescence intensities measured with a plate reader and microscope. A bin of 0 to 100 RFU includes cells of fluorescence intensities less than the detection limit (<100 RFU) and was filtered out before calculating the mean microscope fluorescence intensity. Numbers in panel b (∗) are measured values of each fluorescent cell by ImageJ software.

FIG 4

Concentration-dependent Ψ(t) at 5 h in the presence of reduced l-glutathione (GSH) (a), Upper Mississippi natural organic matter (MNOM), expressed as the thiol concentration (b), glycine (c), urea (d), and l-glyceraldehyde (e). Oxidized l-glutathione at different ratios [brown cross, GSSG/As(III) = 300:1; blue cross, GSSG/As(III) = 3,000:1] was also tested with the GSH experiment. Fixed thiol/As(III) ratios were used for panels a and b, while fixed concentrations of organics were used for panels c to e. No inhibitor compounds were included in the control experiments. Each point represents the mean and standard deviation of four independently grown biological replicates. Ψ(t) varied among different experiments due to variability in the biological reporting system.

FIG 5

Matrix of statistically significant differences in Ψ(t) and r under different experimental conditions with GSH (a) and MNOM (b). Each cell provides a pairwise comparison between a different thiol/As ratio. Solid color blocks represent treatments for which there is a significant decrease in Ψ(t) only (P ≤ 0.05). Hatched color blocks represent significant decrease in Ψ(t) and increase in r through multiple comparisons using Bonferroni-Holm method (P ≤ 0.025 for comparison of Ψ(t); P ≤ 0.05 for comparison of r) (97).

FIG 6

Bar graphs of end points at 5 h from biosensor experiments with different ratios of MNOM to As(III) (a) and Ψ(t) at 5 h from the same experiments (b). The key is expressed as the molar ratio of MNOM-associated thiols to As. A letter over each bar indicates significant difference (P ≤ 0.05) within a group of different MNOM/As ratios at the same As(III) concentration.