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

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.

Atlas of the microbial degradation of fluorinated pesticides.

Fluorine-based agrochemicals have been benchmarked as the golden standard in pesticide development, prompting their widespread use in agriculture. As a result, fluorinated pesticides can now be found in the environment, entailing serious ecological implications due to their harmfulness and persistence. Microbial degradation might be an option to mitigate these impacts, though environmental microorganisms are not expected to easily cope with these fluoroaromatics due to their recalcitrance. Here, we provide an outlook on the microbial metabolism of fluorinated pesticides by analyzing the degradation pathways and biochemical processes involved, while also highlighting the central role of enzymatic defluorination in their productive metabolism. Finally, the potential contribution of these microbial processes for the dissipation of fluorinated pesticides from the environment is also discussed.

Iron isotopic fractionation driven by low-temperature biogeochemical processes.

Iron is geologically important and biochemically crucial for all microorganisms, plants and animals due to its redox exchange, the involvement in electron transport and metabolic processes. Despite the abundance of iron in the earth crust, its bioavailability is very limited in nature due to its occurrence as ferrihydrite, goethite, and hematite where they are thermodynamically stable with low dissolution kinetics in neutral or alkaline environments. Organisms such as bacteria, fungi, and plants have evolved iron acquisition mechanisms to increase its bioavailability in such environments, thereby, contributing largely to the iron cycle in the environment. Biogeochemical cycling of metals including Fe in natural systems usually results in stable isotope fractionation; the extent of fractionation depends on processes involved. Our review suggests that significant fractionation of iron isotopes occurs in low-temperature environments, where the extent of fractionation is greatly governed by several biogeochemical processes such as redox reaction, alteration, complexation, adsorption, oxidation and reduction, with or without the influence of microorganisms. This paper includes relevant data sets on the theoretical calculations, experimental prediction, as well as laboratory studies on stable iron isotopes fractionation induced by different biogeochemical processes.

Adsorption and intracellular uptake of mercuric mercury and methylmercury by methanotrophs and methylating bacteria.

The cell surface adsorption and intracellular uptake of mercuric mercury Hg(II) and methylmercury (MeHg) are important in determining the fate and transformation of Hg in the environment. However, current information is limited about their interactions with two important groups of microorganisms, i.e., methanotrophs and Hg(II)-methylating bacteria, in aquatic systems. This study investigated the adsorption and uptake dynamics of Hg(II) and MeHg by three strains of methanotrophs, Methylomonas sp. strain EFPC3, Methylosinus trichosporium OB3b, and Methylococcus capsulatus Bath, and two Hg(II)-methylating bacteria, Pseudodesulfovibrio mercurii ND132 and Geobacter sulfurreducens PCA. Distinctive behaviors of these microorganisms towards Hg(II) and MeHg adsorption and intracellular uptake were observed. The methanotrophs took up 55-80% of inorganic Hg(II) inside cells after 24 h incubation, lower than methylating bacteria (>90%). Approximately 80-95% of MeHg was rapidly taken up by all the tested methanotrophs within 24 h. In contrast, after the same time, G. sulfurreducens PCA adsorbed 70% but took up <20% of MeHg, while P. mercurii ND132 adsorbed <20% but took up negligible amounts of MeHg. These results suggest that microbial surface adsorption and intracellular uptake of Hg(II) and MeHg depend on the specific types of microbes and appear to be related to microbial physiology that requires further detailed investigation. Despite being incapable of methylating Hg(II), methanotrophs play important roles in immobilizing both Hg(II) and MeHg, potentially influencing their bioavailability and trophic transfer. Therefore, methanotrophs are not only important sinks for methane but also for Hg(II) and MeHg and can influence the global cycling of C and Hg.

Microbial uptake by the respiratory epithelium: outcomes for host and pathogen.

Intracellular occupancy of the respiratory epithelium is a useful pathogenic strategy facilitating microbial replication and evasion of professional phagocytes or circulating antimicrobial drugs. A less appreciated but growing body of evidence indicates that the airway epithelium also plays a crucial role in host defence against inhaled pathogens, by promoting ingestion and quelling of microorganisms, processes that become subverted to favour pathogen activities and promote respiratory disease. To achieve a deeper understanding of beneficial and deleterious activities of respiratory epithelia during antimicrobial defence, we have comprehensively surveyed all current knowledge on airway epithelial uptake of bacterial and fungal pathogens. We find that microbial uptake by airway epithelial cells (AECs) is a common feature of respiratory host-microbe interactions whose stepwise execution, and impacts upon the host, vary by pathogen. Amidst the diversity of underlying mechanisms and disease outcomes, we identify four key infection scenarios and use best-characterised host-pathogen interactions as prototypical examples of each. The emergent view is one in which effi-ciency of AEC-mediated pathogen clearance correlates directly with severity of disease outcome, therefore highlighting an important unmet need to broaden our understanding of the antimicrobial properties of respiratory epithelia and associated drivers of pathogen entry and intracellular fate.

Application of a 15N tracer to simulate and track the fate of atmospherically deposited N in the coastal forests of the Waquoit Bay Watershed, Cape Cod, Massachusetts.

We examined patterns of N retention in the coastal forests of the Waquoit Bay watershed on Cape Cod, Masschusetts using 15N tracer techniques. A solution of 99.6% enriched 15N -NO3-, at a concentration similar to that of background throughfall, was applied to forest plots established along a gradient of soil texture to simulate and track the fate of throughfall NO3- deposition. The tracer solution was applied to replicate plots during both the spring and fall to examine seasonal differences in ecosystem retention. 15N enrichment was subsequently measured in litter, O2 horizon, 0-15 cm mineral soil, fine roots, microbial biomass in the O2 horizon and mineral soil, and lysimeter leachate over a 6 month period following each application. The O2 horizon contained the largest fraction of 15N in all sites immediately following the spring application (19-45%) but was less important following the fall application (10-25%). The mineral soil N pool generally contained the largest fraction of applied 15N (7-28%) in all sites at the end of both 6-month sampling periods. Microbial uptake of applied 15N provided an initial barrier against leaching loss as well as a mechanism for its long-term incorporation into soil organic matter. Microbial processing was less important in the most coarsely textured site, perhaps as a result of lower substrate availability and smaller microbial pool sizes. The highest cumulative leaching losses of applied 15N were observed in the coarse sand site (40, 51%) followed by the fine sand (13, 43%) and loamy sand (4, 19%) sites for the spring and fall applications, respectively. More than 90% of all 15N captured in lysimeters occurred within two days following the applications, and 25-43% of all 15N captured in lysimeters after 2 days was in the form of dissolved organic nitrogen (DON) indicating that it had been assimilated by microbes prior to leaching.

Fate of 14C-labeled dissolved organic matter in paddy and upland soils in responding to moisture.

Soil organic matter (SOM) content in paddy soils is higher than that in upland soils in tropical and subtropical China. The dissolved organic matter (DOM) concentration, however, is lower in paddy soils. We hypothesize that soil moisture strongly controls the fate of DOM, and thereby leads to differences between the two agricultural soils under contrasting management regimens. A 100-day incubation experiment was conducted to trace the fate and biodegradability of DOM in paddy and upland soils under three moisture levels: 45%, 75%, and 105% of the water holding capacity (WHC). (14)C labeled DOM, extracted from the (14)C labeled rice plant material, was incubated in paddy and upland soils, and the mineralization to (14)CO2 and incorporation into microbial biomass were analyzed. Labile and refractory components of the initial (14)C labeled DOM and their respective half-lives were calculated by a double exponential model. During incubation, the mineralization of the initial (14)C labeled DOM in the paddy soils was more affected by moisture than in the upland soils. The amount of (14)C incorporated into the microbial biomass (2.4-11.0% of the initial DOM-(14)C activity) was less affected by moisture in the paddy soils than in the upland soils. At any of the moisture levels, 1) the mineralization of DOM to (14)CO2 within 100 days was 1.2-2.1-fold higher in the paddy soils (41.9-60.0% of the initial DOM-(14)C activity) than in the upland soils (28.7-35.7%), 2) (14)C activity remaining in solution was significantly lower in the paddy soils than in the upland soils, and 3) (14)C activity remaining in the same agricultural soil solution was not significantly different among the three moisture levels after 20 days. Therefore, moisture strongly controls DOM fate, but moisture was not the key factor in determining the lower DOM in the paddy soils than in the upland soils. The UV absorbance of DOM at 280 nm indicates less aromaticity of DOM from the paddy soils than from the upland soils. At any of the moisture levels, much more labile DOM was found in paddy soils (34.3-49.2% of the initial (14)C labeled DOM) compared with that in upland soils (19.4-23.9%). This demonstrates that the lower DOM content in the paddy soil compared with that in the upland soil is probably determined by the less complex components and structure of the DOM.

PLANT AND MICROBIAL CONTROLS ON NITROGEN RETENTION AND LOSS IN A HUMID TROPICAL FOREST.

Humid tropical forests are generally characterized by the lack of nitrogen (N) limitation to net primary productivity, yet paradoxically have high potential for N loss. We conducted an intensive field experiment with 15 NH4 and 15 NO3 additions to highly weathered tropical forest soils in Puerto Rico to determine the relative importance of N retention and loss mechanisms. Over one-half of all the NH4 + produced was rapidly converted to NO3 - via the process of gross nitrification. During the first 24 hours, plant roots took up 28% of the inorganic N produced, dominantly as NH4 + , and were a greater sink for N than soil microbial biomass. Soil microbes were not a significant sink for added 15 NH4 + or 15 NO3 - during the first 24 hours, and only for 15 NH4 + after 7 days. Patterns of microbial community composition, as determined by terminal restriction fragment length polymorphism analysis (TRFLP), were weakly but significantly correlated with nitrification and denitrification to N2 O. Rates of dissimilatory NO3 - reduction to NH4 + (DNRA) were high in this forest, accounting for up to 25% of gross NH4 + production and 35% of gross nitrification. DNRA was a major sink for NO3 - , which may have contributed to the lower rates of N2 O and leaching losses. Despite considerable N conservation via DNRA and plant NH4 + uptake, the fate of ∼45% of the NO3 - produced and 4% of the NH4 + produced were not measured in our fluxes, suggesting that other important pathways for N retention and loss (e.g., denitrification to N2 ) are important in this system. The high proportion of mineralized N that was rapidly nitrified and the fates of that NO3 - highlight the key role of gross nitrification as a proximate control on N retention and loss in humid tropical forest soils. Furthermore, our results demonstrate the importance of the coupling between DNRA and plant uptake of NH4 + as a potential N-conserving mechanism within tropical forests.