Cloacae SLD1a-1 Research Articles

Mass-dependent selenium isotopic fractionation during microbial reduction of seleno-oxyanions by phylogenetically diverse bacteria

Selenium (Se) isotope fractionation has been widely used for constraining redox conditions and microbial processes in both modern and ancient environments, but our knowledge of the controls on fractionation during microbial reduction of Se-oxyanions is based on a limited number of studies. Here we complement and expand the currently available pure culture data for Se isotope fractionation by investigating for the first time six phylogenetically diverse, mesophilic, and non-respiring bacterial strains that reduce Se-oxyanions to elemental Se [Se(0)]. Experiments were performed with either selenate [Se(VI)] or selenite [Se(IV)] at lower, more environmentally-relevant Se (9–47 μM) and carbon (500 μM) concentrations than previously investigated. Enterobacter cloacae SLD1a-1, Desulfitobacterium chlororespirans Co23 and Desulfitobacterium sp. Viet-1 were incubated with Se(VI) and Se(IV). Geobacter sulfurreducens PCA, Anaeromyxobacter dehalogenans FRC-W and Shewanella sp. (NR) were examined for their ability reducing Se(IV) to Se(0). Our data confirm that microbial reduction of both Se-oxyanions is accompanied by large kinetic isotopic fractionation (reported as 82/76ε = 1000×(82/76α-1) in ‰). Under our experimental conditions, microbial reduction of Se(VI) shows consistently greater isotope fractionation (ε = −9.2‰ to −11.8‰) than reduction of Se(IV) (ε = −6.2 to −7.8‰) confirming the difference in metabolic pathways for the reduction of the two Se-oxyanions. For Se(VI), an inverse relationship between normalized cell specific reduction rate (cSRR) and Se isotope fractionation suggests that the kinetic isotope effect for Se(VI) reduction is governed by an enzymatically-specific pathway related to the bacterial strain-specific physiology. In contrast, the lack of correlation between normalized cSRR and isotope fractionation for Se(IV) reduction indicates a non-enzyme specific pathway which is dominantly extracellular. Our study highlights the importance to understand microbially-mediated Se isotope fractionation depending on Se species, and cell-specific reduction rates before Se isotope ratios can become a fully applicable tool to interpret Se isotopic changes in modern and ancient environments.

Modeling the impact of soil aggregate size on selenium immobilization

Abstract. Soil aggregates are mm- to cm-sized microporous structures separated by macropores. Whereas fast advective transport prevails in macropores, advection is inhibited by the low permeability of intra-aggregate micropores. This can lead to mass transfer limitations and the formation of aggregate scale concentration gradients affecting the distribution and transport of redox sensitive elements. Selenium (Se) mobilized through irrigation of seleniferous soils has emerged as a major aquatic contaminant. In the absence of oxygen, the bioavailable oxyanions selenate, Se(VI), and selenite, Se(IV), can be microbially reduced to solid, elemental Se, Se(0), and anoxic microzones within soil aggregates are thought to promote this process in otherwise well-aerated soils. To evaluate the impact of soil aggregate size on selenium retention, we developed a dynamic 2-D reactive transport model of selenium cycling in a single idealized aggregate surrounded by a macropore. The model was developed based on flow-through-reactor experiments involving artificial soil aggregates (diameter: 2.5 cm) made of sand and containing Enterobacter cloacae SLD1a-1 that reduces Se(VI) via Se(IV) to Se(0). Aggregates were surrounded by a constant flow providing Se(VI) and pyruvate under oxic or anoxic conditions. In the model, reactions were implemented with double-Monod rate equations coupled to the transport of pyruvate, O2, and Se species. The spatial and temporal dynamics of the model were validated with data from experiments, and predictive simulations were performed covering aggregate sizes 1–2.5 cm in diameter. Simulations predict that selenium retention scales with aggregate size. Depending on O2, Se(VI), and pyruvate concentrations, selenium retention was 4–23 times higher in 2.5 cm aggregates compared to 1 cm aggregates. Under oxic conditions, aggregate size and pyruvate concentrations were found to have a positive synergistic effect on selenium retention. Promoting soil aggregation on seleniferous agricultural soils, through organic matter amendments and conservation tillage, may thus help decrease the impacts of selenium contaminated drainage water on downstream aquatic ecosystems.

Role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a‐1 and Escherichia coli K12

In this study, we investigated the role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12. A mini-Tn5 transposon mutant of E. cloacae SLD1a-1, designated as 4E6, was isolated that had lost the ability to reduce Se(VI) to Se(0). Genetic analysis of mutant strain 4E6 showed that the transposon was inserted within a menD gene among a menFDHBCE gene cluster that encodes for proteins required for menaquinone biosynthesis. A group of E. coli K12 strains with single mutations in the menF, menD, menC and menE genes were tested for loss of selenate reduction activity. The results showed that E. coli K12 carrying a deletion of either the menD, menC or menE gene was unable to reduce selenate. Complementation using wild-type sequences of the E. cloacae SLD1a-1 menFDHBCE sequence successfully restored the selenate reduction activity in mutant strain 4E6, and E. coli K12 menD and menE mutants. Selenate reduction activity in 4E6 was also restored by chemical complementation using the menaquinone precursor compound 1,4-dihydroxy-2-nathphoic acid. The results of this work suggest that menaquinones are an important source of electrons for the selenate reductase, and are required for selenate reduction activity in E. cloacae SLD1a-1 and E. coli K12.

Enterobacter cloacae SLD1a-1 gains a selective advantage from selenate reduction when growing in nitrate-depleted anaerobic environments

Enterobacter cloacae SLD1a-1 is capable of the complete reduction of selenate to selenium and the initial reaction is catalysed by a membrane-bound selenate reductase. In the present study, continuous culture experiments were employed to investigate the possibility that selenate reduction, via the selenate reductase, might provide sufficient energy to maintain cell viability when deprived of the preferred anaerobic terminal electron acceptor nitrate. The evidence presented indicates that the selenate reductase supports slow growth that retards the wash-out of the culture when switching to nitrate-depleted selenate-rich medium, and provides a proton motive force for sustained cell maintenance. In contrast, a strain of E. cloacae (sub sp. cloacae) that does not readily reduce selenate, cannot sustain cell maintenance when switching to a selenate-rich medium. This work demonstrates for the first time that respiratory linked selenate reduction gives E. cloacae SLD1a-1 a selective advantage when inhabiting selenate-contaminated environments and highlights the suitability of utilising E. cloacae SLD1a-1 when developing selenium remediation strategies.

Chemical Kinetic and Molecular Genetic Study of Selenium Oxyanion Reduction by Enterobacter cloacae SLD1a-1

Microbial processes play an important role in the redox transformations of toxic selenium oxyanions. In this study, we employed chemical kinetic and molecular genetic techniques to investigate the mechanisms of Se(IV) and Se-(VI) reduction by the facultative anaerobe Enterobacter cloacae SLD1a-1. The rates of microbial selenium oxyanion reduction were measured as a function of initial selenium oxyanion concentration (0-1.0 mM) and temperature (10-40 degrees C), and mutagenesis studies were performed to identify the genes involved in the selenium oxyanion reduction pathway. The results indicate that Se(IV) reduction is significantly more rapid than the reduction of Se(VI). The kinetics of the reduction reactions were successfully quantified using the Michaelis-Menten kinetic equation. Both the rates of Se(VI) and Se(IV) reduction displayed strong temperature-dependence with E(a) values of 121 and 71.2 kJ/ mol, respectively. X-ray absorption near-edge spectra collected for the precipitates formed by Se(VI) and Se(IV) reduction confirmed the formation of Se(0). A miniTn5 transposon mutant of E. cloacae SLD1a-1 was isolated that had lost the ability to reduce Se(VI) but was not affected in Se(IV) reduction activity. Nucleotide sequence analysis revealed the transposon was inserted within a tatC gene, which encodes for a central protein in the twin arginine translocation system. Complementation by the wild-type tatC sequence restored the ability of mutant strains to reduce Se(VI). The results suggest that Se(VI) reduction activity is dependent on enzyme export across the cytoplasmic membrane and that reduction of Se(VI) and Se(IV) are catalyzed by different enzymatic systems.

Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: Application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199–211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 μl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane–bound selenate reductase has optimum activity at pH ∼ 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.

Selenate reduction by Enterobacter cloacae SLD1a-1 is catalysed by a molybdenum-dependent membrane-bound enzyme that is distinct from the membrane-bound nitrate reductase

Enterobacter cloacae SLD1a-1 is capable of reducing selenium oxyanions to elemental selenium under both aerobic and anaerobic conditions. In this study the enzyme that catalyses the initial reduction of selenate (SeO 4 2−) to selenite (SeO 3 2−) has been localised to isolated cytoplasmic membrane fractions. Experiments with intact cells have shown that the putative selenate reductase can accept electrons more readily from membrane-impermeable methyl viologen than membrane-permeable benzyl viologen, suggesting that the location of the catalytic site is towards the periplasmic side of the cytoplasmic membrane. Enzyme activity was enhanced by growing cells in the presence of 1 mM sodium molybdate and significantly reduced in cells grown in the presence of 1 mM sodium tungstate. Non-denaturing polyacrylamide gel electrophoresis (PAGE) gels stained for selenate and nitrate reductase activity have revealed that two distinct membrane-bound enzymes catalyse the reduction of selenate and nitrate. The role of this membrane-bound molybdenum-dependent reductase in relation to selenate detoxification and energy conservation is discussed.

Biotransformations of selenium by Enterobacter cloacae SLD1a-1: Formation of dimethylselenide

Whole-cell suspensions of Enterobactercloacae SLD1a-1 produced dimethylselenide(DMSe) from selenate, selenite, elementalselenium, dimethylselenone,seleno-DL-methionine, 6-selenoinosine, and6-selenopurine. Cell-free extracts of thebacterium produced the formation of DMSe fromorganic selenium compounds, includingdimethylselenone, dimethylselenoniopropionate,seleno-DL-methionine, seleno-DL-ethionine, and6-selenoguanosine. The highest rate of DMSeproduction occurred from whole-cell suspensionsand cell-free extracts containingdimethylselenone. DMSe was also produced bycell-free extracts containing selenite orelemental selenium and methylcobalamin. Cell-free extracts did not produce DMSe frominorganic selenium when S-adenosyl-L-methionine was present. Additionally, DL-homocysteine and L-methioninewere found to inhibit selenium volatilization. These findings suggest the formation of DMSefrom inorganic selenium occurs through thetransfer of a methyl group frommethylcobalamin.

Factors affecting the volatilization of dimethylselenide by Enterobacter cloacae SLD1a-1

The facultative anaerobic bacterium Enterobacter cloacae SLD1a-1 (ATCC 700258), capable of reducing selenate (SeO 4 2−) and selenite (SeO 3 2−) to elemental selenium (Se 0), was found to volatilize dimethylselenide (DMSe) in the presence of SeO 3 2−. The effect of temperature, pH and electrical conductivity (EC) on the ability of E. cloacae SLD1a-1 to methylate selenium (Se) were investigated in a liquid medium of tryptic soy broth containing 10 μM SeO 3 2−. Optimum Se volatilization occurred at a pH, temperature and EC of 6.5, 35°C and 11 dS m −1, respectively. Volatilization of Se was also found to be concentration dependent, as E. cloacae SLD1a-1 produced 11.3 times more DMSe at a SeO 3 2− concentration of 10 μM than at 1.0 μM. By determining the optimum environmental conditions which stimulate Se volatilization, it may be possible to design a strategy to remediate seleniferous water.

Reduction of Selenium Oxyanions by Enterobacter cloacae SLD1a-1: Isolation and Growth of the Bacterium and Its Expulsion of Selenium Particles

A facultative bacterium capable of removing the selenium (Se) oxyanions selenate (SeO(inf4)(sup2-)) and selenite (SeO(inf3)(sup2-)) from solution culture in flasks open to the atmosphere was isolated and studied with the goal of assessing its potential for use in bioremediation of seleniferous agricultural drainage water. Elemental Se (Se(sup0)) was confirmed as a product of the reaction. The organism, identified as Enterobacter cloacae and designated strain SLD1a-1 (ATCC 700258), removed from 61.5 to 94.5% of added SeO(inf4)(sup2-) (the primary species present in agricultural drainage water) at concentrations from 13 to 1,266 (mu)M. Equimolar amounts of nitrate (NO(inf3)(sup-)), which interferes with SeO(inf4)(sup2-) reduction in some organisms, did not influence the reaction in growth experiments but had a slight inhibitory effect in a washed-cell suspension. Washed-cell suspension experiments also showed that (i) SeO(inf3)(sup2-) is a transitory intermediate in reduction of SeO(inf4)(sup2-), being produced and rapidly reduced concomitantly; (ii) NO(inf3)(sup-) is also reduced concomitantly and at a much higher rate than SeO(inf4)(sup2-); and (iii) although enzymatic, reduction of either oxyanion does not appear to be an inducible process. Transmission electron microscopy revealed that precipitate particles are <0.1 (mu)m in diameter, and these particles were observed free in the medium. Evidence indicates that SLD1a-1 uses SeO(inf4)(sup2-) as an alternate electron acceptor and that the reaction occurs via a membrane-associated reductase(s) followed by rapid expulsion of the Se particles.