Environ. Sci. Technol.

2007, 41, 7795-7801

Chemical Kinetic and Molecular Genetic Study of Selenium Oxyanion Reduction by Enterobacter cloacae SLD1a-1
JINCAI MA, DONALD Y. KOBAYASHI, AND N A T H A N Y E E * , Department of Environmental Sciences, and Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901

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 (1040 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 Ea 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.

Introduction
The release of selenium into the environment from anthropogenic activities is a widespread environmental problem. Industrial and agricultural practices such as water irrigation, fossil fuel combustion, petroleum refining, and mining operations have resulted in elevated Se concentrations at numerous sites worldwide (1, 2). Elevated concentrations of selenium oxyanions in contaminated waters presents a significant threat to aquatic wildlife and can result in death and teratogenic deformities of fish and waterfowl (3-6).
* Corresponding author e-mail: nyee@envsci.rutgers.edu. Department of Environmental Sciences. Department of Plant Biology and Pathology.
10.1021/es0712672 CCC: $37.00 Published on Web 10/12/2007 2007 American Chemical Society

Examples of selenium contamination include Belews Lake (U.S.A.), where selenium-laden wastewater from a coal-fired power plant caused severe reproductive failures in fish (7), and the San Joaquin Valley and Green River basin (U.S.A.), where selenium poisoning caused acute teratogenesis in indigenous bird populations (1, 8, 9). The toxicity and bioavailability of selenium is strongly affected by its chemical speciation (10). As such, there is significant interest in understanding the natural processes that control selenium speciation in contaminated environments (e.g., 11-14). Microorganisms play an important role in catalyzing the reduction of selenium in soils and aquatic sediments (1519), although abiotic reduction of selenium can also occur on reactive Fe(II) mineral surfaces such as green rust (20). A diverse range of microorganisms can transform the soluble selenium oxyanions selenate [Se(VI), SeO42-] and selenite [Se(IV), SeO32-] to insoluble elemental selenium [Se(0)] (e.g., 21-28). Bacteria that are able to reduce Se(VI) and Se(IV) to Se(0) appear to be ubiquitous in aquatic and terrestrial environments (29). Because elemental selenium is insoluble and less toxic than the dissolved selenium oxyanions, the ability of Se-reducing bacteria to precipitate Se(0) has been proposed as a possible bioremediation strategy for Secontaminated waters (30-32). However, the kinetics and mechanisms of microbial selenium oxyanion reduction are poorly understood. To accurately predict the activity of Se-reducing bacteria in contaminated waters, knowledge of the molecular pathways for microbial selenium oxyanion reduction is required. Enterobacter cloacae SLD1a-1 is a facultative anaerobe that has been employed as a model organism for understanding the biochemistry of microbial selenium reduction (33, 34). Recently, we have demonstrated that E. cloacae SLD1a-1 is genetically tractable and amenable to molecular characterization (35). This heterotrophic bacterium was originally isolated from Se-contaminated agricultural drainage water in the San Joaquin Valley, CA (24), and is able to catalyze the reduction of both Se(VI) and Se(IV). Unlike obligate anaerobic Se-reducing bacteria, which cannot be cultivated under ambient air conditions, E. cloacae can be handled aerobically and readily grown to high cell densities and, therefore, is well suited for practical bioremediation purposes. In this study, we performed chemical kinetic and molecular genetic studies of E. cloacae SLD1a-1 to investigate the mechanisms of microbial selenium oxyanion reduction. We provide chemical kinetic data that demonstrate significant differences in rates of Se(VI) and Se(IV) reduction. Transposon mutagenesis resulted in a mutant blocked in the Tat secretion pathway that was devoid of Se(VI) reduction activity. Notably, the mutants ability to catalyze Se(IV) reduction was not affected. The results of this work offer novel and important insights into the kinetics and mechanisms of microbial selenium oxyanion reduction.

Materials and Methods

Growth and Preparation of Cell Culture. All bacteria were grown in Luria Broth LB medium (Difco) unless otherwise noted. Escherichia coli strains were grown at 37 C, and E. cloacae cells were grown at 30 C. After 24 h of growth, cells were harvested by centrifugation at 9000 rpm for 15 min and were resuspended in a solution containing 10 mM HEPES buffer and 20 mM sodium lactate. The pH of the suspension was then adjusted to 7.2 ( 0.1 using 0.1 HCl and 0.1 M NaOH, and it was diluted to an optical density (OD600 nm) of 0.80 ( 0.05 (0.5 g cells/L, dry wt) using 10 mM HEPES buffer containing 20 mM lactate. Antibiotics were added as suppleVOL. 41, NO. 22, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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ments to the medium when appropriate at the following concentrations: tetracycline (Tc), 12.5 g/mL; kanamycin (Km), 50 g/mL; and ampicillin (Ap), 50 g/mL. Se(VI)- and Se(IV)-Reduction Experiments. The rate of selenium oxyanion reduction was quantified as a function of initial selenate or selenite concentration (0-1.0 mM) and temperature (10-40 C). To initiate the reaction, a solution with a known amount of either selenate or selenite was added to the cell suspension. The reaction vessel was continuously shaken and maintained at constant temperature. Standard plate counts were performed to measure the changes in cell numbers during the experiment. Samples were taken periodically with a sterile syringe and filtered using a 0.1 m nylon disposable filter to separate the aqueous solution from biomass and precipitated Se(0). The concentration of aqueous selenate remaining in solution was determined using an ICS1500 ion chromatography system (Dionex, USA) equipped with an ion-exchange column (IonPac AS18). The aqueous concentration of selenite remaining in the Se(IV) reduction experiments was determined using atomic absorption spectroscopy. Calibration standards were prepared using matrix matching background solution containing 10 mM HEPES and 20 mM sodium lactate. In control experiments, several treatments including live cells with lactate, live cells without lactate, lactate without cells, and heat-deactivated cells (100 C, 10 min) were tested for selenate reduction activity. Modeling Approach. Microbial reduction of Se(VI) and Se(IV) followed Michaelis-Menton enzyme kinetics, with the corresponding rate law
d[SeO2 4 ]

FIGURE 1. Se(VI) reduction by E. cloacae compared to experimental controls. The data represent the averages and standard deviations for three replicates experiments. Se(VI) reduction was measured at pH 7.2 and a temperature of 22 C. Tn5lacZ1 element according to the procedure described in the literature (36). Mutants were screened for selenate and selenite reduction activity by plating onto LB agar supplemented with 50 g/mL kanamycin and 10 mM sodium Se(VI) or 0.5 mM sodium Se(IV), respectively, and observing for red precipitate formation. Restriction enzyme digests, electrophoresis, ligations, and Southern hybridizations were performed using standard procedures (37). To determine the insertion site of the miniTn5lacZ1 element in strain 15H1, a genomic library of the mutant was constructed in the cosmid vector pLAFR3 (38), and several overlapping cosmid clones (25 kb) containing the mini-Tn5lacZ1 insertion were directly selected by plating cells on LB agar containing Km and Tc. The 8 kb EcoRI fragment containing the mini-Tn5lacZ1 insertion element and flanking DNA originating from E. cloacae SLD1a-1 was subcloned into pBluescript SK(+), yielding pECE8. Because of primer binding problems, only the sequence of the region upstream of the mini-Tn5lacZ1 insertion was obtainable in pECE8. Therefore, a primer set was constructed that consisted of forward primer SLDTatB-F (5-CCCGGTGGTGAAAAATAGTGAAG-3) positioned 203 bp upstream of the tatC gene based on sequence data obtained of SLD1a-1 DNA from pECE8, and reverse primer 638TatD-R, based on Enterobacter 638 genomic sequence data (GenBank AAVF01000014) (5-GATGGACTCCCGCCGTTGAC-3) positioned 199 bp downstream of the start codon for the tatD gene and predicted to be homologous to the region in SLD1a1. A product of the predicted size of 1.2 kb was amplified from SLD1a-1 chromosomal DNA and cloned into pGEM-T Easy (Invitrogen, La Jolla, CA), resulting in plasmid, pECT12. Cloning of the full tatABC operon for sequence analysis and functional complementation of mutant 15H1 was performed by PCR amplification using the primers UbiB-F (5-GAATTCAATGGCTGCCGGAATAGTGGTCTG-3) positioned 128 bp upstream of tatA, based on sequence data obtained from pECE8 and TatD-R (5-AAGCTTGCTCGCTTTCATGCAGGTTGGTG-3), positioned 149 bp downstream of the tatC, based on sequence from pECT12. Italic sequences for each primer indicate the added restriction enzyme sites EcoRI for UbiB-F and HindIII for TatD-R to facilitate subcloning for complementation purposes. A 1.9 kb PCR product was amplified from SLD1a-1 chromosomal DNA and cloned into pGEM-T Easy. The resulting plasmid pECT19 was used for complete sequence analysis of the tatABC operon in SLD1a-

[X]dt
d[SeO2 3 ]

SeO4 [SeO2 kmax 4 ] 4 KSeO + [SeO2 m 4 ] SeO3 kmax [SeO2 3 ] 3 KSeO + [SeO2 m 3 ]
222-

2-

(1)

[X]dt

(2)

where Km is the half velocity constant (mM), kmax is the apparent maximum reduction rate (mol/min/g), and [X] is the cell concentration (g/L). The effect of temperature on the reduction rate of Se oxyanions varied according to the Arrhenius equation

( )
k kref

) Ae-Ea/RT

(3)

where k is reaction rate measured at a temperature of T, kref is the maximum reduction rate, Ea is activation energy (kJ/ mol/g cell), A is the pre-exponential factor that is relatively independent of temperature, and T is temperature in kelvin. X-ray Absorption Spectroscopy. The precipitates formed in the Se(VI) and Se(IV) reduction experiments were analyzed using X-ray absorption spectroscopy to verify the formation of elemental selenium. The X-ray absorption near-edge structure (XANES) measurements were carried out at the National Synchrotron Light Source at Brookhaven National Laboratory beamline X-11B. Se K-edge XANES spectra was collected from 12.400 to 12.800 keV for centrifuged pellets containing both cells and the selenium particles and compared with a range of reference standards including sodium selenate, sodium selenite, elemental selenium, zinc selenide, and selenocystine powders. The K-edge data was calibrated by defining the inflection point of the first feature in foil spectra for selenium. The foil spectra were measured between sample runs. Analysis of the XANES data was performed using WinXAS version 3.0. Transposon Mutagenesis and Molecular Characterization of the tatABC operon in E. cloacae SLD1a-1. Random mutagenesis of E. cloacae was conducted using the mini7796
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FIGURE 2. The rate of microbial selenium oxyanion reduction measured as a function of initial selenium oxyanion at 22 C. The solid lines represent the best fit curve for each data set determined by the Michaelis-Menten kinetic equation: (A) Se(VI) reduction (b), (B) Se(IV) reduction (O), and (C) comparison of Se(VI) and Se(IV) reduction rates (solid line is Se(VI) reduction, and the dash line is Se(IV) reduction). 1. For 15H1 mutant complementation, the 1.9 kb fragment in pECT19 was excised as an EcoRI-HindIII fragment and cloned into the broad host range plasmid pRK415 to form pECR19. pECR19 was transformed into E. coli S17-1 and conjugally mated into mutant strain 15H1 in a manner similar to the procedure described in the literature (35). DNA sequencing was conducted by GENEWIZ, Inc. (South Plainfield, NJ). DNA sequence analysis was performed using DNAStar software (Lasergene, Madison, WI). Database searches were conducted with identified open reading frames (ORFs) by using the BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST). The DNA sequence of the tatABC operon from E. cloacae SLD1a-1 has been deposited in GenBank under the accession number EF633682.

FIGURE 3. Effect of temperature on the reduction rate of microbial selenium oxyanion reduction. The solid line represents the best fit curve determined by the Arrhenius equation: (A) Se(VI) reduction (b), (B) Se(IV) reduction (O), and (C) Arrhenius plot of the microbial selenium oxyanion reduction reaction (Se(VI) reduction (b) and Se(IV) reduction (O)). that Se(VI) reduction does not occur when Se(VI) is reacted with lactate in the absence of live cells and that it also does not occur in the presence of live cells without lactate. Se(VI) reduction was observed only when both live cells and lactate were present. Maximum reduction rates of Se oxyanions occurred within the first 6 h following initiation of the reaction, where the decrease in substrate concentration was approximately linear with time. The reduction of Se(VI) was concurrent with the solution turning bright red, indicating the formation of a reaction precipitate indicative of the selenium precipitation. Experiments conducted with various initial Se(VI) concentrations showed that the rate of reduction increased within increasing initial Se(VI) concentrations (Figure 2a). A similar trend was also observed for Se(IV) reduction (Figure 2b). The rates of Se(VI) and Se(IV) reduction were modeled using the Michaelis-Menten kinetics eqs 1 and 2, respectively. A nonlinear least-square analysis of the data yielded a Km value of 3.1 mM and a kmax of 1.7 mol/min/g for Se(VI) reduction
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Results
Rates of Selenium Oxyanion Reduction. Figure 1 shows the rate of Se(VI) reduction by E. cloacae compared to that of the experimental controls. The experimental data demonstrate

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FIGURE 4. XANES spectra of the red precipitate formed by Se(VI) and Se(IV) reduction. Also shown are the selenium reference standards: Se(VI) (Na2SeO4), Se(IV) (Na2SeO3), and Se(0) (abiotic elemental selenium). and Km value of 0.72 mM and kmax of 1.3 mol/min/g for Se(IV) reduction. The model curves generated by the kinetic parameters provide an excellent fit to the experimental data (Figure 2a and b). Comparison of the Se(VI) and Se(IV) reduction data shows that Se(IV) reduction is significantly more rapid than Se(VI) reduction over the concentration range studied (Figure 2c). Interestingly, the kmax values indicate that the maximum reduction velocity for Se(VI) is slightly higher than that of Se(IV), implying that Se(VI) reduction rates exceeds the rate of Se(IV) reduction at high selenium concentrations (>11 mM). Figure 3 illustrates the effect of temperature on the reduction rate constants. In both the Se(VI) and Se(IV) reduction experiments, the measured kmax increased with increasing temperature between 10 and 36 C and decreased at temperatures above 40 C. The data suggests an optimal temperature for Se(VI) and Se(IV) reduction of approximately 36 C. An Arrhenius plot of the data collected between 10 and 36 C result in a linear relationship between ln k and 1/T (Figure 3c) and yield Ea values for Se(VI) and Se(IV) reduction equal to 121 and 71.2 kJ/mol, respectively. These results indicate that the activation energy required for selenate reduction is higher than that of selenite reduction. Combined with the reduction rate constants, the kinetic data suggest different enzymatic systems are responsible for the catalysis of Se(IV) and Se(VI) reduction. XANES Analysis of Reduction Products. XANES spectra were collected for the red precipitate formed during the Se(VI) and Se(IV) reduction experiments. The results show that the energy position of the Se K-edge shifts to lower energies with a decrease in oxidation state (Figure 4). The position of the Se K-edge measured for Se +VI, +IV, and 0 were 12.665, 12.662, and 12.658 keV respectively. These Se K-edge values are in good agreement with previous XANES measurements of selenium reference compounds (11, 20). Comparison of the XANES spectra collected for the Se(VI) and Se(IV) reduction products confirm the red precipitate is composed of Se(0). Isolation and Characterization of an E. cloacae Mutant Defective in Se(VI) Reduction. Mini-Tn5lacZ1 transposon mutagenesis was employed to obtain genetic information to determine if separate enzyme systems control the reduction of Se(VI) and Se(IV) in E. cloacae SLD1a-1. A single mutant, designated 15H1, was isolated that had lost the ability to transform Se(VI) to Se(0) as determined by the inability to form a red precipitate indicative of elemental selenium on agar medium containing sodium selenate (Figure 5). Measurement of Se(VI) reduction activity in liquid media confirmed that the mutant had lost the ability to reduce Se7798
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FIGURE 5. Selenium oxyanion reduction activity of the E. cloacae wild type strain and the mutant strain (designated as 15H1). The data represent the averages and standard deviations for three replicates experiments. The images show the wild type strain and the mutant strain grown on selenate and selenite containing agar. Red colonies indicate the precipitation of elemental selenium, and white colonies indicate the strain does not precipitate Se(0). (VI). In contrast, strain 15H1 formed a red precipitate when plated on agar supplemented with sodium selenite, indicating its ability to still transform Se(IV) to Se(0) (Figure 5). In liquid media, the Se(IV) reduction activity of the mutant strain was identical to that of the wild-type strain. Southern hybridization analysis of mutant strain 15H1 chromosomal DNA using the mini-Tn5lacZ1 as a probe indicated that the transposon resided in an 8 kb EcoRI fragment. A 1.9 kb fragment corresponding to the region surrounding the mini-Tn5lacZ1 insertion in 15H1 was amplified by PCR from the wild-type E. cloacae strain SLD1a-1 chromosomal DNA. Sequence analysis of a portion of the 8 kb EcoRI fragment from 15H1 and the entire 1.9 kb PCR product from SLD1a-1 indicated the mini-Tn5lacZ1 element had inserted 200 bp downstream of the start codon for tatC,

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FIGURE 6. Map of the 8 kb EcoRI fragment with mini-Tn5lacZ1 transposon insertion in tatC gene. The triangle indicates the location where mini-Tn5 element was inserted: ubiB, putative ubiquinone biosynthesis protein; tatABC, twin arginine translocation operon; tatD encodes for a cytoplasmic protein with DNase activity and is not required in sec-independent protein export. The hatched arrow fillings indicate the genes that belong to a separate operon. Se(VI) reduction by metabolically active cells (Km ) 3.1 mM) falls between the Km values reported for cell membrane fractions (Km ) 6.25 mM) and for the purified enzyme (Km ) 2.1 mM) from E. cloacae (33, 34). The Km values for Se(VI) reduction by E. cloacae is also comparable to the Km derived for the anaerobic Se-respiring bacterium S. barnesii (Km ) 4.1 mM) (18). The kinetic constants determined for E. cloacae indicate that the Km value for Se(IV) reduction occurs at nearly four times lower selenium concentrations than that for Se(VI) reduction. The difference in Km values for Se(IV) and Se(VI) suggests that E. cloacae is more efficient at reducing selenite than selenate. The difference in reduction kinetics is correlated to the activation energies measured for the reduction reactions. Temperature-dependent reduction rates indicate that higher activation energies are required to reduce Se(VI) than are required for the reduction of Se(IV). This result suggests that selenite reduction is kinetically more favorable than selenate reduction. The ability of E. cloacae to reduce Se(IV) at faster rates than Se(VI) can be attributed to differences in enzyme concentration, or alternatively, to the catalytic efficiency of the reductase proteins. Mechanisms of Selenium Oxyanion Reduction. Genetic analysis of the mutant strain 15H1 show that the twin-arginine translocation (Tat) pathway is involved in the Se(VI) reduction process. In bacteria, the physiological role of the Tat system is to transport folded proteins from the cytoplasm to periplasm across the inner cytoplasmic membrane. Redox enzymes such as hydrogenases, formate dehydrogenases, nitrate reductase, trimethlylamine N-oxide (TMAO) reductases, and dimethyl sulfoxide (DMSO) reductases are known to be excreted via the Tat pathway (42). The Tat system acts separately from the general secretory (Sec) pathway, which is responsible for the export of Fe(III) reductase enzymes in metal-reducing bacteria (43). However, similar to the Sec pathway, mutation of Tat system disrupts the transport of reductase enzymes to their active locations (e.g., 44). Our results provide strong evidence that the enzyme conferring selenate reductase activity in E. cloacae is exported to the periplasm through the Tat pathway and that mutation of this protein transport system results in complete loss of selenate reduction activity. This enzyme export mechanism is consistent with the findings of Ridley et al. (34), which showed that the terminal selenate reductase enzyme in E. cloacae SLD1a-1 is imbedded in the cytoplasmic membrane with the active site facing the periplasmic space. In addition to E. cloacae, the Tat system may also control the secretion and activity of the selenate reductase in other microorganisms. Because proteins that are exported by the Tat system contain a twin-arginine signal sequence, we can survey known selenate reductase enzymes for the signal peptides. A review of database entries shows that only one bacterial selenate reductase has been molecularly characterized. In the anaerobic bacterium Thauera selenatis, Se(VI) reduction is catalyzed by SerABC, which is a substrate-specific reductase that reduces selenate but does not reduce nitrate, nitrite, chlorate, or sulfate. The SerA subunit contains an apparent leader peptide with a twin-arginine motif (GeneBank Accession CAB53372), suggesting it is exported across the
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FIGURE 7. Selenate reduction activity of the E. cloacae wild type strain, mutant strain 15H1, and the wild type sequence complemented mutant strain 15H1. The data represent the averages and standard deviations for three replicates experiments. The images show the corresponding colonies grown on selenate containing agar. Red colonies indicate the precipitation of elemental selenium, and white colonies indicate the strain does not precipitate Se(0). a gene that is part of the tatABC operon encoding for components of a twin-arginine translocation (Tat) system (Figure 6). The predicted protein sequence for TatA and TatB from E. cloacae SLD1a-1 shares 90 and 77% identities with Enterobacter 638, while TatC shares 93% identity with the same strain. This is consistent with the previous report which showed that TatC is the most conserved of the Tat proteins and that sequence conservation is particularly strong within the transmembrane domains (39). The structural organization of the tatABC operon in E. cloacae is consistent with that described for E. coli (40), in which the tatABC operon is preceded by the ubiB gene and the tatD gene is located immediately downstream of the operon. Also consistent with transcriptional analysis of the tatABC operon in E. coli, the 1.9 kb PCR fragment in pECR19 was capable of complementing the mutation in 15H1. Complementation of the mutated gene restored the mutants ability to reduce Se(VI) therefore confirming that tat genes are essential to selenate reduction activity in E. cloacae SLD1a-1 (Figure 7).

Discussion
Selenium Oxyanion Reduction Kinetics. The experimental data indicate that Se(VI) reduction by E. cloacae occurs only with metabolically active cells. Unlike passive selenate reduction that has been observed with metal-reducing bacterium Shewanella (41), E. cloacae requires an external electron donor to catalyze Se(VI) reduction. The kinetic experiments demonstrated that the rates of Se(VI) and Se(VI) reduction by E. cloacae can be successfully described using the Michaelis-Menten kinetic equation. The Km for

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TABLE 1. Bacterial Strains and Plasmidsa

strain or plasmid relevant characteristics source or ref

strains
E. cloacae SLD1a-1 (wild type) 15H1, mutant E. coli DH5R S17-1
pLAFR3 pUTmini-Tn5lacZ1 pRK415 pBluescript II SK(() pGEM-T Easy pECE8 pECT12 pECT19 pECR19
a

selenate reductase positive, Rifampicin tagged selenate reductase negative, Kmr general laboratory strain, selenate reductase positive general laboratory strain, selenate reductase negative

Losi and Frankenberger Jr.25 this study Gibco-BRL Simon et al.50 Staskawicz et al.38 Kobayashi et al.51 Keen et al.52 Strategene Promega this study this study this study this study

plasmids
cosmid cloning vector; Tcr transposon carrying vector, Kmr broad-host-range cloning vector, Tcr PCR cloning vector; Apr Cloning vector; Apr 8-kb EcoRI fragment containing transposon cloned into pBluescript II SK(() 1.2-kb PCR product cloned into pGEM-T Easy 1.9-kb PCR product cloned into pGEM-T Easy tatABC operon from pECT19 cloned into pRK415

Tcr, tetracycline resistance; Kmr, kanamycin resistance; Apr, ampicillin resistance.

inner membrane by the Tat pathway. The presence of the twin-arginine signal peptide in the phylogentically distinct selenate reductase of T. selenatis also suggests that the Tat system may play an important role in selenate reduction in different species of bacteria. Our results show that the disruption of the Tat system in E. cloacae does not affect selenite reduction activity, suggesting that the enzyme catalyzing reduction of Se(IV) is not dependent on secretion via the Tat pathway. On the basis of this observation, we propose that selenate reduction and selenite reduction are mediated by two separate enzymatic systems. Selenate reduction by E. cloacae has been reported to be catalyzed by a membrane-bound protein complex with an apparent molecular mass of 600 kDa (34). The selenate reductase in E. cloacae is a molybdo enzyme containing heme and nonheme iron as prosthetic constituents and a b-type cytochrome in the active complex. We recently demonstrated that the selenate reductase activity occurs under suboxic conditions and is controlled by the global anaerobic regulator FNR (fumarate nitrate reduction regulator) (35). Biochemical studies have shown that, in addition to Se(VI), the selenate reductase of E. cloacae also displays activity toward chlorate and bromate, but not toward nitrate, TMAO, or DMSO (33, 34). In contrast to selenate reduction, mechanisms for selenite reduction remain poorly understood. The enzyme that catalyzes the reduction of selenite in E. cloacae has not been characterized. Klonowska et al. (45) showed that selenite reduction by the facultative anaerobe Shewanella oneidensis occurs under anaerobic conditions. In Se-respiring bacteria, T. selenatis mutants defective in nitrite reductase activity are unable to sustain Se(IV) reduction (46), suggesting that selenite reduction is catalyzed by the nitrite reductase or a component of the nitrite respiratory system (22, 47). In addition to nitrite reductases, other enzymes such as sulfite reductase and glutathione reductase have also been linked to microbial Se(IV) reduction. The sulfite reductase in Clostridium pasteurianum can catalyze the reduction of selenite (48), and glutathione in the non-sulfur bacteria Rhodospirillum rubrum and Rhodobacter capsulatus promotes selenite reduction activity (49). Determination of the occurrence and activity of these enzymes in E. cloacae will further elucidate the Se(IV) reduction mechanism. The results of this study suggest that the overall reduction of Se(IV) to Se(0) by E. cloacae SLD1a-1 is a two-step process, where selenate is first reduced to selenite and then selenite is further reduced to elemental selenium. The kinetic and molecular evidence support the hypothesis that selenate and
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selenite reduction by E. cloacae are catalyzed by separate and distinct enzymatic systems. The chemical kinetic data shows that selenite reduction is more rapid than the reduction of selenate. Because the rates of microbial selenate and selenite reduction are ultimately controlled by the underlying molecular reactions, additional molecular investigations are required to accurately constrain the reduction kinetics. Identification of the Tat system in the selenate reduction process advances our understanding of microbial selenium reduction toward a mechanistic molecular model.

Acknowledgments
We thank D. Mack and M. Burk for their help with preliminary experiments. This work was supported by the USDA-NRI research grant to N.Y. under contract number 2005-3510716230.

Literature Cited
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Received for review May 29, 2007. Revised manuscript received August 17, 2007. Accepted September 4, 2007. ES0712672

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