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Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3 RONALD S

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Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3 RONALD S APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 3011-3019 Vol. 60, No. 8 0099-2240/94/$04.00+0 Copyright X 1994, American Society for Microbiology Isolation, Growth, and Metabolism of an Obligately Anaerobic, Selenate-Respiring Bacterium, Strain SES-3 RONALD S. OREMLAND,* JODI SWITZER BLUM, CHARLES W. CULBERTSON, PIETER T. VISSCHER, LAURENCE G. MILLER, PHILLIP DOWDLE, AND FRANCES E. STROHMAIER U.S. Geological Survey, Menlo Park, Califomia 94025 Received 6 April 1994/Accepted 24 May 1994 A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO2 with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Seo). Hence, SES-3 can carry out a complete reduction of selenate to Seo. Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of ['4C] lactate to '4Co2 coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m-chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Seo by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [75Se] selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Seo. This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Seo may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions. The bacterial reduction of selenate ions to elemental sele- and strain SES-1 constituted the only reports of DSeR in pure nium (Se') represents an important aspect of the selenium culture. We now report on the characteristics of a novel cycle by which this toxic element is sequestered into sediments freshwater isolate, designated strain SES-3, a strictly anaerobic (30). The phenomenon is widespread in nature and includes vibrio which grows by DSeR coupled with the oxidation of contaminated as well as pristine sediments, and activity in lactate to acetate plus carbon dioxide. assayed sediment samples occurs without a noticeable lag (32, 40). Selenate reduction to Seo represents an important quan- MATERIALS AND METHODS titative sink for selenium oxyanions in shallow, lotic environ- ments, such as waste treatment ponds (31), and may be Isolation and cultivation. Strain SES-3 was isolated from an exploited for bioremediation purposes (9, 20, 27, 28). Although acetate-oxidizing, selenate-respiring enrichment (39) recov- much is known about assimilation of selenium in its role as an ered from Massie Slough, a freshwater marsh in the Stillwater enzyme cofactor (38), relatively little is known about bacteria Wildlife Management Area of western Nevada. Ambient which are capable of reducing selenate in sufficient quantities selenate concentrations in Massie Slough waters were about 51 to contribute to the biogeochemical cycling of this element. nM (32). In surveys of different sediment types, Massie Slough Two facultative anaerobes which have the ability to bio- material had the highest potential DSeR rates (40) and the chemically reduce selenate to Seo have been isolated. These most rapid rates of in situ selenate turnover (32). The enrich- include a strain of Pseudomonas stutzeri (14) and a novel ment was streaked onto 2% agar plates composed of the same species, Thauera selenatis (21, 22). P. stutzeri seems to reduce mineral salts medium (20 mM acetate plus 20 mM selenate). selenium oxyanions solely for the purpose of detoxification, All manipulations were performed in an anaerobic glove box. while in T. selenatis, dissimilatory selenate reduction (DSeR) After the enrichment was streaked, the plates were sealed in an to selenite is a mode of anaerobic respiration capable of anaerobic jar and incubated at room temperature. After a few sustaining growth (19). With regard to strict anaerobes, after weeks, red colonies were abundant, and isolated colonies were adaptation to selenium oxyanions, Wolinella succinogenes can picked and transferred into crimp-seal 25-ml culture tubes precipitate Seo from selenate or selenite but cannot use containing 10 ml of liquid medium. The tubes were sealed selenium oxyanions for respiratory growth (44). A strictly under either 4:1 N2-C02 or 4:1 H2-CO2. No growth was anaerobic, gram-negative coccus (strain SES-1) was isolated detected in the tubes with acetate under N2-C02; however, from estuarine sediments which grew via DSeR to Seo by using growth, evident as turbidity and the formation of red elemental acetate as the electron donor (30). Unfortunately, SES-1 was Seo, occurred in the tubes with H2. The H2 requirement may lost before it could be studied definitively. Thus, T. selenatis have been due to the initial selective pressure caused by the presence of -1% H2 in the sealed petri-incubator jars. We determined that the isolate could grow on acetate plus H2, on without * Corresponding author. Mailing address: U.S. Geological Survey, pyruvate or lactate under N2-C02, but not on acetate ms 465, 345 Middlefield Rd., Menlo Park, CA 94025. Phone: (415) H2. For convenience, we worked with a lactate-based medium, 329-4482. Fax: (415) 329-4463. Electronic mail address: roremlan@ and all subsequent growth experiments were conducted with rcamnl.wr.usgs.gov. lactate as the electron donor. The medium contained the 3011 3012 OREMLAND ET AL. APPL. ENVIRON. MICROBIOL. following: K2HPO4 (0.225), KH2PO4 (0.225), NaCl (0.46), lactate and 5 mM Na selenite (or 1 mM nitrite). The effect of (NH4)2SO4 (0.225), MgSO4* 7H20 (0.117), yeast extract (1.0), various electron acceptors (sulfate, selenate, sulfite, thiosul- Na lactate (2.24), Na2SeO4 (3.78) or NaNO3 (1.7), NaHCO3 fate, nitrate, and nitrite; 5 mM each) upon reduction of 2.5 (4.2), Na2S * 9H20, (0.1), and cysteine-HCl (0.1) (all in grams mM selenite was monitored in a tracer experiment with per liter); SL10 trace element solution (1.0 ml) (47); Na2WO4 [75Se]selenite (Amersham Inc., Arlington Heights, Ill.; specific (3 pug/liter); and vitamin solution (10 ml). With the exception activity, 757 mCi mmol-1). Cell suspensions of selenate-grown for quantification of growth on nitrate, ammonium sulfate was cells (30 or 70 ml) received 1.5 or 3.0 ,uCi, respectively, of the included routinely when cells were grown with nitrate as the radioisotope, and loss of [75Se]selenite from solution and the electron acceptor. The vitamin solution contained the follow- precipitation of 75Se0 was determined by expressing 0.5 ml of ing (in milligrams per liter): p-aminobenzoic acid (5), biotin the suspension through disposable 0.2-,im-pore-size filters (5), folic acid (2), pyridoxine-HCl (1), riboflavin (5), thiamine (13-mm diameter; Alltech Inc.). The filters were rinsed by (5), nicotinic acid (5), pantothenic acid (5), thiotic acid (5), and injection with an additional 0.5 ml of unlabeled buffer, and the vitamin B12 (0.1). The basal salts were dispensed into serum filters and the filtrate were counted separately (see below). bottles (usually with 30 ml in 59-ml bottles but, if proportion- Nitrate-grown cells were tested in a similar fashion for their ally scaled up, with as much as 900 ml in special 2-liter serum ability to reduce [75Se]selenite. The ability of selenate- or bottles) or into 10 ml of medium in 25-ml culture tubes, crimp nitrate-grown cells to oxidize [U-14C]lactate (New England sealed under N2-CO2, and autoclaved. After the bottles were Nuclear, Boston, Mass.; specific activity, 177 mCi mmol-') to cooled, the vitamins, lactate, electron acceptors, reducing 14Co2 with various electron acceptors (NOf3, SeO42-, MnO2, agents, and bicarbonate solutions were added by injection from FeOOH, HAs042-, fumarate, S032-; 5 mmol liter-') or with anaerobic, filter-sterilized stock solutions. The final pH was various ionophores or inhibitors was examined. Cell suspen- 7.3. Cultures were incubated statically at 30°C. Gentle shaking sions were incubated with 1 mM Na lactate plus 2.2 ,uCi of was found to inhibit growth of the enrichment (38a), and [14C]lactate for time periods ranging from 2 to 19.25 h (see therefore, the pure cultures were not shaken. Subsamples of text) at 30°C with slow rotary shaking (-100 rpm). Activity was the liquid and gas phases were taken by syringe (after hand stopped by injection of 1 ml of 6 N HCl, and bottles were swirling to achieve uniform dispersement) for determination of shaken (250 rpm) for -20 h after which the headspace was cell density and the concentrations of substrates and interme- analyzed for 14Co2 (see below). Carbonyl cyanide m-chloro- diates. The volume withdrawn was replaced with N2-CO2. phenylhydrazone (CCCP) and 2,4-dinitrophenol were added Washed cell suspensions. Two liters of late-log-phase cells from 10 mM stock solutions prepared in acetone. Corrections grown with either selenate or nitrate as the electron acceptor were made for inhibition caused by acetone or ethanol addi- were dispensed into 10 200-ml centrifuge bottles and harvested tion without the inhibitors. Azide and cyanide were added by centrifugation at 10,000 x g for 20 min at 4°C. After the from 1 M and 50 mM stock solutions in water.
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