APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p. 5607–5609 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.9.5607–5609.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 9
Selenite and Tellurite Reduction by Shewanella oneidensis Agnieszka Klonowska,1 Thierry Heulin,1 and Andre´ Vermeglio2* Laboratoire d’Ecologie Microbienne de la Rhizosphe`re1 and Laboratoire de Bioe´nerge´tique Cellulaire CEA/Cadarache,2 DSV-DEVM-UMR 6191 CNRS-CEA-Aix-Marseille II, 13108 Saint Paul lez Durance Cedex, France Received 1 February 2005/Accepted 3 April 2005
Shewanella oneidensis MR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways. Selenium, an element widely distributed on the earth’s crust and required for the synthesis of the essential amino acid selenocysteine, is highly toxic at M concentrations (1). In aerated environments, selenium occurs predominantly in the high-valence soluble selenate (SeO42⫺, ⫹VI) and selenite (SeO32⫺, ⫹IV) forms, while the dominant species in anaerobic sediments is the insoluble elemental selenium
[Se(0)]. Microorganisms are involved in the geochemical cycle of selenium due to their ability to enzymatically reduce Se(IV) and Se(VI) (10). Depending on the considered species, the microbial reduction functions as a detoxification mechanism (4, 6), maintains the redox poise (23), or is part of a respiratory electron chain (2, 9, 17, 20). Oxyanions of tellurium, an element of the same group (XVI) in the peri-
FIG. 1. Microscopic analysis of S. oneidensis MR-1 cells grown in LB medium supplemented with 2 mM selenite (A, B, and C) or 0.5 mg liter⫺1 tellurite (D). (A) Laser scanning confocal microscope, (B) scanning electron microscope, and (C and D) TEM micrograph images. The selenium and tellurium deposits are indicated by arrows.
* Corresponding author. Mailing address: Laboratoire de Bioe´nerge´tique Cellulaire CEA/Cadarache, DSV-DEVM-UMR 6191 CNRSCEA-Aix-Marseille II, 13108 Saint Paul lez Durance Cedex, France. Phone: 33 442254630. Fax: 33 442254701. E-mail:
[email protected]. 5607
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FIG. 2. Aerobic (A) and anaerobic (B) growth of S. oneidensis MR-1 in LB medium supplemented from the beginning with different concentrations of selenite (open circles, no selenite; closed circles, 0.2 mM; squares, 1 mM; and triangles, 2 mM). Anaerobic growth was performed in LB medium supplemented by lactate and fumarate. (C) Depletion of selenite from an aerobic culture when selenite (2 mM) was present from the beginning or added at different times of culture growth. Open triangles, selenite added at the beginning of the culture; circles, selenite added after 3 h (optical density at 600 nm [OD600] of 0.6); squares, selenite added after 4 h (OD600 of 1.0); and closed triangles, selenite added after 5 h (OD600 of 2.0). (D) Depletion of selenite from LB anaerobic medium amended from the beginning with 1 mM selenite in the presence of lactate.
odic table as selenium, are also reduced by various bacterial species (5, 21). Shewanella oneidensis (formerly Shewanella putrefaciens) MR-1 (22), a facultatively anaerobic ␥-proteobacterium, possesses remarkably diverse respiratory capacities, including the ability to reduce metals like Fe(III) and Mn(IV) and radionuclides (7, 8, 12, 13, 16). In this report, we analyze the reduction process of selenite and tellurite by this bacterium. The liquid cultures of S. oneidensis MR-1 grown either aerobically or anaerobically in the presence of Se(IV) or Te(IV) turned red (18) or black (5), respectively, proving the ability of this bacterium to reduce these oxyanions to their elemental forms. To assess the cellular localization of the reduced deposits, bacterial cells were analyzed by means of three different microscopes (laser scanning confocal microscope, environmental scanning electron microscope [ESEM], and transmission electron microscope [TEM]). In the case of selenite, spherical deposits, identified as Se(0) by energy dispersion of X rays (EDX) (data not shown), were detected in the medium or attached to the cells (Fig. 1A through C), suggesting that selenite reduction occurs at the surface of the cell. Reduction at the cell surface, related to the electron transfer capacity of cytochrome c (11) on the outer membrane, has already been reported in the case of insoluble Mn(IV) and Fe(III) oxides (14). In the case of tellurite, needle-like inclusions identified as Te(0) by EDX analysis were localized in the cytoplasm or near the cytoplasmic membrane
(Fig. 1D) as already reported for other tellurite-reducing bacteria (21). The influence of Se(IV) on the bacterial growth was measured under both aerobic and anaerobic conditions (Fig. 2A and B). Under aerobic conditions, Se(IV) concentrations higher than 0.2 mM affected both the growth rate and the final cell yield. The bacteria appeared to be more sensitive to Se(IV) addition under anaerobic conditions, as shown by the decreases of 48% and 52% in growth rate and the final cell yield, respectively, observed for 0.2 mM Se(IV) (Fig. 2B). While Se(IV) reduction occurred only in the late stationary phase under aerobic conditions (Fig. 2C), this process followed roughly the growth curve under anaerobic conditions (Fig. 2D). Interestingly, the addition of 2 mM selenite to aerobic cultures at different times during the growth was followed by an immediate reduction (Fig. 2C). This observation and the relative fast reduction observed under anaerobiosis suggest that the selenite reduction process was highly dependent on the O2 concentration in the culture medium as previously reported for Stenotrophomonas maltophilia (3). We have therefore determined the influence of the O2 concentration, from 0 to 250 M, on the yield of Se(IV) reduction. The Se(IV) reduction was maximal under anaerobic conditions and decreased strongly in the presence of O2, reaching values as low as 5 to 7% for O2 concentrations greater than 120 M. This Se(IV) reduction capability was induced by anaerobic conditions and not due to inactivation of some electron carriers or
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enzymatic activities by the presence of O2 as shown by the low yield of Se(IV) reduction (4%), measured under anaerobic conditions, for cells previously grown under aerobic conditions. Under anaerobic conditions, Se(IV) reduction activity was highly dependent upon the nature of the electron donor. The best reduction was obtained for cells incubated in Luria-Bertani (LB) medium or in the presence of yeast extract. Much lower reduction yields were obtained with electron donors (lactate, formate, and pyruvate) and Casamino Acids or Bacto tryptone in phosphate buffer. The best reduction yield [13% of the maximal amount of Se(IV) reduced in LB medium] was obtained with lactate. Se(IV) reduction yield was also influenced by the presence of different terminal electron acceptors (fumarate, nitrate, nitrite, TMAO [trimethylamine-N-oxide], and dimethyl sulfoxide). Addition of these different electron acceptors resulted in almost 95% inhibition of Se(IV) reduction. Se(IV) reduction occurred, however, after a long period of growth, probably after complete reduction of the added terminal electron acceptor. The Te(IV) reduction was also inhibited by the addition of fumarate, nitrate, and nitrite but was not affected by dimethyl sulfoxide or TMAO. The various electron acceptors used are the substrates for periplasmic terminal reductases of S. oneidensis, which are all supplied with electrons by the membranebound tetracytochrome c, CymA (19). We have therefore examined the role of CymA in the oxyanion reduction capability. A mutant from which cymA is deleted (15) is still able to reduce selenite or tellurite (data not shown). We therefore conclude that competition for electrons between Se(IV) reduction and the various electron acceptors tested occurs upstream from the electron carrier CymA, possibly at the quinone pool level. The difference in the localizations of Se(0) and Te(0) deposits (Fig. 1), together with the differential effect of terminal electron acceptors, suggests that the reduction mechanisms of Se(IV) and Te(IV) are distinct processes. Further proof was obtained by comparing the effects of various inhibitors on these processes. For example, 2-n-heptyl-4-hydroxyquinolineN-oxide (HQNO), antimycin A, pCMB, and potassium cyanide inhibited the Se(IV) reduction process, while the Te(IV) reduction was affected only by the two former inhibitors. In conclusion, despite their close positions in the periodic table in the XVI group, the two oxyanions, selenite and tellurite, are reduced by two distinct pathways in S. oneidensis. Clearly, additional work is needed to describe in more detail the molecular mechanisms of the reduction of these two oxyanions by S. oneidensis. We are grateful to M. Lesourd for the contribution to TEM observations and EDX analysis and to I. Felines for the contribution to ESEM observations of S. oneidensis. We acknowledge B. M. Tebo and R. Bencheikh (Scripps Institution of Oceanography, University of California—San Diego, La Jolla, Calif.) for providing us the mutant cymA strain. We also offer special thanks to G. De Luca for friendly discussions and encouragement. The work was supported by a grant from the “Programme Toxicologie Nucle´aire” of the French Atomic Energy Commission (CEA). REFERENCES 1. Conde, J. E., and A. M. Sanz. 1997. Selenium concentrations in natural and environmental waters. Chem. Rev. 97:1979–2004.
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