Reduction of molybdate by sulfate-reducing bacteria
- First Online:
- Cite this article as:
- Biswas, K.C., Woodards, N.A., Xu, H. et al. Biometals (2009) 22: 131. doi:10.1007/s10534-008-9198-8
- 400 Views
Molybdate is an essential trace element required by biological systems including the anaerobic sulfate-reducing bacteria (SRB); however, detrimental consequences may occur if molybdate is present in high concentrations in the environment. While molybdate is a structural analog of sulfate and inhibits sulfate respiration of SRB, little information is available concerning the effect of molybdate on pure cultures. We followed the growth of Desulfovibrio gigas ATCC 19364, Desulfovibrio vulgaris Hildenborough, Desulfovibrio desulfuricans DSM 642, and D. desulfuricans DSM 27774 in media containing sub-lethal levels of molybdate and observed a red-brown color in the culture fluid. Spectral analysis of the culture fluid revealed absorption peaks at 467, 395 and 314 nm and this color is proposed to be a molybdate–sulfide complex. Reduction of molybdate with the formation of molybdate disulfide occurs in the periplasm D. gigas and D. desulfuricans DSM 642. From these results we suggest that the occurrence of poorly crystalline Mo-sulfides in black shale may be a result from SRB reduction and selective enrichment of Mo in paleo-seawater.
KeywordsMolybdateMolybdenum disulfideTransition metalsDissimilatory metal reductionSulfate-reducing bacteria
Sulfate-reducing bacteria (SRB) are anaerobic chemolithotrophic bacteria that characteristically derive energy for growth by coupling electron transport from electron donors to the reduction of sulfate by a process known as dissimilatory sulfate reduction. From recent reviews (Barton et al. 2003; Bruschi et al. 2007) it is apparent that SRB also have the capability of diverting electrons to several oxidized metals including Fe(III), Cr(VI)O42−, U(VI)O42−, Mn(IV)O2, Te(VI)O42−, and Se(VI)O42−. Large quantities of these oxidized metals are reduced by SRB and this process is termed dissimilatory metal reduction. Unlike the reduction of sulfate which is a cytoplasmic activity, the reduction of metals occurs at the surface of the cells with most of the reduced metal released into the extracellular fluid. Transition metals, including molybdenum, are important to SRB for the synthesis of enzymes involved in redox reactions (Moura et al. 2007; Barton and Fauque 2009); however, at elevated levels molybdate it is an inhibitor of sulfate metabolism and toxic to SRB.
Anaerobic sulfidogenic bacteria of the genus Desulfovibrio are capable of removing molybdate from the extracellular chemical environment through a reduction process. Previously, it was reported that at sub-inhibitory concentrations, molybdate, Mo(VI), was converted to Mo(IV) by D. desulfuricans DSM 642 with the production of insoluble MoS2 (Tucker et al. 1997). This paper presents molybdate metabolism by SRB, reports the conditions required for Mo(VI) reduction, and discusses a possible role of SRB for enrichment of reduced Mo in natural black shale environments containing clay minerals, organic matter and sulfide minerals.
Molybdoproteins present in SRB
Although there had not been a systematic analysis of molybdoenzymes present in SRB, several molybdoproteins have been isolated. The NapA segment of the periplasmic nitrate reductase from D. desulfuricans ATCC 27774 contains Mo and has been extensively studied (Moura et al. 2007). Unique molybdoproteins of unknown physiological activity have been isolated from Desulfovibrio africanus (Hatchikian and Bruschi 1979) and Desulfovibrio salexigens (Czechowski et al. 1986). Additionally, the aldehyde dehydrogenase from D. gigas and several strains of D. desulfuricans has been purified and characterized (Moura and Barata 1994; Rebelo et al. 2000; Moura et al. 2004).
Enzymes demonstrated in SRB that are characterized as molybdoproteins in other bacteria include: carbon monoxide dehydrogenase in D. vulgaris Hildenborough (Voordouw 2002), ethylbenzene dehydrogenase in SRB strain EbS7 (Kniemeyer et al. 2003), dimethyl sulfoxide reductase in D. desulfuricans strain PA2805 (Jonkers et al. 1996), and formate dehydrogenase in Desulfovibrio alaskensis (Brondino et al. 2004). Several strains of Desulfovibrio including D. vulgaris Hildenborough, D. gigas, and D. desulfuricans display nitrogenase activity (Postgate and Kent 1985; Lespinat et al. 1987).
Molybdate transport systems
The uptake of molybdate into cells and the formation of molybdopterin, the molybdenum cofactor, is well characterized (Mendel 2005). To satisfy the requirement for molybdenum, D. vulgaris Hildenborough has an active transport system that is similar to other bacteria. Analysis of the D. vulgaris Hildenborough genome reveals that genes for high affinity molybdate uptake by an ABC transporter are clustered in an operon. The periplasmic binding protein for molybdate is encoded on modA, the transmembrane permease lipoprotein is a product of modB, and the cytoplasmic ATP–binding protein is produced from modC. These genes (e.g., modA, modB, modC) are located at the gene locus of DVU0177, DVU0181 and DVU180, respectively. Several bacteria have an additional regulatory protein, ModE, which regulates the synthesis of the molybdate uptake operon. In the presence of molybdate, ModE binds to a specific sequence in the operator/promoter region of the operon of the ABC-molybdate transporter and interferes with the reading of the modA, B, C, and D genes (Grunden and Shanmugam 1997). This ModE regulator prevents synthesis of the proteins for the molybdate transporter system under conditions where molybdate uptake exceeds encorporation of molybdate into molybdopterin. In some nitrogen-fixing bacteria that do not have this tight couple between molybdate uptake and utilization, there appears to be a molybdenum storage protein that prevents accumulation of free intracellular levels of molybdate (Grunden and Shanmugam 1997).
Homeostasis of transition metals, including molybdate, is important because elevated levels of free metal ions would be toxic to the cell. How bacteria of the Desulfovibrio species achieve molybdenum homeostasis is not established but it may be expected to be similar to the metal regulatory systems used by other bacteria. Zn, Mn and Ni homeostasis in Escherichia coli is proposed to be under the regulation of the FUR system (Lee and Helmann 2007) and Fe homeostasis in D. vulgaris is proposed to be under the FUR system (He et al. 2006). Export of molybdate may be attributed to transporters similar to the P-type ATPase established for metals in prokaryotes (Cooms and Barkay 2005); however, a P-type ATPase for molybdate has not been reported in SRB.
Molybdate inhibition of sulfate activation
As reported by Peck (1959), MoO42− inhibits ATP sulfurylase, the first enzyme in sulfate activation. The mechanism of inhibition by molybdate and other Group VI anions (SeO42−, WO42− and CrO42−) is the formation of an unstable molecule equivalent to APS (Peck 1961). The result from using an anion that was a competitive inhibitor for sulfate in the ATP sulfurylase reaction was that energy was consumed but an appropriate electron acceptor was not generated.
Molybdate is a competitive inhibitor for the sulfate uptake system of enteric bacteria (Kredich 1987). In D. vulgaris Hildenborough, the uptake of sulfate is by the sulfate permease (SulP) family at the gene locus of DVU0053, DVU0279, DVU1999. Molybdate at elevated concentrations would prevent inhibit sulfate import in D. vulgaris. Newport and Nedwell (1988) propose that there are several sites of action for molybdate inhibition of SRB and provide evidence for molybdate inhibition of sulfate transport in several SRB including Desulfovibrio sp. For some time, chromate was used to control the growth of SRB in cooling tower waters and other environments but due to the high toxicity of chromate, molybdate was commonly employed to inhibit the production of H2S from sulfate. To control the growth of SRB and generation of hydrogen sulfide by sulfidogenic bacteria, additions of 20 mM molybdate were effective in salt marsh sediments (Banat et al. 1981) and mangrove forest sediments (Lyimo et al. 2002). The addition of 3 mM molybdate to distillery wastes was found to inhibit production of H2S from sulfate respiration for several days (Ranade et al. 1999).
Molybdate inhibition with pure cultures
Reduction of molybdate
Several bacterial species have been reported to reduce molybdate, Mo(VI), to molybdenum blue which has a mean oxidation state between +5 and +6 for Mo (Williams and da Silva 2002). Cells of Serratia marcescens in the presence of sucrose and ammonium sulfate reduced molybdate to molybdenum blue, a polymer of phosphate and molybdate, which has maximum absorption at 865 and 700 nm (Shukor et al. 2008). Anaerobically grown cells of E. coli will reduce molybdate in the presence of phosphate and glucose to produce molybdenum blue (Campbell et al. 1985).
Reduction of molybdate by cultures of SRB is by a process that does not involve the production of molybdenum blue. Tucker et al. (1997) indicate that D. desulfuricans DSM 624 reduced molybdate to the mineral molybdenite (MoS2). Reduction of Mo(VI) by D. desulfuricans was demonstrated to be an enzymatic process requiring viable bacterial cells plus an electron donor of either lactate or H2. With the addition of 1–3 mM molybdate to the culture medium, reduction of Mo(VI) coincided with sulfate reduction and production of H2S. Columns constructed with D. desulfuricans immobilized in acrylamide simultaneously reduced molybdate, chromate, selenate and uranyl ions when lactate and sulfate was in the feed solution along with the oxy-anions (Tucker et al. 1998). In these experiments dealing with MoS2, formation there was no indication of molybdenum blue being formed but the culture media containing molybdate had a red-brown color (Tucker et al. 1997).
In a distinctly different experiment, Chen et al. (1998) added elemental molybdenum to a culture of D. desulfuricans ATCC 7757 and they observed the formation of an orange color which had adsorption peaks at 314, 396, and 468 nm. Through a series of experiments, they determined that in the appropriate chemical environment, sulfide reduced Mo(VI)O42− to Mo(V)-S producing the orange solution and that Mo(V)-S is analogous to Mo(V)-cysteine and Mo(V)-thiocyanate. Using X-ray photoelectron spectroscopy, Chen et al. (1998) demonstrated that molybdenum disulfide was produced by D. desulfuricans in media amended with elemental molybdenum.
Cell associated reduction of molybdate
Mo sulfide in black shale
Mo(VI)O42− reduction to Mo(IV)S2 is facilitated by an environment containing sulfide with SRB having an important role in the process. The reduction with molybdate added to the culture media of SRB is similar to the reactions described for the production of MoS2 following the addition of elemental Mo(s) to the culture media (Chen et al. 1998). The reduction of Mo(VI)O42− is a two step process. The initial step is a chemical reaction requiring a sulfide environment with the production of Mo(V) in the formation of some type of a molybdate-sulfide compound. This reduction of molybdate-sulfide appears to occur in the periplasm of cell and the reduced molybdenum sulfide accumulates extracellularly or at the surface of the cells. The process observed here with molybdate reduction may involve electron flow activities similar to that observed with U(VI) and Cr(VI) reduction in SRB (Barton et al. 2007, Bruschi et al. 2007) and other dissimilatory metal reducers. It appears that molybdate is not the molecule reduced directly by SRB but rather it is a molybdate-sulfide complex. The poorly crystalline Mo-sulfides in black shale could result from microbial reduction and selective enrichment of Mo in paleo-seawater via SRB.
This research was supported in part by grants from DOE-WERC, MARC and IMSD grants from National Institute of Health, and NASA Astrobiology Institute (N07-5489). Support also was provided by Delaware EPSCoR through the Delaware Biotechnology Institute with funds from the National Science Foundation Grant EPS-0447610 and the State of Delaware. Genome analysis was from the Institute for Genomic Research Comprehensive Microbial Database at www.tigr.org.