Genomic insight into iron acquisition by sulfate-reducing bacteria in microaerophilic environments.

Historically, sulfate-reducing bacteria (SRB) have been considered to be strict anaerobes, but reports in the past couple of decades indicate that SRB tolerate exposure to O2 and can even grow in aerophilic environments. With the transition from anaerobic to microaerophilic conditions, the uptake of Fe(III) from the environment by SRB would become important. In evaluating the metabolic capability for the uptake of iron, the genomes of 26 SRB, representing eight families, were examined. All SRB reviewed carry genes (feoA and feoB) for the ferrous uptake system to transport Fe(II) across the plasma membrane into the cytoplasm. In addition, all of the SRB genomes examined have putative genes for a canonical ABC transporter that may transport ferric siderophore or ferric chelated species from the environment. Gram-negative SRB have additional machinery to import ferric siderophores and ferric chelated species since they have the TonB system that can work alongside any of the outer membrane porins annotated in the genome. Included in this review is the discussion that SRB may use the putative siderophore uptake system to import metals other than iron.

Hydrogen Availability Is Dependent on the Actions of Both Hydrogen-Producing and Hydrogen-Consuming Microbes.

Hydrogen gas (H2) is produced by H2-producing microbes in the gut during polysaccharide fermentation. Gut microbiome also includes H2-consuming microbes utilizing H2 for metabolism: methanogens producing methane, CH4, and sulfate-reducing bacteria producing hydrogen sulfide, H2S. H2S is not measured in the evaluation of gaseous byproducts of microbial fermentation. We hypothesize that the availability of measured H2 depends on both hydrogen producers and hydrogen consumers by measuring H2 in vitro and in vivo. In the in vitro study, groups were Bacteroides thetaiotaomicron (B. theta, H2 producers), Desulfovibrio vulgaris (D. vulgaris, H2 consumers), and D. vulgaris + B. theta combined. Gas samples were collected at 2 h and 24 h after incubation and assayed for H2, CH4, and H2S. In the in vivo study Sprague-Dawley rats were gavaged with suspended bacteria in four groups: B. theta, D. vulgaris, combined, and control. Gas was analyzed for H2 at 60 min. In the in vitro experiment, H2 concentration was higher in the combined group (188 ± 93.3 ppm) compared with D. vulgaris (27.17 ± 9.6 ppm) and B. theta groups (34.2 ± 29.8 ppm; P < 0.05); H2S concentration was statistically higher in the combined group (10.32 ± 1.5 ppm) compared with B. theta (0.19 ± 0.03 ppm) and D. vulgaris group (3.46 ± 0.28 ppm; P < 0.05). In the in vivo study, H2 concentrations were significantly higher in the B. theta group (44.3 ± 6.0 ppm) compared with control (31.8 ± 4.3) and the combined group (34.2 ± 8.7, P < 0.05). This study shows that sulfate-reducing bacteria could convert available H2 to H2S, leading to measured hydrogen levels that are dependent on the actions of both H2 producers and H2 consumers.

Bismuth(III) interactions with Desulfovibrio desulfuricans: inhibition of cell energetics and nanocrystal formation of Bi2S3 and Bi0.

Sulfate-reducing bacteria have been suggested to have an etiological role in the development of inflammatory bowel diseases and ulcerative colitis in humans. Traditionally. bismuth compounds have been administered to alleviate gastrointestinal discomfort and disease symptoms. One mechanism by which this treatment occurs is through binding bacterial derived hydrogen sulfide in the intestines. With the addition of bismuth-deferiprone, bismuth-citrate and bismuth subsalicylate to reactions containing cells of D. desulfuricans ATCC 27774, the oxidation of H2 with sulfate as the electron acceptor was inhibited but H2 oxidation with nitrate, nitrite and sulfite was not reduced. Our research suggests that a target for bismuth inhibition of D. desulfuricans is the F1 subunit of the ATP synthase and, thus, dissimilatory sulfate reduction does not occur. At sublethal concentrations, bismuth as Bi(III) is precipitated by hydrogen sulfide produced from respiratory sulfate reduction by D. desulfuricans. Nanocrystals of bismuth sulfide were determined to be Bi2S3 through the use of high resolution transmission electron microscopy imaging with X-ray energy-dispersive spectroscopy analysis. In the absence of sulfate, D. desulfuricans oxidizes H2 with the reduction of Bi(III) to Bi0 and this was also established by X-ray energy-dispersive spectroscopy analysis.

Sulfur Cycling and the Intestinal Microbiome.

In this review, we focus on the activities transpiring in the anaerobic segment of the sulfur cycle occurring in the gut environment where hydrogen sulfide is produced. While sulfate-reducing bacteria are considered as the principal agents for hydrogen sulfide production, the enzymatic desulfhydration of cysteine by heterotrophic bacteria also contributes to production of hydrogen sulfide. For sulfate-reducing bacteria respiration, molecular hydrogen and lactate are suitable as electron donors while sulfate functions as the terminal electron acceptor. Dietary components provide fiber and macromolecules that are degraded by bacterial enzymes to monomers, and these are fermented by intestinal bacteria with the production to molecular hydrogen which promotes the metabolic dominance by sulfate-reducing bacteria. Sulfate is also required by the sulfate-reducing bacteria, and this can be supplied by sulfate- and sulfonate-containing compounds that are hydrolyzed by intestinal bacterial with the release of sulfate. While hydrogen sulfide in the intestinal biosystem may be beneficial to bacteria by increasing resistance to antibiotics, and protecting them from reactive oxygen species, hydrogen sulfide at elevated concentrations may become toxic to the host.

Bismuth(III) deferiprone effectively inhibits growth of Desulfovibrio desulfuricans ATCC 27774.

Sulfate-reducing bacteria have been implicated in inflammatory bowel diseases and ulcerative colitis in humans and there is an interest in inhibiting the growth of these sulfide-producing bacteria. This research explores the use of several chelators of bismuth to determine the most effective chelator to inhibit the growth of sulfate-reducing bacteria. For our studies, Desulfovibrio desulfuricans ATCC 27774 was grown with nitrate as the electron acceptor and chelated bismuth compounds were added to test for inhibition of growth. Varying levels of inhibition were attributed to bismuth chelated with subsalicylate or citrate but the most effective inhibition of growth by D. desulfuricans was with bismuth chelated by deferiprone, 3-hydroxy-1,2-dimethyl-4(1H)-pyridone. Growth of D. desulfuricans was inhibited by 10 µM bismuth as deferiprone:bismuth with either nitrate or sulfate respiration. Our studies indicate deferiprone:bismuth has bacteriostatic activity on D. desulfuricans because the inhibition can be reversed following exposure to 1 mM bismuth for 1 h at 32 °C. We suggest that deferiprone is an appropriate chelator for bismuth to control growth of sulfate-reducing bacteria because deferiprone is relatively nontoxic to animals, including humans, and has been used for many years to bind Fe(III) in the treatment of ß-thalassemia.

Influence of Methylobacterium on iron translocation in plants.

Iron metabolism in plants is essential to maintain optimal growth and iron nutrition is dependent on uptake of iron from the environment and movement of iron in the plant tissues. We have examined the translocation of iron in plant leaves following foliar application of FeEDTA to Vicia faba and Zea mays. Using radiolabeled iron, we observed that iron translocation is stimulated by products of Methylobacterium mesophylicum and by the cytokinin, kinetin. When cytokinins were applied to leaves along with (55)FeEDTA, the rate of iron translocation was greater than in controls without cytokinin addition. Since recent studies indicate that M. mesophylicum is widely distributed in the environment as a pyllospheric bacterium, this organism may have an important role in enhancing translocation of nutrients in plant leaves.

Selenium respiration in anaerobic bacteria: Does energy generation pay off?

Selenium (Se) respiration in bacteria was revealed for the first time at the end of 1980s. Although thermodynamically-favorable, energy-dense and documented in phylogenetically-diverse bacteria, this metabolic process appears to be accompanied by a number of challenges and numerous unanswered questions. Selenium oxyanions, SeO42- and SeO32-, are reduced to elemental Se (Se0) through anaerobic respiration, the end product being solid and displaying a considerable size (up to 500 nm) at the bacterial scale. Compared to other electron acceptors used in anaerobic respiration (e.g. N, S, Fe, Mn, and As), Se is one of the few elements whose end product is solid. Furthermore, unlike other known bacterial intracellular accumulations such as volutin (inorganic polyphosphate), S0, glycogen or magnetite, Se0 has not been shown to play a nutritional or ecological role for its host. In the context of anaerobic respiration of Se oxyanions, biogenic Se0 appears to be a by-product, a waste that needs proper handling, and this raises the question of the evolutionary implications of this process. Why would bacteria use a respiratory substrate that is useful, in the first place, and then highly detrimental? Interestingly, in certain artificial ecosystems (e.g. upflow bioreactors) Se0 might help bacterial cells to increase their density and buoyancy and thus avoid biomass wash-out, ensuring survival. This review article provides an in-depth analysis of selenium respiration (model selenium respiring bacteria, thermodynamics, respiratory enzymes, and genetic determinants), complemented by an extensive discussion about the evolutionary implications and the properties of biogenic Se0 using published and original/unpublished results.

PbS biomineralization using cysteine: Bacillus cereus and the sulfur rush.

Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and phylogenetically classified. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is degraded intracellularly to hydrogen sulfide (H2S). H2S is then exported to the extracellular environment to react with Pb(II), yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and methionine) were not reduced to hydrogen sulfide. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate of 2.03 µg L-1 h-1 (∼8.7 µM L-1 h-1). The mineralogy of biogenic PbS was characterized and confirmed by XRD, HRTEM and EDX. Electron microscopy and electron diffraction identified crystalline PbS nanoparticles with a diameter <10 nm,  localized in the extracellular matrix and on the surface of the cells. This is the first study demonstrating the use of cysteine in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden industrial effluents.

Biochemistry, physiology and biotechnology of sulfate-reducing bacteria.

Chemolithotrophic bacteria that use sulfate as terminal electron acceptor (sulfate-reducing bacteria) constitute a unique physiological group of microorganisms that couple anaerobic electron transport to ATP synthesis. These bacteria (220 species of 60 genera) can use a large variety of compounds as electron donors and to mediate electron flow they have a vast array of proteins with redox active metal groups. This chapter deals with the distribution in the environment and the major physiological and metabolic characteristics of sulfate-reducing bacteria (SRB). This chapter presents our current knowledge of soluble electron transfer proteins and transmembrane redox complexes that are playing an essential role in the dissimilatory sulfate reduction pathway of SRB of the genus Desulfovibrio. Environmentally important activities displayed by SRB are a consequence of the unique electron transport components or the production of high levels of H(2)S. The capability of SRB to utilize hydrocarbons in pure cultures and consortia has resulted in using these bacteria for bioremediation of BTEX (benzene, toluene, ethylbenzene and xylene) compounds in contaminated soils. Specific strains of SRB are capable of reducing 3-chlorobenzoate, chloroethenes, or nitroaromatic compounds and this has resulted in proposals to use SRB for bioremediation of environments containing trinitrotoluene and polychloroethenes. Since SRB have displayed dissimilatory reduction of U(VI) and Cr(VI), several biotechnology procedures have been proposed for using SRB in bioremediation of toxic metals. Additional non-specific metal reductase activity has resulted in using SRB for recovery of precious metals (e.g. platinum, palladium and gold) from waste streams. Since bacterially produced sulfide contributes to the souring of oil fields, corrosion of concrete, and discoloration of stonework is a serious problem, there is considerable interest in controlling the sulfidogenic activity of the SRB. The production of biosulfide by SRB has led to immobilization of toxic metals and reduction of textile dyes, although the process remains unresolved, SRB play a role in anaerobic methane oxidation which not only contributes to carbon cycle activities but also depletes an important industrial energy reserve.

Reduction of molybdate by sulfate-reducing bacteria.

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.

A New Combination of Substrates: Biogas Production and Diversity of the Methanogenic Microorganisms.

Agriculture, food industry, and manufacturing are just some of the areas where anaerobic technology can be used. Currently, anaerobic technologies are mainly used for wastewater treatment, solid waste treatment, or for the production of electrical and thermal energy from energy crops processing. However, a clear trend is towards more intensive use of this technology in biomass and biodegradable waste processing and hydrogen or biomethane production. An enormous number of anaerobic digesters are operating worldwide but there is very little information about the effect of different substrate combinations on the methanogens community. This is due to the fact that each of the anaerobic digesters has its own unique microbial community. For the most effective management of anaerobic processes it would be important to know the composition of a consortium of anaerobic microorganisms present in anaerobic digesters processing different input combinations of raw material. This paper characterizes the effect of the input raw materials on the diversity of the methanogen community. Two predominant microorganisms in anaerobic digesters were found to be 99% identity by the sequences of the 16S rRNA gene to the Methanoculleus and Thermogymnomonas genera deposited in GenBank.

Sulfate-reducing bacteria impairs working memory in mice.

The ability of gut microbes to bi-directionally communicate with the brain and vice versa form the basis of the gut microbiome-central nervous system axis. It has been shown that inoculation with pathogenic gut bacteria alters the behavior of mice; however, it is not known whether or not non-pathogenic resident microbes have similar effects. In this study, we tested the hypothesis that the administration of sulfate-reducing bacteria (SRB), a specific group of resident gut bacteria that generate hydrogen sulfide (H2S), impair learning and memory performance in mice tested in an 8-arm radial maze and Morris water maze. We found that mice spent more time in the center of the maze when they were gavaged with live SRB as compared to mice given saline (control), lactulose+mannitol (L/M), or killed SRB. SRB-gavaged mice were also tested using the Morris water maze and were found to take longer to complete the test, spend more time further from the platform, and have a longer path length to reach the platform. This effect of SRB on maze performance was associated with a higher concentration of H2S in the small intestine and cecum. We conclude that SRB, a specific resident gut bacterial species, could impair cognitive function in mice.

Hydrogen sulfide: a toxic gas produced by dissimilatory sulfate and sulfur reduction and consumed by microbial oxidation.

Sulfur is an essential element for the synthesis of cysteine, methionine, and other organo-sulfur compounds needed by living organisms. Additionally, some prokaryotes are capable of exploiting oxidation or reduction of inorganic sulfur compounds to energize cellular growth. Several anaerobic genera of Bacteria and Archaea produce hydrogen sulfide (H2S), as a result of using sulfate (SO(4)(2 -) ), elemental sulfur (S(0)), thiosulfate (S2O(3)(2 -)), and tetrathionate (S(4)O(6)(2 -)) as terminal electron acceptors. Some phototrophic and aerobic sulfur bacteria are capable of using electrons from oxidation of sulfide to support chemolithotrophic growth. For the most part, biosulfur reduction or oxidation requires unique enzymatic activities with metal cofactors participating in electron transfer. This review provides an examination of cytochromes, iron-sulfur proteins, and sirohemes participating in electron movement in diverse groups of sulfate-reducing, sulfur-reducing, and sulfide-oxidizing Bacteria and Archaea.

Suitability of fluorescence measurements to quantify sulfate-reducing bacteria.

Fluorescence activity has been used to identify Desulfovibrio and has been termed the 'desulfoviridin test'. This fluorescence is attributed to the prosthetic group of bisulfite reductase, a key enzyme in dissimilatory sulfate reduction. We have pursued the use of fluorescence measurements to quantify sulfate-reducing bacteria. Cells of D. desulfuricans and D. gigas were treated with NaOH and produced two fluorescence spectra: one with maximum fluorescence with an excitation at 395 nm and an emission at 605 nm and another with an excitation at 320 nm and emission at 360 nm. Using the fluorescence with excitation at 395 nm and emission at 605 nm, we explored a series of parameters to measure Desulfovibrio in pure cultures and environmental samples. Fluorescence measurements are reliable provided the cells are treated with 1.75 N NaOH and the chromophore released from the cells is not exposed to strong light intensity, and is not exposed to temperatures greater than 20 °C, and measurements are done within a few minutes of extraction. Bleaching of fluorescence was attributed to metal ions in solution which was not observed until metal concentrations reached 1.5mM. We propose that D. desulfuricans is appropriate as the reference organism for measurement of sulfate-reducing bacteria by fluorescence and by using fluorescence intensity, 10(5) cells/ml can be readily detected in environmental samples.

Hemoproteins in dissimilatory sulfate- and sulfur-reducing prokaryotes.

Dissimilatory sulfate and sulfur reduction evolved billions of years ago and while the bacteria and archaea that use this unique metabolism employ a variety of electron donors, H(2) is most commonly used as the energy source. These prokaryotes use multiheme c-type proteins to shuttle electrons from electron donors, and electron transport complexes presumed to contain b-type hemoproteins contribute to proton charging of the membrane. Numerous sulfate and sulfur reducers use an alternate pathway for heme synthesis and, frequently, uniquely specific axial ligands are used to secure c-type heme to the protein. This review presents some of the types and functional activities of hemoproteins involved in these two dissimilatory reduction pathways.

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite.

The measurement of elemental selenium (Se(0)) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se(0) that is rapid and can be employed to measure the quantity of Se(0) produced by bacterial cultures. This method employs the use of 1M Na(2)S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se(0) by Moraxella bovis, and by bacterial consortia in soil and water samples.

The bacterial metallome: composition and stability with specific reference to the anaerobic bacterium Desulfovibrio desulfuricans.

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe(1)Mn(0.3)Zn(0.26)Cu(0.03)Co(0.03)Mo(0.03). Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c3 and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.