Desulfovibrio desulfuricans and its derived metabolites confer resistance to FOLFOX through METTL3.

BACKGROUND: Chemoresistance is a critical factor contributing to poor prognosis in clinical patients with cancer undergoing postoperative adjuvant chemotherapy. The role of gut microbiota in mediating resistance to tumour chemotherapy remains to be investigated. METHODS: Patients with CRC were categorised into clinical benefit responders (CBR) and no clinical benefit responders (NCB) based on chemotherapy efficacy. Differential bacterial analysis using 16S rRNA sequencing revealed Desulfovibrio as a distinct microbe between the two groups. Employing a syngeneic transplantation model, we assessed the effect of Desulfovibrio on chemotherapy by measuring tumour burden, weight, and Ki-67 expression. We further explored the mechanisms underlying the compromised chemotherapeutic efficacy of Desulfovibrio using metabolomics, western blotting, colony formation, and cell apoptosis assays. FINDINGS: In comparison, Desulfovibrio was more abundant in the NCB group. In vivo experiments revealed that Desulfovibrio colonisation in the gut weakened the efficacy of FOLFOX. Treatment with Desulfovibrio desulfuricans elevates serum S-adenosylmethionine (SAM) levels. Interestingly, SAM reduced the sensitivity of CRC cells to FOLFOX, thereby promoting the growth of CRC tumours. These experiments suggest that SAM promotes the growth and metastasis of CRC by driving the expression of methyltransferase-like 3 (METTL3). INTERPRETATION: A high abundance of Desulfovibrio in the intestines indicates poor therapeutic outcomes for postoperative neoadjuvant FOLFOX chemotherapy in CRC. Desulfovibrio drives the manifestation of METTL3 in CRC, promoting resistance to FOLFOX chemotherapy by increasing the concentration of SAM. FUNDING: This study is supported by Wuxi City Social Development Science and Technology Demonstration Project (N20201005).

Initial stage of the biofilm formation on the NiTi and Ti6Al4V surface by the sulphur-oxidizing bacteria and sulphate-reducing bacteria.

The susceptibility to the fouling of the NiTi and Ti6Al4V alloys due to the adhesion of microorganisms and the biofilm formation is very significant, especially in the context of an inflammatory state induced by implants contaminated by bacteria, and the implants corrosion stimulated by bacteria. The aim of this work was to examine the differences between the sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB) strains in their affinity for NiTi and Ti6Al4V alloys. The biofilms formed on alloy surfaces by the cells of five bacterial strains (aerobic SOB Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans, and anaerobic SRB Desulfovibrio desulfuricans-3 strains) were studied using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The protein concentrations in liquid media have also been analyzed. The results indicate that both alloys tested may be colonized by SOB and SRB strains. In the initial stage of the biofilm formation, the higher affinity of SRB to both the alloys has been documented. However, the SOB strains have indicated the higher (although differentiated) adaptability to changing environment as compared with SRB. Stimulation of the SRB growth on the alloys surface was observed during incubation in the liquid culture media supplemented with artificial saliva, especially of lower pH (imitated conditions under the inflammatory state, for example in the periodontitis course). The results point to the possible threat to the human health resulting from the contamination of the titanium implant alloys surface by the SOB (A. thiooxidans and A. ferrooxidans) and SRB (D. desulfuricans).

Preparation of metal-resistant immobilized sulfate reducing bacteria beads for acid mine drainage treatment.

Novel immobilized sulfate-reducing bacteria (SRB) beads were prepared for the treatment of synthetic acid mine drainage (AMD) containing high concentrations of Fe, Cu, Cd and Zn using up-flow anaerobic packed-bed bioreactor. The tolerance of immobilized SRB beads to heavy metals was significantly enhanced compared with that of suspended SRB. High removal efficiencies of sulfate (61-88%) and heavy metals (>99.9%) as well as slightly alkaline effluent pH (7.3-7.8) were achieved when the bioreactor was fed with acidic influent (pH 2.7) containing high concentrations of multiple metals (Fe 469 mg/L, Cu 88 mg/L, Cd 92 mg/L and Zn 128 mg/L), which showed that the bioreactor filled with immobilized SRB beads had tolerance to AMD containing high concentrations of heavy metals. Partially decomposed maize straw was a carbon source and stabilizing agent in the initial phase of bioreactor operation but later had to be supplemented by a soluble carbon source such as sodium lactate. The microbial community in the bioreactor was characterized by denaturing gradient gel electrophoresis (DGGE) and sequencing of partial 16S rDNA genes. Synergistic interaction between SRB (Desulfovibrio desulfuricans) and co-existing fermentative bacteria could be the key factor for the utilization of complex organic substrate (maize straw) as carbon and nutrients source for sulfate reduction.

Proteomics of Desulfovibrio desulfuricans and X-ray absorption spectroscopy to investigate mercury methylation in the presence of selenium.

The effects of mercury added as Hg(2+) and selenium as selenite to cultures of the sulfate reducing bacterium Desulfovibrio desulfuricans were investigated under controlled laboratory conditions. There was no significant difference in the growth curves in comparison to control except in the 0.5 µM Hg-6.3 µM Se combined system in which Hg methylation was significantly reduced. A significant decrease in the production of methylmercury indicates a disruption of the methylation process due to the presence of the relatively high concentrations of Se in the system, suggesting a modification of the biological pathway. The results of detailed 2D gel electrophoresis in combination with mass spectrometry confirmed that the Hg methylation process should certainly be influenced when the protein Dde_1198 protein-glutamate O-methyltransferase was totally suppressed in a culture containing 0.5 µM Hg and 6.3 µM Se. Since this protein plays an important role in the methylation process, its suppression in the presence of Se brings a possible explanation for the antagonism between Se and Hg in natural systems. The experiment involving the determination of Hg and Se in membrane proteins separated by 1D gel thin-layer isoelectric focusing revealed that when both elements were present in a culture, the concentration of Hg in the separated proteins was significantly lower in comparison to those without added Se to the culture and vice versa. Finally, near-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure were used to corroborate the presence of a very inert solid HgSe in the cell membrane obtained from the culture containing 0.5 µM Hg and 6.3 µM Se. This confirms the protective effect of Se against Hg assimilation at the molecular level and reinforces the findings of our research group in numerous field and laboratory studies.

In-situ subaqueous capping of mercury-contaminated sediments in a fresh-water aquatic system, Part II-evaluation of sorption materials.

The function and longevity of traditional, passive, isolation caps can be augmented through the use of more chemically active capping materials which have higher sorptive capacities, ideally rendering metals non-bioavailable. In the case of Hg, active caps also mitigate the rate and extent of methylation. This research examined low cost, readily available, capping materials for their ability to sequester Hg and MeHg. Furthermore, selected capping materials were evaluated to inhibit the methylation of Hg in an incubation study as well as the capacity of a selected capping material to inhibit translocation of Hg and MeHg with respect to ebullition-facilitated contaminant transport in a column study. Results indicated that bauxite had a better capacity for mercury sorption than the other test materials. However, bauxite as well as soil capping materials did not decrease methylation to a significant extent. Materials with larger surface areas, higher organic matter and acid volatile sulfide (AVS) content displayed a larger partitioning coefficient. In the incubation experiments, the presence of a carbon source (lactate), electron acceptor (sulfate) and the appropriate strains of SRB provided the necessary conditions for Hg methylation to occur. The column study showed effectiveness in sequestering Hg and MeHg and retarding transport to the overlying water column; however, disturbances to the soil capping material resulting from gas ebullition negated its effectiveness.

The reversibility of dissimilatory sulphate reduction and the cell-internal multi-step reduction of sulphite to sulphide: insights from the oxygen isotope composition of sulphate.

Dissimilatory sulphate reduction (DSR) leads to an overprint of the oxygen isotope composition of sulphate by the oxygen isotope composition of water. This overprint is assumed to occur via cell-internally formed sulphuroxy intermediates in the sulphate reduction pathway. Unlike sulphate, the sulphuroxy intermediates can readily exchange oxygen isotopes with water. Subsequent to the oxygen isotope exchange, these intermediates, e.g. sulphite, are re-oxidised by reversible enzymatic reactions to sulphate, thereby incorporating the oxygen used for the re-oxidation of the sulphur intermediates. Consequently, the rate and expression of DSR-mediated oxygen isotope exchange between sulphate and water depend not only on the oxygen isotope exchange between sulphuroxy intermediates and water, but also on cell-internal forward and backward reactions. The latter are the very same processes that control the extent of sulphur isotope fractionation expressed by DSR. Recently, the measurement of multiple sulphur isotope fractionation has successfully been applied to obtain information on the reversibility of individual enzymatically catalysed steps in DSR. Similarly, the oxygen isotope signature of sulphate has the potential to reveal complementary information on the reversibility of DSR. The aim of this work is to assess this potential. We derived a mathematical model that links sulphur and oxygen isotope effects by DSR, assuming that oxygen isotope effects observed in the oxygen isotopic composition of ambient sulphate are controlled by the oxygen isotope exchange between sulphite and water and the successive cell-internal oxidation of sulphite back to sulphate. Our model predicts rapid DSR-mediated oxygen isotope exchange for cases where the sulphur isotope fractionation is large and slow exchange for cases where the sulphur isotope fractionation is small. Our model also demonstrates that different DSR-mediated oxygen isotope equilibrium values are observed, depending on the importance of oxygen isotope exchange between sulphite and water relative to the re-oxidation of sulphite. Comparison of model results to experimental data further leads to the conclusion that sulphur isotope fractionation in the reduction of sulphite to sulphide is not a single-step process.

Effect of uranium (VI) on two sulphate-reducing bacteria cultures from a uranium mine site.

This work was conducted to assess the impact of uranium (VI) on sulphate-reducing bacteria (SRB) communities obtained from environmental samples collected on the Portuguese uranium mining area of Urgeiriça. Culture U was obtained from a sediment, while culture W was obtained from sludge from the wetland of that mine. Temperature gradient gel electrophoresis (TGGE) was used to monitor community changes under uranium stress conditions. TGGE profiles of dsrB gene fragment demonstrated that the initial cultures were composed of SRB species affiliated with Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Desulfomicrobium spp. (sample U), and by species related to D. desulfuricans (sample W). A drastic change in SRB communities was observed as a result of uranium (VI) exposure. Surprisingly, SRB were not detected in the uranium removal communities. Such findings emphasize the need of monitoring the dominant populations during bio-removal studies. TGGE and phylogenetic analysis of the 16S rRNA gene fragment revealed that the uranium removal consortia are composed by strains affiliated to Clostridium genus, Caulobacteraceae and Rhodocyclaceae families. Therefore, these communities can be attractive candidates for environmental biotechnological applications associated to uranium removal.

Thioredoxin is involved in U(VI) and Cr(VI) reduction in Desulfovibrio desulfuricans G20.

A transposon insertion mutant has been identified in a Desulfovibrio desulfuricans G20 mutant library that does not grow in the presence of 2 mM U(VI) in lactate-sulfate medium. This mutant has also been shown to be deficient in the ability to grow with 100 microM Cr(VI) and 20 mM As(V). Experiments with washed cells showed that this mutant had lost the ability to reduce U(VI) or Cr(VI), providing an explanation for the lower tolerance. A gene encoding a cyclic AMP (cAMP) receptor protein (CRP) was identified as the site of the transposon insertion. The remainder of the mre operon (metal reduction) contains genes encoding a thioredoxin, thioredoxin reductase, and an additional oxidoreductase whose substrate has not been predicted. Expression studies showed that in the mutant, the entire operon is downregulated, suggesting that the CRP may be involved in regulating expression of the whole operon. Exposure of the cells to U(VI) resulted in upregulation of the entire operon. CdCl(2), a specific inhibitor of thioredoxin activity, inhibits U(VI) reduction by washed cells and inhibits growth of cells in culture when U(VI) is present, confirming a role for thioredoxin in U(VI) reduction. The entire mre operon was cloned into Escherichia coli JM109 and the transformant developed increased U(VI) resistance and the ability to reduce U(VI) to U(IV). The oxidoreductase protein (MreG) from this operon was expressed and purified from E. coli. In the presence of thioredoxin, thioredoxin reductase, and NADPH, this protein was shown to reduce both U(VI) and Cr(VI), providing a mechanism for the cytoplasmic reduction of these metals.

Pseudosymmetry, high copy number and twinning complicate the structure determination of Desulfovibrio desulfuricans (ATCC 29577) flavodoxin.

The crystal structure of oxidized flavodoxin from Desulfovibrio desulfuricans (ATCC 29577) was determined by molecular replacement in two crystal forms, P3(1)21 and P4(3), at 2.5 and 2.0 A resolution, respectively. Structure determination in space group P3(1)21 was challenging owing to the presence of pseudo-translational symmetry and a high copy number in the asymmetric unit (8). Initial phasing attempts in space group P3(1)21 by molecular replacement using a poor search model (46% identity) and multi-wavelength anomalous dispersion were unsuccessful. It was necessary to solve the structure in a second crystal form, space group P4(3), which was characterized by almost perfect twinning, in order to obtain a suitable search model for molecular replacement. This search model with complementary approaches to molecular replacement utilizing the pseudo-translational symmetry operators determined by analysis of the native Patterson map facilitated the selection and manual placement of molecules to generate an initial solution in the P3(1)21 crystal form. During the early stages of refinement, application of the appropriate twin law, (-h, -k, l), was required to converge to reasonable R-factor values despite the fact that in the final analysis the data were untwinned and the twin law could subsequently be removed. The approaches used in structure determination and refinement may be applicable to other crystal structures characterized by these complicating factors. The refined model shows flexibility of the flavin mononucleotide coordinating loops indicated by the isolation of two loop conformations and provides a starting point for the elucidation of the mechanism used for protein-partner recognition.

Uranium immobilization by sulfate-reducing biofilms grown on hematite, dolomite, and calcite.

Biofilms of sulfate-reducing bacteria Desulfovibrio desulfuricans G20 were used to reduce dissolved U(VI) and subsequently immobilize U(IV) in the presence of uranium-complexing carbonates. The biofilms were grown in three identically operated fixed bed reactors, filled with three types of minerals: one noncarbonate-bearing mineral (hematite) and two carbonate-bearing minerals (calcite and dolomite). The source of carbonates in the reactors filled with calcite and dolomite were the minerals, while in the reactor filled with hematite it was a 10 mM carbonate buffer, pH 7.2, which we added to the growth medium. Our five-month study demonstrated that the sulfate-reducing biofilms grown in all reactors were able to immobilize/reduce uranium efficiently, despite the presence of uranium-complexing carbonates.

Palladium and gold removal and recovery from precious metal solutions and electronic scrap leachates by Desulfovibrio desulfuricans.

Biomass of Desulfovibrio desulfuricans was used to recover Au(III) as Au(0) from test solutions and from waste electronic scrap leachate. Au(0) was precipitated extracellularly by a different mechanism from the biodeposition of Pd(0). The presence of Cu(2+) ( approximately 2000 mg/l) in the leachate inhibited the hydrogenase-mediated removal of Pd(II) but pre-palladisation of the cells in the absence of added Cu(2+) facilitated removal of Pd(II) from the leachate and more than 95% of the Pd(II) was removed autocatalytically from a test solution supplemented with Cu(II) and Pd(II). Metal recovery was demonstrated in a gas-lift electrobioreactor with electrochemically generated hydrogen, followed by precipitation of recovered metal under gravity. A 3-stage bioseparation process for the recovery of Au(III), Pd(II) and Cu(II) is proposed.

Toxic effects of uranium on Desulfovibrio desulfuricans G20.

The toxic effects of U(VI) were studied using Desulfovibrio desulfuricans G20 in a medium containing bicarbonate or 1,4-piperazinediethane sulfonic acid disodium salt monohydrate (PIPES) buffer (each at 30 mM and pH 7). Uranium(VI) toxicity was dependent on the medium buffer and was observed in terms of longer lag times and, in some cases, no measurable growth. The minimum inhibiting concentration was 140 microM U(VI) in PIPES-buffered medium. This is 36-fold lower than that reported previously for D. desulfuricans. For all cases in which D. desulfuricans G20 grew in the presence of U(VI), the final cell protein yield was equivalent to that of the U(VI)-free control. In 24 h, D. desulfuricans G20 (total cell protein, 40 mg/L) removed 50 FiM U(VI) from solution in PIPES buffer, as compared to 96 microM U(VI) in bicarbonate buffer under anaerobic, nongrowth conditions. Even though the solubility of U(VI) was significantly lower in PIPES buffer than in bicarbonate buffer, U(VI) was much more toxic in PIPES buffer than in bicarbonate buffer. Analysis of thin sections of D. desulfuricans G20 treated with 90 microM U(VI) in medium containing PIPES buffer revealed that only a very small fraction of cells had reduced U precipitates in the periplasmic spaces. In the presence of bicarbonate buffer, however, reduced U was observed not only in the periplasm but also in the cytoplasm. Selected-area electron diffraction patterns and crystallographic analysis of transmission-electron microscopic lattice fringe images confirmed the structure of precipitated U in the cell periplasm and cytoplasm as being that of uraninite. These results suggest that U(VI) toxicity and the detoxification mechanisms of D. desulfuricans G20 depend greatly on the chemical forms of U(VI) that are present.

Reoxidation of reduced uranium with iron(III) (hydr)oxides under sulfate-reducing conditions.

In cultures of Desulfovibrio desulfuricans 620 the effects of iron(III) (hydr)oxides (hematite, goethite, and ferrihydrite) on microbial reduction and reoxidation of uranium (U) were evaluated under lactate-limited sulfate-reducing conditions. With lactate present, G20 reduced U(VI) in both 1,4-piperazinediethanesulfonate (PIPES) and bicarbonate buffer. Once lactate was depleted, however, microbially reduced U served as an electron donor to reduce Fe(III) present in iron(III) (hydr)oxides. With the same initial amount of Fe(III) (10 mmol/L) for each iron(III) (hydr)oxide, reoxidation of U(IV) was greater with hematite than with goethite orferrihydrite. As the initial mass loading of hematite increased from 0 to 20 mmol of Fe(III)/L, the rate and extent of U(IV) reoxidation increased. Subsequent addition of hematite [15 mmol of Fe(III)/L] to stationary-phase cultures containing microbially reduced U(IV) also resulted in rapid reoxidation to U(VI). Analysis by U L3-edge X-ray absorption near-edge spectroscopy (XANES) of microbially reduced U particles yielded spectra similar to that of natural uraninite. Observations by high-resolution transmission electron microscopy, selected area electron diffraction, and energy-dispersive X-ray spectroscopic analysis confirmed that precipitated U associated with cells was uraninite with particle diameters of 3-5 nm. By the same techniques, iron sulfide precipitates were found to have a variable Fe and S stoichiometry and were not associated with cells.

Interaction between uranium and the cytochrome c3 of Desulfovibrio desulfuricans strain G20.

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.