Mechanism of Reduction of Aqueous U(V)-dpaea and Solid-Phase U(VI)-dpaea Complexes: The Role of Multiheme c-Type Cytochromes

The biological reduction of soluble U(VI) complexes to form immobile U(IV) species has been proposed to remediate contaminated sites. It is well established that multiheme c-type cytochromes (MHCs) are key mediators of electron transfer to aqueous phase U(VI) complexes for bacteria such as Shewanella oneidensis MR-1. Recent studies have confirmed that the reduction proceeds via a first electron transfer forming pentavalent U(V) species that readily disproportionate. However, in the presence of the stabilizing aminocarboxylate ligand, dpaea2– (dpaeaH2=bis(pyridyl-6-methyl-2-carboxylate)-ethylamine), biologically produced U(V) persisted in aqueous solution at pH 7. We aim to pinpoint the role of MHC in the reduction of U(V)-dpaea and to establish the mechanism of solid-phase U(VI)-dpaea reduction. To that end, we investigated U-dpaea reduction by two deletion mutants of S. oneidensis MR-1–one lacking outer membrane MHCs and the other lacking all outer membrane MHCs and a transmembrane MHC–and by the purified outer membrane MHC, MtrC. Our results suggest that solid-phase U(VI)-dpaea is reduced primarily by outer membrane MHCs. Additionally, MtrC can directly transfer electrons to U(V)-dpaea to form U(IV) species but is not strictly necessary, underscoring the primary involvement of outer membrane MHCs in the reduction of this pentavalent U species but not excluding that of periplasmic MHCs.

: Fe(II) concentration measured by ferrozine test in the incubation supernatants of MR-1 (pink), ∆OMC (blue), ∆OMC∆MtrA (yellow) and a no-cell control (black) from 0 to 48h of incubation with A. 2000µM Fe(III)-citrate, B. 2000µM ferrihydrite. Figure S2. Cell viability of MR-1(pink dots), ∆OMC (blue dots) and ∆OMC∆MtrA (yellow dots) over the experimental time in incubations with A. Fe(III)-citrate, B. Ferrihydrite. Figure S3. Cell viability of MR-1 (pink dots), ∆OMC (blue dots) and ∆OMC∆MtrA (yellow dots) over the experimental time in incubations with A. solid phase U(VI)-dpaea and B. aqueous U(VI)-dpaea. C. Concentration of U(VI)-dpaea in solution for a no-cell control (black) and MR-1 (pink). Figure S4: U oxidation state in the solid phase (cell pellet) and in the aqueous phase (supernatant) of A. incubations with S. oneidensis MR-1; B. and no-cell controls, incubated with 20µM aqueous U(VI)dpaea, at pH 7.3, OD600 =1. Figure S5. Initial rate of reaction for aqueous U(VI)-dpaea incubated with WT S. oneidensis MR-1 (pink), ∆OMC (blue) and ∆OMC∆MtrA (yellow). The data are only considered for the first 7h of the experiment. Figure S6. Initial rate of reaction of aqueous U(V)-dpaea incubated with WT S. oneidensis MR-1 (pink), ∆OMC (blue) and ∆OMC∆MtrA (yellow). The data are only considered for the first 48h of the experiment. Figure S7. Percentage of U(IV) obtained by ion exchange chromatography of the reaction between U(V)-dpaea and either oxidized (blue) or reduced (green) MtrC after 20s, 2h and 4h of reaction. U(V)dpaea (pink) was used as a control to ensure that no spontaneous disproportionation occurred during the experimental time. Figure S8. Incubation of the WT MR-1 strain (pink) and a mutant strain of MR-1 lacking the flavin transporter system (dark blue) with A. solid U(VI)-dpaea, B. aqueous U(V)-dpaea. Figure S9. A. Percentage of U(IV) obtained by ion exchange chromatography of the reaction between 100µM U(IV)-citrate and 100µM oxidized MtrC (light blue) after 2 min of reaction. U(IV)-citrate in buffer A (dark blue) was used as a control for the U oxidation state. B. UV-vis spectra of the hemes of MtrC before (dotted black) and after (light blue) reaction (2 hours) with U(IV) citrate. Table S1. Primers used for PCR and sequencing for the ∆mtrC/omcA/mtrF construct. Table S2. Primers used for PCR and sequencing for the ∆mtrC/omcA/mtrF/mtrA construct. Table S3. First-order kinetic constants for incubations of aqueous U(VI)-dpaea or U(V)-dpaea with WT, ∆OMC and ∆MtrA S. oneidensis MR-1 strains. Table S4. Summary of the experimental results describing the reactions of U(V)-dpaea with either oxidized or reduced MtrC after 2 min, and also with the dialysis control (no protein control) obtained by dialyzing the amount of sodium dithionite used to reduce MtrC. Additionally, the reaction of U(IV)citrate with oxidized MtrC is reported. We also included the results from the reaction between reduced MtrC and solid or soluble U(VI)-dpaea. Table S5. Summary of the experimental results corresponding to Figure S7 describing the reactions of U(V)-dpaea with either oxidized or reduced MtrC over 4h. Table S6. U(VI) and U(IV) concentrations and percentage obtained by ion exchange chromatography (two left columns) and proposed derived U(IV) and U(V) concentrations and percentage (two right columns) in the reduction of solid and aqueous U(VI)-dpaea, and aqueous U(V)-dpaea by purified and reduced MtrC. Table S7. Apparent formation rates of U(IV) species upon reduction of solid U(VI)-dpaea, aqueous U(VI)-dpaea, and aqueous U(V)-dpaea by purified and reduced MtrC. Table S8. Primers used for PCR and sequencing for the ∆bfe construct. Table S9. Riboflavins profile over 72h measured by HPLC for both the WT and ∆bfe when incubated with 4mM U(VI)-dpaea.

S3
Text S1. Description of the mtrC/omcA/mtrF deletion mutant Regions flanking mtrF (SO_1780) in Shewanella oneidensis ∆omcA∆mtrC were amplified by PCR with primers mtrF_5'O/mtrF_5'I and mtrF_3'I/mtrF_3'O(DD) (Table S 1), fused by overlap extension PCR and cloned into suicide plasmid pMQS 1 . The resulting plasmid, pMQS-mtrF(DD), was introduced into Shewanella oneidensis ∆omcA∆mtrC by conjugation from E. coli strain WM3064. Colonies with single crossover plasmid insertions were selected on LB agar plates containing kanamycin, purified once on agar plates with kanamycin and resistant colonies were subsequently grown overnight in LB (containing no NaCl) without antibiotic. Double crossover mutants were selected on LB agar plates (containing no NaCl) supplemented with 10% sucrose. Sucrose resistant and kanamycin sensitive colonies were checked by colony PCR for gene deletion using primers flanking the deleted region (mtrF_FO + mtrF_RO(DD)) ( Table S1). Selected clones were purified, genomic DNA isolated, and the region containing the deleted gene was amplified by PCR and the deletion verified by Sanger sequencing (Table S1). Henceforth, for simplicity, the ∆mtrC/omcA/mtrF deletion mutant will be referred as ∆OMC. 'OMC' stands for outermembrane c-type cytochromes.

Text S2. Description of the mtrC/omcA/mtrF/mtrA deletion mutant
Regions flanking mtrA (SO_1777) in Shewanella oneidensis ∆mtrC/omcA/mtrF were amplified by PCR with primers mtrA_5'O/mtrA_5'I and mtrA_3'I/mtrA_3'O (Table S2), fused by overlap extension PCR and cloned into suicide plasmid pMQS 1 . The resulting plasmid, pMQS-mtrA, was introduced into Shewanella oneidensis ∆mtrC/omcA/mtrF by conjugation from E. coli strain WM3064. Colonies with single crossover plasmid insertions were selected on LB agar plates containing kanamycin, purified once on agar plates with kanamycin and resistant colonies were subsequently grown overnight in LB (containing no NaCl) without antibiotic. Double crossover mutants were selected on LB agar plates (containing no NaCl) S4 supplemented with 10% sucrose. Sucrose resistant and kanamycin sensitive colonies were checked by colony PCR for gene deletion using primers flanking the deleted region (mtrA_FO + mtrA_RO) (Table S2). Selected clones were purified, genomic DNA isolated, and the region containing the deleted gene was amplified by PCR and the deletion verified by Sanger sequencing (Table S2). Henceforth, for simplicity, the ∆mtrC/omcA/mtrF/mtrA deletion mutant will be referred as ∆OMC∆MtrA.

Text S3. Strain and growth conditions
In addition to WT S. oneidensis MR-1, this study also includes the newly generated mutants,

Text S8: Reduction of MtrC
In an MBraun glovebox, purified MtrC (protocol described text S8) was reduced using sodium dithionite (Na2S2O4). Sodium dithionite was added gradually, until the hemes were fully reduced. Their redox status was monitored by UV-vis spectrophotometry (UV-2501P, Shimadzu, Kyoto Japan) at a wavelength range of 500-580 nm. An anaerobic quartz cuvette (Msscientific, Berlin, Germany) was used for this purpose. Reduced MtrC is characterized by two peaks at 522 nm and 552 nm (β and α Soret absorption peaks), whereas oxidized MtrC displays a maximum at 530 nm in this spectral region. In order to remove the potential excess of sodium dithionite, which could react with U, the reduced protein was dialyzed for about 18h using dialysis cassettes (Side-A-Lizer ®, ThermoFisher Scientific, Waltham MA USA) in the glovebox. The buffer (buffer A) used was composed of 100 mM HEPES and 50 mM NaCl, and the pH was adjusted to a value of 7.5. The redox status of the hemes was probed after dialysis in order to ensure that they were still fully reduced. The concentration was measured again with the Bicinchoninic acid (BCA) protein assay. To ensure that dialysis effectively removed excess sodium dithionite from the protein solution, sodium dithionite was prepared in buffer A at the same initial concentration as that used to reduce the cytochromes. The solution was dialyzed following the method described above and post-recovery, it was reacted with U(V)-dpaea. We did not observe reduction of U(V)-dpaea, suggesting that the dialysis time was sufficiently long for the complete diffusive removal of the reducing agent. This robustness S7 of the dialysis treatment was demonstrated by the reproducibility of the rate of reduction of U(V)-dpaea by reduced MtrC.

Text S9: Reaction of MtrC with solid phase U(VI)-dpaea and aqueous U(VI)-dpaea
In the glovebox, four reactions were initiated as follows: (i) solid U(VI)-dpaea in buffer A; (ii) solid U(VI)-dpaea in buffer A with reduced MtrC; (iii) aqueous U(VI)-dpaea in buffer A; (iv) aqueous phase U(VI)-dpaea in buffer A with reduced MtrC. Reactions (i) and (iii) served to control the initial oxidation state of U. Solid U(VI)-dpaea and protein solutions were prepared at a starting concentration of 300 µM. The initial U concentration in the solution of aqueous U(VI)-dpaea was measured to be 23 µM. The reactions were initiated by mixing equal volumes of U and MtrC. Timepoints were collected by removing an aliquot from the reaction mixture, and immediately loading it onto IEC resins to separate U(VI) from U(IV), as done previously 2 .
The heme redox status was probed before and after the reaction by UV-vis spectrophotometry to evaluate how they were influenced by U. Both U(IV) and U(VI) fractions were quantified by ICP-MS.

Text S10. Description of the bfe deletion mutant
Regions flanking bfe (SO_0702) in Shewanella oneidensis MR-1 were amplified by PCR with primers bfe_5'O/bfe_5'I and bfe_3'I/bfe_3'O (Table S9), fused by overlap extension PCR and cloned into suicide plasmid pMQS as a KpnI-BamHI fragment 5 . The resulting plasmid, pMQS-bfe, was introduced into Shewanella oneidensis MR-1 by conjugation from E. coli strain WM3064. Colonies with single crossover plasmid insertions were selected on LB agar plates containing kanamycin, purified once on agar plates with kanamycin and resistant colonies were subsequently grown overnight in LB (containing no NaCl) without antibiotic. Double crossover mutants were selected on LB agar plates (containing no NaCl) supplemented with 10% sucrose. Sucrose resistant and kanamycin sensitive colonies were checked by colony PCR for gene deletion using primers flanking the deleted region (bfe_FO S8 + bfe_RO) (Table S9). Selected clones were purified, genomic DNA isolated, and the region containing the deleted gene was amplified by PCR and the deletion verified by Sanger sequencing (Table S9). The flavin secretion profile was compared to that of the WT MR-1 by analyzing aliquots of both the bfe deletion mutant (∆bfe) and the WT incubations with 4mM U(VI)-dpaea by, in triplicate, after 0h, 24h, 72h. HPLC was used to quantify the flavins following the protocol described by D. E. Ross al. 6 . Only riboflavins were detected and to a lesser extent in the ∆bfe strain over 72h, confirming that the strain is less efficient in the transport of flavins to the extracellular medium (Table S10).  In the controls with no cell, U(VI)-dpaea remains in the aqueous phase over the experimental time With MR-1, we observed that some U is found in the cell pellet, up to 70% of the total U after 72h, however it is mostly solid U(IV) in dark green (about 50%). The solid U(VI) in yellow associated with cells likely represents U(V) (20%). Indeed, upon acidification of the samples before the IEC tests U(V) disproportionates. The remaining U in the aqueous phase S14 appears as a mixture of aqueous U(VI) in light blue and little U(IV) in green. We interpret this as a mixture of 20% unreduced soluble U(VI)-dpaea and 10% reduced aqueous U(V). Hence it is reduction which is responsible for the decrease of U observed Figure 1.B.. S15 Figure S5: Initial rate of reaction for aqueous U(VI)-dpaea incubated with WT S. oneidensis MR-1 (pink), ∆OMC (blue) and ∆OMC∆MtrA (DMtrA, for short, in yellow). The data are only considered for the first 7h of the experiment. The corresponding regression equation and the R 2 coefficient are displayed using the same color code. In the reaction between oxidized MtrC and U(IV)-citrate, no change was observed in U oxidation state (Figure 4.A., Table S5), nor heme redox status (Figure 4.B.). Hence, we concluded that no electron transfer occurred from U(IV)-citrate to oxidized MtrC.