An elusive electron shuttle from a facultative anaerobe
- Harvard Medical School, United States
- Lawrence Berkeley National Laboratory, University of California, Berkeley, United States
- Southeast University, China
Figures
Figure 1 with 4 supplements
Functional analysis and structures of electron shuttles.
(A) Wild type S. oneidensis MR-1 was inoculated on the horizontal line and menC mutant was inoculated on the vertical lines (x2). Reduction of AQDS (colorless) to AHQDS (yellow) is only observed by m…
Figure 1—figure supplement 1
Purification scheme of ACNQ from spent supernatant.
(A) Optimized workflow for the purification of ACNQ. Cultures were grown for 72 hr under aerobic conditions at 30°C, 0 rpm. The cells were pelleted by centrifugation (7,000 rpm, 30 min) and the …
Figure 1—figure supplement 2
Comparison of positive mode fragmentation pattern of ACNQ isolated from MR-1 spent supernatant (bottom) and synthesized material (top).
All fragmentation matched and both metabolites eluted at the same time thus confirming the structure of the natural product.
Figure 1—figure supplement 3
Complete deuterium exchange on the LCMS, using mixtures of D2O/acetonitrile as the mobile phase, revealed that the active component (ACNQ) has three exchangeable protons.
(A) EIC of ACNQ in D2O; (A1) Observed ion for ACNQ at [M + D]+ 222.0702; (B) EIC of ACNQ in H2O;( B1) Observed ion for ACNQ at [M + H]+ 218.0450 indicated a mass difference of 3 Da, thus three …
Figure 1—figure supplement 4
Comparison of negative mode fragmentation pattern of ACNQ isolated from MR-1 spent supernatant (top) and synthesized material (bottom).
All fragmentation matched and both metabolites eluted at the same time thus confirming the structure of the natural product.
Figure 2 with 15 supplements
Production of and biosynthetic pathway for ACNQ.
(A) Relative production levels of ACNQ and menaquinones by S. oneidensis MR-1 (MR1), E. coli, V. cholerae, S. oneidensis menA, and menC mutants. The ability of E. coli to produce ACNQ is in contrast …
Figure 2—figure supplement 1
Production of ACNQ by V. cholerae V52, Lactococcus lactis lactis (ATCC 25285), E. coli K12, and Bacteroides fragilis (DSM 20481).
https://doi.org/10.7554/eLife.48054.009
Figure 2—figure supplement 2
Various bacterial strains that can recover reduction of AQDS by S. oneidensis menC::Tn10.
Each of the different bacterial strains was inoculated on the horizontal line and menC::Tn10 was inoculated on the vertical lines (x2). Reduction of AQDS (colorless) to AHQDS (yellow) is only …
Figure 2—figure supplement 3
Absolute quantification of ACNQ in MR-1 culture using 15N-ACNQ as an internal standard to determine recovery yields.
Standard curves were generated using 4-point calibration curves with biological triplicates.
Figure 2—figure supplement 4
Simulation of ACNQ production and diffusion in an agarose gel.
Calculated local concentration of ACNQ (shown in colormap) as a function of time and distance from a population of Shewanella oneidensis MR-1 that produces ACNQ. Since the diffusion of ACNQ is quite …
Figure 2—figure supplement 5
Production of ACNQ by various E. coli K12 men mutants.
Production was only observed in the WT and ΔmenA mutant, consistent with the MR-1 data.
Figure 2—figure supplement 6
Production of ACNQ by S. oneidensis ΔglmS.
Production level in ΔglmS mutant is consistent with WT when grown under the same condition (GlcNAc supplement).
Figure 2—figure supplement 7
DHNA conversion to ACNQ in the same minimal media that was used to culture MR-1.
No DHNA was observed after 24 hr of incubation at room temperature under aerobic conditions.
Figure 2—figure supplement 8
DHNA conversion to ACNQ in ammonium phosphate, pH 8.
No DHNA was observed after 24 hr of incubation at room temperature under aerobic conditions.
Figure 2—figure supplement 9
DHNA conversion to ACNQ in minimal media with amino acids as the only nitrogen source.
No DHNA was observed after 6 hr of incubation.
Figure 2—figure supplement 10
Production of ACNQ when grown with (A) NH4SO4, (B) NH4SO4 and amino acid mixture and (C) amino acid mixture as the sole nitrogen source.
ACNQ production was observed under each growth condition. [Amino acid mixture (10x mixture): L-arginine: 1.26 g/L, L-cystine: 0.24 g/L, L-histidine HCl: 0.42 g/L, L-isoleucine: 0.52 g/L, L-leucine: …
Figure 2—figure supplement 11
Absolution quantification of menaquinone (MK) in MR-1, V. cholerae V52, and E. coli K12 cultures.
Menaquinone-4 was used to generate the standard curve (4-point). The error bars in the top graph represent variance in biological replicates. (EC – E. coli K12, VC – V. cholerae V52, MR1 – S. …
Figure 2—figure supplement 12
Proposed mechanism for the non-enzymatic conversion of DHNA to ACNQ.
Initiated by oxidation of DHNA then conjugate addition of ammonium at the C3 position followed by enol formation and subsequent oxidation of the hydroquinone.
Figure 2—figure supplement 13
Menaquinone analysis of S. oneidensis menC::Tn10 (menC) and S. oneidensis MR-1 (MR-1) cell pellets when grown with 0.05, 0.5, and 1 μM of ACNQ supplemented to the media under anaerobic conditions with fumarate as the sole TEA.
No evidence of ACNQ being converted to menaquinone or being prenylated to form an amino-modified menaquinone was observed.
Figure 2—figure supplement 14
Menaquinone analysis of S. oneidensis menC::Tn10 (menC) and S. oneidensis MR-1 (MR-1) cell pellets when grown with 0.05 μM of ACNQ supplemented to the media under anaerobic conditions with TMAO as the sole TEA.
No evidence of ACNQ being converted to menaquinone or being prenylated to form an amino-modified menaquinone was observed.
Figure 2—figure supplement 15
Menaquinone analysis of S. oneidensis menC::Tn10 (menC) and S. oneidensis MR-1 (MR-1) cell pellets when grown with 0.05, 0.5, and 1 μM of ACNQ supplemented to the media under aerobic conditions.
No evidence of ACNQ being converted to menaquinone or being prenylated to form an amino-modified menaquinone was observed.
Figure 3 with 3 supplements
ACNQ acts as an extracellular electron transfer mediator for MR-1 in a bioelectrochemical system.
(A) The current production of an established MR-1 biofilm over time in the presence of different media. Oxidation current drops after the introduction of fresh medium (gray trace), but recovers …
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Figure 3—source data 1
Source Data for Figure 3C.
- https://doi.org/10.7554/eLife.48054.028
Figure 3—figure supplement 1
ACNQ acts as an electron shuttle and does not affect cytochrome c protein expression.
(A) Differential pulse voltammetry (DPV) of ACNQ in M9 media. (B) The relationship between ACNQ redox peak current and CV scan rate from non-turnover MR-1 biofilm. (C) Chronoamperometry (−0.1 VAg/AgC…
Figure 3—figure supplement 2
ACNQ acts as an extracellular electron transfer mediator for S. oneidensis Δbfe and Δmtr.
(A) Chronoamperometry of S. oneidensis Δbfe. (B) Chronoamperometry of S. oneidensis Δmtr. (C) CV scan of the S. oneidensis Δbfe. (D) CV scan of the S. oneidensis Δmtr.
Figure 3—figure supplement 3
Menaquinone analysis of S. oneidensis menC::Tn10 (menC) and S. oneidensis MR-1 (MR-1) cell pellets when grown with 0.05, 0.5, and 1 μM of ACNQ supplemented to the media under aerobic conditions.
No evidence of ACNQ being converted to menaquinone or being prenylated to form an amino-modified menaquinone was observed.
Additional files
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Supplementary file 1
(A) Acquisition of non-E. coli CGCS bacterial strains. (B) E. coli strains purchased from Coli Genetic Stock Center.
- https://doi.org/10.7554/eLife.48054.029
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Supplementary file 2
Statistical report for Figure 3C.
- https://doi.org/10.7554/eLife.48054.030
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Transparent reporting form
- https://doi.org/10.7554/eLife.48054.031
