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Riboflavin biosynthesis protein RibF

Two extracellular electron transfer pathways have been identified in the reduction of graphene oxide (GO) by Shewanella oneidensis. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material. It has been proposed that flavins may act as electron shuttles in the reduction of extracellular material by Shewanella oneidensis. These flavins can exit the cell as Flavin adenine dinucleotide (FAD) where they are then reconverted to riboflavin. RibF converts riboflavin to FAD to be shuttled out. Increasing expression of RibF should increase the conversion of riboflavin to FAD and would therefore increase the electron donors available for reduction.

Usage and Biology

Shewanella oneidensis MR-1 are gram-negative bacteria at the center of studies of microbial reduction due to their ability to transfer electrons extracellularly to reduce materials such as graphene oxide (GO)[1]. Such characteristics have made S. oneidensis MR-1 an organism of interest in microbial fuel cells for bioelectricity generation and potential applications in bioremediation[2]. Two extracellular electron transfer pathways have been identified in the reduction of GO by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material[3]. It has been proposed that flavins may act as electron shuttles in the reduction of extracellular material by S. oneidensis MR-1.[4] These flavins can exit the cytoplasm as flavin adenine dinucleotide (FAD) where they are then converted to riboflavin in the periplasm or in the extracellular space. ribf codes for the riboflavin biosynthesis protein which catalyzes the phosphorylation of riboflavin to flavin mononucleotide (FMN) then adenylation of FMN to FAD.[5] This FAD can then be transported into the periplasm by inner membrane flavin transporters. From the periplasm, FAD is converted to FMN where it is shuttled out of the cell then converted to riboflavin. Riboflavin has been seen to be the dominant flavin involved in reduction of extracellular material where it acts as a reducing electron shuttle eventually transferring its electrons to the terminal electron acceptor. Riboflavin has also been shown to be a key component in the transfer of electrons from biofilm in contact with electrodes as the terminal electron acceptor.[6] It was thought that increasing expression of ribf would increase the conversion of riboflavin to FAD and would therefore increase the amount of FAD shuttled into the periplasm to be converted to riboflavin for a faster rate of reduction.

We therefore synthesized the ribf gene from the S. oneidensis MR-1 genome and inserted it into the kanamycin resistant vector pcD8 under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin)[7].

Results

Optical density measurements (O.D.₆₀₀)

Previous studies have shown that the magnitude of reduction of graphene oxide can be measured using optical density measurements at a wavelength of 600 nm. It has also been shown that the presence of graphene oxide has no significant effect on the growth rate of S. oneidensis MR-1.[8] So, we utilized this technique to measure and compare the magnitude of reduction with our different transformed strains and the wildtype.

Expression of our genes in our IPTG-inducible vector, pcD8, was initially induced with 1.5mM IPTG. Immediately following induction, reduction of graphene oxide was carried out under constant shaking at 200 rpm, and the O.D.₆₀₀ was measured every hour for 48 hours for TSB only, graphene oxide only, graphene oxide and TSB only, and graphene oxide and TSB each with ribf and the wildtype. Here, the TSB only , graphene oxide only, and graphene oxide and TSB only are our negative controls, and wild type MR-1 is the positive control to which all our transformed strains are compared. The O.D.₆₀₀ measurements obtained from these experiments reflect both bacterial absorbance as well as reduced graphene oxide absorbance. So, the absolute O.D.₆₀₀ due to reduced graphene oxide is obtained by correcting for O.D.₆₀₀ bacteria and TSB only.

Figure 1: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (brown) for the reduction period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 1 shows the microbial reduction of graphene oxide with the transformed strain, ribf. It represents the combined O.D.₆₀₀ measurements for the reduced graphene oxide absorbance and bacterial absorbance. The figure shows that transformation of S. oneidensis MR-1 with ribf still allows for reduction following a lag from the initiation of reduction as compared to the wildtype and pcD8. However, at its steepest point, the O.D.₆₀₀ curve, which signifies the increase in reduction, is comparable to that of the wildtype and the empty vector, pcD8.

Figure 2: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to ribf (deep purple) over a 48-hour period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 3: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (brown)adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, time zero reflects the start of induction with 1.5mM IPTG.

The bacterial growth over the 48-hour period was measured by means of O.D.₆₀₀ to determine the impact of ribf expression on bacterial growth. Figure 2 shows that bacteria expressing the ribf gene (deep purple) had a delay in growth for the first 20 hours as compared to the wildtype (dark gray) and the other expressed genes. Beyond that point, the bacterial growth evens out to be about the same as the wildtype and pcD8.

To get the absolute O.D.₆₀₀, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.₆₀₀ was corrected for the O.D.₆₀₀ for bacterial growth in TSB only. Figure 3 shows that after correction for bacterial growth ribf , at the steepest point on its curve, had a comparable rate of reduction with the wildtype and pcD8. The lag in reduction with ribf may be due to its initial delay in growth when compared to the other strains.

Since ribf showed slower growth than wild-type MR-1 (Figure 2), we hypothesized that the induced gene may have been slightly toxic to the cells. We then observed the growth curve when the concentration of IPTG was decreased to 1.0mM to reduce the amount of ribf expression.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (brown). Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 5: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to ribf (deep purple) over a 48-hour period. Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 6: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (brown) adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 4 shows that with 1.0mM IPTG induction, microbial reduction of graphene oxide with the transformed strain ribf still had the initial lag in reduction but after the 5 hour mark, its reduction curve was comparable to wild-type MR-1 and the empty vector, pcD8. From the figure the slope of the reduction curve of ribf after 5 hours looks as steep as the slope for the curve of the wildtype without corrections for bacterial growth.

Figure 5 shows an increase in the delay in the growth for ribf for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2). However, with 1.0mM IPTG the exponential growth following the delay is faster than that of 1.5mM IPTG induction.

Figure 6 shows that with the correction for bacterial growth, microbial reduction with ribf with 1.0mM IPTG induction at the steepest region of its curve had a greater rate of reduction as compared to the wildtype and pcD8.

We carried out an induction time of 5 hours prior to reduction to allow ribf to already have the its enhanced electron transport pathway fully active before we began the reduction reaction. This was carried out with either 1.5mM or 0.75mM of IPTG under the same conditions of shaking at 200 rpm.

Figure 7: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (light blue) for the reduction period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

From Figure 7, the microbial reduction of graphene oxide with the transformed strain ribf shows a comparable rate of reduction to that of the wildtype after its initial delay without any corrections to the O.D.₆₀₀ measurements for bacterial growth. It also shows a comparable rate of reduction to that of pcD8.

Figure 8: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (red) compared to ribf (deep purple) for the 48-hour period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

The bacterial growth measured by O.D.₆₀₀ in Figure 8 shows that bacteria expressing the ribf gene (deep purple) still had the initial delay with the 5hr induction, however it is greater in magnitude as compared to the delay in the 1.5mM IPTG and 1.0mM IPTG 0hr induction (Figure 2,5). It also has a greater exponential increase in growth and exceeds the growth of the wildtype and pcD8 after 24 hours.

Figure 9: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (light blue) adjusting for the O.D.600 values due to bacterial growth. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

After corrections for bacterial growth, Figure 9 shows that ribf has a faster rate of microbial reduction at the steepest region of its curve as compared to the curves for wildtype and pcD8.

Figure 10: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (light blue) for the reduction period. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 10 shows that the microbial reduction of graphene oxide with the transformed strain ribf at 0.75mM IPTG 5 hr induction has comparable rate of reduction with that of the wildtype and pcD8 following its initial delay in reduction without any corrections to the O.D.₆₀₀ measurements for bacterial growth.

Figure 11: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (red) compared to ribf (deep purple) for the 48-hour period. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 11, shows that the delay in growth in ribf (deep purple) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG for 5hr induction (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of ribf still lags behind the wildtype and pcD8.

Figure 12: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ribf (light blue) adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, reduction was initiated following a 5 hour induction with 0.75mM IPTG (5hr induction).

Figure 12, shows that the microbial reduction of graphene oxide with the transformed strain ribf at 0.75mM IPTG 5 hr induction has a comparable rate of reduction with that of the wildtype and pcD8 following its initial delay in reduction after adjusting for the bacterial growth.

The growth curves of ribf show stochastic patterns for each of the IPTG conditions (Figure 2,5,8,11). It is thought that such patterns are observed due to the production of biofilm where increased biofilm production may cause bacteria to adhere to the sides of the plate wells, reducing the amount of ribf cells in the TSB for O.D.₆₀₀ measurements.

The slopes at the steepest point on the curves were obtained for each induction condition to give the maximum reduction rate measured during the reduction period, and this was compared to that of wild-type MR-1.

Figure 13: Maximum rates of the bacterial reduction curves adjusted for bacterial growth at the IPTG induction conditions of 1.5mM and 1.0 mM IPTG with 0hr induction and 1.5mM and 0.75mM IPTG with 5 hour induction.

The results show that after a 5hr induction with 1.5mM IPTG as well as 0.75mM IPTG, ribf has a faster rate of reduction as compared to wild-type MR-1 (Figure 9,12). Figure 13 shows that the maximum rate obtained for ribf at these conditions were 8.836 hr^-1 and 8.468 hr^-1, respectively, whereas the wildtype had a rate of 7.732 hr^-1. There are comparable reduction rates with 1.5mM and 1.0mM IPTG for reduction immediately following induction (Figure 3,6). The reduction with ribf appears to be more similar to wild-type MR-1 at the lower IPTG concentration, 1.0mM (Figure 7).

The negative controls (TSB only, GO only, and GO and TSB only) show an insignificant change in O.D.₆₀₀ over time indicating that the bacteria are responsible for the increase in O.D.₆₀₀, that is, reduction.

Raman spectroscopy

Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by ribf over the 48 hour period. Sequence and Features

References

[1] Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Microbial Reduction of Graphene Oxide by Shewanella. Nano Research 2011, 4, 563–570.
[2] Schwalb, C.; Chapman, S. K.; Reid, G. A. The Tetraheme Cytochrome Cyma Is Required for Anaerobic Respiration with Dimethyl Sulfoxide and Nitrite in Shewanella Oneidensis. Biochemistry 2003, 42, 9491–9497.
[3] Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella Oneidensis-Reduced Graphene Oxide Biohybrid with Enhanced Biosynthesis and Transport of Flavins Enabled a Highest Bioelectricity Output in Microbial Fuel Cells.
[4] Kouzuma, A.; Kasai, T.; Hirose, A.; Watanabe, K. Catabolic and Regulatory Systems in Shewanella Oneidensis MR-1 Involved in Electricity Generation in Microbial Fuel Cells. Frontiers in Microbiology 2015, 6.
[5] UniProt Consortium European Bioinformatics Institute Protein Information Resource SIB Swiss Institute of Bioinformatics. Riboflavin biosynthesis protein.
[6] Marsili, E.; Baron, D. B.; Shikhare, I. D.; Coursolle, D.; Gralnick, J. A.; Bond, D. R. Shewanella Secretes Flavins That Mediate Extracellular Electron Transfer. Proceedings of the National Academy of Sciences 2008, 105, 3968–3973.
[7] Dundas, C. M.; Walker, D. J. F.; Keitz, B. K. Tuning Extracellular Electron Transfer by Shewanella Oneidensis Using Transcriptional Logic Gates. ACS Synthetic Biology 2020, 9, 2301–2315.
[8] Lehner, B. A.; Janssen, V. A.; Spiesz, E. M.; Benz, D.; Brouns, S. J.; Meyer, A. S.; van der Zant, H. S. Creation of Conductive Graphene Materials by Bacterial Reduction Using Shewanella Oneidensis. ChemistryOpen 2019, 8, 888–895.