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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.[6] 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 ydeh 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 of graphene oxide and TSB only and bacteria and TSB only.

Figure 1: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (deep blue) 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, ydeh. It shows that transformation of S. oneidensis MR-1 with ydeh still allows for reduction at a rate comparable to that of the wildtype. This figure represents the combined O.D.₆₀₀ measurements for the reduced graphene oxide absorbance and bacterial absorbance.

Figure 2: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to ydeh (black) 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 ydeh (deep blue)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 ydeh expression on bacterial growth. Figure 2 shows that bacteria expressing the ydeh gene (black) had less overall growth over the 48-hour period and showed a more variable growth curve as compared to the wildtype (dark gray) and the other expressed genes.

To get the absolute O.D.₆₀₀, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.₆₀₀ were corrected for the O.D.₆₀₀ for bacterial growth in TSB only. Figure 3 shows that after correction for bacterial growth ydeh had a comparable rate of reduction with the wildtype where it appeared to be slightly slower.

Since ydeh 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 ydeh expression.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (deep blue). 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 ydeh (black) 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 ydeh (deep blue) 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 ydeh had comparable reduction rates to wild-type MR-1 and the empty vector, pcD8. From the figure it is seen that the rate of reduction of ydeh without corrections for bacterial growth is slightly greater than that of the wildtype.

Figure 5 shows that there was no significant alteration in the growth for ydeh for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2).

Figure 6 shows that with the correction for bacterial growth, microbial reduction with ydeh with 1.0mM IPTG induction had a similar rate of reduction to the wildtype and a faster rate of reduction than pcD8 for the first 5 hours.

We carried out an induction time of 5 hours prior to reduction to allow ydeh to already have the biofilm production fully active before we began the reduction reactions expression. 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 ydeh (deep blue) for the reduction period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

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

Figure 9: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (deep 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)

The bacterial growth measured by O.D.₆₀₀ (Figure 9) shows that bacteria expressing the ydeh gene (black) still had less overall growth over the 48-hour period and more variability in its growth curve as compared to the wildtype and the other expressed genes.

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

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

Figure 12: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to ydeh (green) 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 delay in growth in ydeh (green) with 0.75mM IPTG is decreased from that seen with 1.5mM IPTG (Figure 8). However, even with the concentration of IPTG reduced to half from 1.5mM, the growth curve of ydeh still lags behind the wildtype and pcD8.

The growth curves of ydeh show stochastic patterns for each of the IPTG conditions (Figure 3,6,9,12). 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 ydeh 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, ydeh 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 ydeh 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 ydeh 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 ydeh 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.