Difference between revisions of "Part:BBa K3724009"

Latest revision as of 04:33, 17 December 2021

Tetraheme cytochrome c protein/ quinol dehydrogenase cymA

cyma in Shewanella oneidensis MR-1 encodes the tetraheme cytochrome c protein cymA.

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]. The DET pathway involves c-type cytochromes within the bacterial membrane including the tetraheme cytochrome c protein cymA. cymA is an inner membrane protein that is essential for the reduction of extracellular materials. It accepts electrons from the cytoplasmic menaquinone pool where these electrons are then shuttled to decaheme c-type cytochromes in the outer membrane or to electron shuttles in the periplasm for extracellular reduction [4]. Studies of the role of cymA in reduction have shown that cymA has many different terminal reductases making it a key component in the electron transfer pathway for the reduction of many different extracellular materials [2]. However, there has been some contention about whether cymA is critical for the reduction of GO or not. Recent studies have shown that cymA mutant strains of S. oneidensis MR-1 have no GO reducing ability deeming cymA an essential protein in the microbial reduction of GO [5]. With this information, we sought to overexpress the cyma gene in S. oneidensis MR-1 to increase the rate of reduction of GO. It was thought that the higher the density of the electron transport protein, cymA, in the inner membrane, the more electrons get shuttled to the outer membrane and to graphene oxide.

We therefore, synthesized the cyma gene, optimized for S. oneidensis MR-1, and inserted it into the kanamycin resistant vector pcD8 under the control of an IPTG-inducible promoter (Keitz lab, University of Texas Austin) [6].

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 [7]. 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 cymA 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 bacteria and TSB only.

Figure 1: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to cyma (yellow) 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, cyma. It shows that transformation of S. oneidensis MR-1 with cyma 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 cyma (light blue) 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 cyma (yellow)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 cyma expression on bacterial growth. Figure 2 shows that bacteria expressing the cyma gene (light blue) had a similar growth curve to the wildtype (dark gray) and the empty vector pcD8. This indicated that the expression of cyma in our transformed strain was likely not toxic to the cells.

To get the absolute O.D.₆₀₀, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.₆₀₀ values were corrected for the O.D.₆₀₀ for bacterial growth in TSB only. Figure 3 shows that after correction for bacterial growth cyma had a slightly slower rate of reduction as compared to wildtype but had a comparable rate of reduction with the empty vector, pcD8.

Since ydeh (BBa_K3724008), ribf (BBa_K3724010) and oprf (BBa_K3724011) 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 gene expression for all of our transformed strains including cyma.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to cyma (yellow). 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 cyma (light blue) 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 cyma (yellow) 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 cyma had generally comparable reduction rates to wild-type MR-1 and the empty vector, pcD8 where its rate is slightly lower.

Figure 5 shows that there was no significant alteration in the growth for cyma for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2) as was expected. The curve showed similar bacterial growth as compared to the wildtype and the empty vector, pcD8.

Figure 6 shows that with the correction for bacterial growth, microbial reduction with cyma for 1.0mM IPTG induction had slightly lower rates of reduction than the wildtype and the empty vector, pcD8.

We carried out an induction time of 5 hours prior to reduction to allow cyma to already have the inner membrane protein production fully active before we began the reduction reactions. This was carried out with 1.5mM IPTG as IPTG concentration had no effect on cyma growth curves (Figure 2,5). The reductions were carried out 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 cyma (yellow) 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 cyma shows a comparable rate of reduction to that of the wildtype without any corrections to the O.D.₆₀₀ measurements for bacterial growth. It also shows a slightly slower 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 cyma (brown) 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 bacterial growth for the transformed strain expressing the cyma gene (brown) is still similar to the wildtype and the empty vector, pcD8, over the 48-hour period, with the 5 hour induction. This indicates that expression of cyma overtime is not likely to be toxic to the cells.

Figure 9: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to cyma (yellow) 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 cyma had a rate of reduction comparable to that of wild type and the empty vector, pcD8 where the reduction rate was slightly slower.

The growth curves of cyma show similar bacterial growth compared to the wildtype and pcD8 for each of the IPTG conditions (Figure 2,5,8). The rates of reduction of our transformed strain with cyma were also similar to the wildtype and pcD8, only lagging slightly behind.

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 the maximum rate of reduction for cyma varied across the different IPTG induction conditions. From Figure 13, the greatest maximum rate, 8.168 hr^-1, occurs at the 1.5mM IPTG 0 hour induction condition and compares to the max rate of wild type (8.936 hr^-1) while it is greater than the max rate of the empty vector, pcD8 (7.66 hr^-1) at that same condition.

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 which is the measure of reduction of graphene oxide in these experiments.

Raman spectroscopy

Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by cyma after the 48 hour period. We primarily relied on the D/G ratio, which is the intensity of the D peak divided by the intensity of the G peak. The D peak is associated with double-bonded carbon, and the G peak is associated with single-bonded carbon.

We used chemically reduced GO as our positive control as chemical reduction with ascorbic acid results in a much greater degree of reduction than seen in microbial reduction. We also had two negative controls: GO only and GO and TSB media only.

After reduction, we compared the D/G ratios to determine how well our transformed strain had done in reducing the number of Sp² carbons.

Figure 14: D/G ratio for rGO produced under microbial conditions and chemical conditions.

As Figure 14 shows, the chemically reduced GO was by far the most reduced, with an average D/G ratio of around 1.2. The microbial reduction with cyma resulted in a D/G ratio of 0.99 which is similar to the D/G ratio of wildtype. This demonstrates that our part worked as intended and was able to reduce the GO to expected levels of reduction during the reduction period.

To investigate the degree of reduction at an earlier time point, we took Raman spectra for cyma at the 12 hour time point.

Figure 15: D/G ratio for rGO produced under microbial conditions after 12 hours

As figure 15 shows, not all of the bacteria reduced the GO at the same rate. After 12 hours, reduced GO with cyma was far more reduced than the pcD8 and wildtype strains. This shows that this modified bacteria strain is faster at reducing the GO than either the wild type strain or the strain with the empty expression vector.

Conclusion

We were able to successfully transform the tetraheme cytochrome c protein, cymA, into S. oneidensis MR-1. This transformed strain reduced the graphene oxide to levels expected with microbial reduction for S. oneidensis MR-1. It also had the highest degree of reduction at 12 hours. This indicates that expression of cyma in S. oneidensis MR-1 ,can decrease the time it takes for graphene oxide to be reduced to expected reduction levels for microbial reduction.

Sequence and Features

References

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[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. Nano Energy 2018, 50, 639–648.
[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] Jiao, Y.; Qian, F.; Li, Y.; Wang, G.; Saltikov, C. W.; Gralnick, J. A. Deciphering the Electron Transport Pathway for Graphene Oxide Reduction by Shewanella Oneidensis MR-1. Journal of Bacteriology 2011, 193, 3662–3665.
[6] 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.
[7]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.