Design overview

City of London iGEM’s Design page describes our motivation for and process of developing the project we chose to pursue for the 2023-2024 iGEM season. On this page, we delve into specifics of the catalytic decomposition of nitrous oxide by nitrous oxide reductase (N2OR) as well as our exploration of comR up-regulation in order to down-regulate comC to increase the membrane permeability of the bacteria to copper. We also explore our decisions regarding chassis and reductase selection in addition to a brief overview of plasmid construction.

Nitrous oxide reductase: the breakdown

Nitrous oxide reductase (N2OR) is known to be the only naturally occurring enzyme found in a range of denitrifying bacteria which catalyses the reduction of nitrous oxide ( N2O ) to nitrogen and water, using copper ions as its cofactor. This 2 electron reduction is shown below: (note: Zhang, L., Wüst, A., Prasser, B., Müller, C. and Einsle, O. (2019). Functional assembly of nitrous oxide reductase provides insights into copper site maturation. Proceedings of the National Academy of Sciences, 116(26), pp.12822–12827. doi:https://doi.org/10.1073/pnas.1903819116. )

N2O + 2H+ + 2 e− → N2 + H2O ΔG0′ = −339.5 kJ·mol−1

Coded by the gene nosZ, N2OR is a metalloprotein that is found in the periplasm of bacteria and has two distinct copper centres called CuA and CuZ which are both involved in the reaction mechanism. While CuA is a binuclear mixed valent [Cu1.5+:Cu1.5+] copper site which is able to accept and transfer one electron, CuZ is a tetranuclear [4Cu:2S] site which is coordinated by an asymmetric histidine heptad that binds and therefore activates the N2O for catalysis. (note: Zhang, L., Wüst, A., Prasser, B., Müller, C. and Einsle, O. (2019). Functional assembly of nitrous oxide reductase provides insights into copper site maturation. Proceedings of the National Academy of Sciences, 116(26), pp.12822–12827. doi:https://doi.org/10.1073/pnas.1903819116. ) In fact, the CuZ copper site is the site at which the 2 electron reduction takes place. (note: Bennett, S.P., Soriano-Laguna, M.J., Bradley, J.M., Svistunenko, D.A., Richardson, D.J., Gates, A.J. and Le, N.E. (2019). NosL is a dedicated copper chaperone for assembly of the CuZcenter of nitrous oxide reductase. Chemical Science, 10(19), pp.4985–4993. doi:https://doi.org/10.1039/c9sc01053j. )

For many years, nosZ has been considered the only enzyme involved in N2O reduction and such enzymes are nowadays termed “Clade I NosZ”. (note: Hein, S. and Simon, J. (2019) 'Bacterial nitrous oxide respiration: electron transport chains and copper transfer reactions,' Advances in Microbial Physiology/Advances in Microbial Physiology, pp. 137–175. https://doi.org/10.1016/bs.ampbs.2019.07.001. ) However there are in fact two distinct clades of N2OR-containing bacteria and archaea that have been identified. Clade II members act as an N2O sink. Clade I members such as α-, β- and γ-proteobacteria, are able to produce and remove N2O under optimum conditions. (note: Bennett, S.P., Soriano-Laguna, M.J., Bradley, J.M., Svistunenko, D.A., Richardson, D.J., Gates, A.J. and Le, N.E. (2019). NosL is a dedicated copper chaperone for assembly of the CuZcenter of nitrous oxide reductase. Chemical Science, 10(19), pp.4985–4993. doi:https://doi.org/10.1039/c9sc01053j. ) Since nitrous oxide reductase is periplasmic it must be transported to the periplasm from the cytoplasm. This is done via a TAT system. For Clade I organisms the system must be periplasmic. Any mutations in the TAT leader system of nitrous oxide reductase means that the protein is still in the cytoplasm where it is folded but does not contain copper. (note: Bennett, S.P., Soriano-Laguna, M.J., Bradley, J.M., Svistunenko, D.A., Richardson, D.J., Gates, A.J. and Le, N.E. (2019). NosL is a dedicated copper chaperone for assembly of the CuZcenter of nitrous oxide reductase. Chemical Science, 10(19), pp.4985–4993. doi:https://doi.org/10.1039/c9sc01053j. )

Although nosZ codes for the main structure of the enzyme of N2OR, nosZ is part of a larger cluster of genes, called the nos gene cluster, however the number of genes in the nos gene cluster is dependent on the species of denitrifying bacteria. For example, while Paracoccus denitrificans, a gram negative α-proteobacterium, has 8 genes in the nos gene cluster (nosCRZDFYLX), Pseudomonas Stutzeri has 6 main genes (nosRZDFYL). (note: Bennett, S.P. et al. (2020) 'nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase,' Microbiology, 166(10), pp. 909–917. https://doi.org/10.1099/mic.0.000955. )

The table below outlines the function of all the genes in the nos gene cluster. (note: Bennett, S.P. et al. (2020) 'nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase,' Microbiology, 166(10), pp. 909–917. https://doi.org/10.1099/mic.0.000955. )

Table 1

N.B: P. stutzeri does not code for nosX, however this species of bacteria codes for a homologue called ApbE which is a FAD+ binding flavinyl transferase that donates flavin to NosR which then activates nitrous oxide reductase. (note: Bennett, S.P. et al. (2020) 'nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase,' Microbiology, 166(10), pp. 909–917. https://doi.org/10.1099/mic.0.000955. )

Which reductase did we choose?

We picked P. stutzeri (Clade I) ZoBell ATCC 14405 as our N2OR source, due to the favourable kinetics displayed by P. stutzeri and past experimental success when using this strain.

Kinetics of the reductase in P. Stutzeri

Under anaerobic conditions, P. stutzeri TR2 (clade I N2OR) had the highest V_{max} (8.37 ± 0.81 μM/s/OD), whereas G. aurantiaca T-27 (clade II N2OR) had the lowest V_{max} (0.13 ± 0.02 μM/s/OD). (note: Wang, Z., Vishwanathan, N., Kowaliczko, S. and Ishii, S. (2023). Clarifying Microbial Nitrous Oxide Reduction under Aerobic Conditions: Tolerant, Intolerant, and Sensitive. Microbiology Spectrum, [online] 11(2). doi:https://doi.org/10.1128/spectrum.04709-22. ) In another study, P. stutzeri strain DCP-Ps1 exhibited the highest V_{max} of 4.16 ± 0.44 μmol min⁻¹ mg biomass⁻¹, a value at least five times greater than the V_{max} values of any other organism tested. As implicated from its low growth rate on N₂O, the lowest V_{max} value (0.0171 ± 0.0024 μmol min⁻¹ mg biomass⁻¹) was observed for A. dehalogenans strain 2CP-C. (note: Yoon, S., Nissen, S., Park, D., Sanford, R.A. and Löffler, F.E. (2016). Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ. Applied and Environmental Microbiology, 82(13), pp.3793–3800. doi:https://doi.org/10.1128/aem.00409-16. ) Since V_{max} reflects how fast an enzyme can catalyse reactions, it is evident that N2OR in P. stutzeri displays greater enzymatic activity according to these studies.

Past experimental success

A study proved that expression of the full gene cluster was required for the production, maturation and activity maintenance of the enzyme from P. stutzeri ZoBell in E. coli. (note: Zhang, L., Wüst, A., Prasser, B., Müller, C. and Einsle, O. (2019). Functional assembly of nitrous oxide reductase provides insights into copper site maturation. Proceedings of the National Academy of Sciences, 116(26), pp.12822–12827. doi:https://doi.org/10.1073/pnas.1903819116. ) The work was based on plasmid pPR6hE that contains all nos genes of P. stutzeri strain ZoBell. All experiments were undertaken in anaerobic conditions, though the enzyme showed some stability in an aerobic environment.

Problems regarding N2OR: how can we solve them?

Despite being the only known enzyme to break down N2O, N2OR has its setbacks. Put simply, N2O emissions would not be as much of a global issue if the denitrifying bacteria in the soil were properly functioning and reducing the N2O to N2 and H2O at a fast enough rate. In order to solve these problems we aimed to propose a 2-part solution:

1. Genetically modify E. coli to express the nos gene cluster from P. stutzeri, as well as up-regulating the gene cluster in E. coli, so that there is more enzyme available to catalyse the breakdown of nitrous oxide
2. Increase E. coli membrane permeability to copper ions (N2OR’s cofactor) to increase periplasmic copper levels, as it has been hypothesised that copper is in fact the limiting factor for N2OR activity.

Part 1

Upregulating the nos gene cluster in E. coli:

Originally, we tried researching different external factors which could potentially increase the transcription of the nos gene cluster when inserted into E. coli, instead of solely using a high copy plasmid vector to allow for elevated enzyme expression. One study we found investigated how copper levels affect the expression of the gene nosZ. (note: Black, A., Hsu, P.L., Hamonts, K.E., Clough, T.J. and Condron, L.M. (2016). Influence of copper on expression of nirS ,norB and nosZ and the transcription and activity of NIR , NOR and N 2 OR in the denitrifying soil bacteria Pseudomonas stutzeri. Microbial Biotechnology, 9(3), pp.381–388. doi:https://doi.org/10.1111/1751-7915.12352. ) This study researched into how increasing Cu concentrations could affect expression and activity of nitrite reductase (NIR), NOR and N2OR (nosZ) in P. stutzeri, however found that the expression of nosZ did not respond to differing Cu concentration. Surprisingly, the level of nosZ transcription decreased at 0.50 mM of Cu onward, suggesting that Cu could have inhibitory effects. (note: Black, A., Hsu, P.L., Hamonts, K.E., Clough, T.J. and Condron, L.M. (2016). Influence of copper on expression of nirS ,norB and nosZ and the transcription and activity of NIR , NOR and N 2 OR in the denitrifying soil bacteria Pseudomonas stutzeri. Microbial Biotechnology, 9(3), pp.381–388. doi:https://doi.org/10.1111/1751-7915.12352. ) However, this contrasts the results of a study conducted by Felgate et al. which found that transcription of nosZ of P. denitrificans was found to be upregulated at high Cu concentration, such as 2 mM. (note: Felgate, H., Giannopoulos, G., Sullivan, M.J., Gates, A.J., Clarke, T.A., Baggs, E., Rowley, G. and Richardson, D.J. (2012). The impact of copper, nitrate and carbon status on the emission of nitrous oxide by two species of bacteria with biochemically distinct denitrification pathways. Environmental Microbiology, 14(7), pp.1788–1800. doi:https://doi.org/10.1111/j.1462-2920.2012.02789.x. ) It must be noted that both studies use different species of denitrifying bacteria, however since the results between these studies were so different, we decided to simplify our approach and instead place our nos gene inserts (see contributions page) into either psB1C3 or psB4K5 plasmid vectors, as they are high copy plasmids that would allow for high levels of nosZ transcription.

Part 2

How do we know that copper is a limiting factor?

Several studies have suggested that copper is a limiting factor for the functioning of nitrous oxide reductase. The Cu acts as a cofactor and the Cu requirement for the active dimeric form of N2OR requires the bacterium to have an adequate supply of Cu. An absence of Cu in some culture studies has resulted in a rise in N2O emissions. (note: Granger, J. and Ward, B.B. (2003). Accumulation of nitrogen oxides in copper-limited cultures of denitrifying bacteria. Limnology and Oceanography, 48(1), pp.313–318. doi:https://doi.org/10.4319/lo.2003.48.1.0313. ) Figure 2 While a Cu-deficient denitrifying bacterial community can still remain viable, it is likely that they will release much higher levels of N2O. In one study, Cu-limitation had a major effect on the nosRZDFYL gene cluster, required for the functional N2O reductase system, the expression of which decreased by 8- to 25-fold in the Cu-limited cultures. This result demonstrates that nos genes are subject to regulation by Cu. (note: Sullivan, M.J., Gates, A.J., Appia-Ayme, C., Rowley, G. and Richardson, D.J. (2013). Copper control of bacterial nitrous oxide emission and its impact on vitamin B 12 -dependent metabolism. Proceedings of the National Academy of Sciences, 110(49), pp.19926–19931. doi:https://doi.org/10.1073/pnas.1314529110. ) In another study, Paracoccus denitrificans were grown under anaerobic batch culture conditions with NO3− as electron acceptor in a medium containing 13 µmol/L (Cu-H) and 0.5 µmol/L (Cu-L) copper. The key difference between the two was an accumulation of N2O in the Cu-L culture that was not observed in the Cu-H culture. This accumulation reached a maximum of around 2 mmol N⋅N2O. These results suggest that in the Cu-L cultures the catalytic capacity of the Cu-dependent Nos system is transiently exceeded by the rate of the reactions that generate nitrous oxide (i.e., NO3−, NO2−, and NO reduction) and is consistent with other observations that Cu limitation can lead to nitrous oxide release by denitrifying bacteria. A study focusing on the effects of inorganic versus organic copper on denitrification in agricultural soil also proved that activity of N2OR is lowered under conditions of Cu limitation. (note: Wang, Q., Burger, M., Doane, T.A., Horwath, W.R., Castillo, A.R. and Mitloehner, F.M. (2013). Effects of inorganicv. organic copper on denitrification in agricultural soil. Advances in Animal Biosciences, 4(s1), pp.42–49. doi:https://doi.org/10.1017/s2040470013000307. ) Another study looking at treatment of sediments with different copper concentrations revealed that much lower Cu concentrations inhibited the N2O reduction step in denitrification. (note: Granger, J. and Ward, B.B. (2003). Accumulation of nitrogen oxides in copper-limited cultures of denitrifying bacteria. Limnology and Oceanography, 48(1), pp.313–318. doi:https://doi.org/10.4319/lo.2003.48.1.0313. ) Figure 2

Increase E.coli membrane permeability to copper ions: ComC/R

When researching methods to increase the availability of copper in the cell, we came across a study which used a lux-based biosensor to monitor intracellular copper levels in situ, and a transposon mutagenesis approach to identify genes involved in copper entry into cells. They isolated mutant strains (called low-glowers) with reduced luminescence when exposed to copper, indicating lower intracellular copper levels. One of these mutants had a transposon insertion in the comR gene, which encodes the ComR protein which is a TetR-like transcription regulator. comR did not regulate its own expression, but was required for copper-induction of the neighbouring comC gene, as shown by real-time quantitative PCR and with a promoter-lux fusion. The purified ComR regulator bound to the promoter region of the comC gene in vitro and was released by copper. By membrane fractionation, ComC was shown to be present in the outer membrane. Cells lacking ComC showed increased copper accumulation in the periplasm and cytoplasm, evident due to the activation of CusRS (periplasmic) and CueR (cytoplasmic).Thus, ComC is an outer membrane protein which lowers the permeability of the outer membrane to copper. The expression of comC is controlled by ComR, a novel, TetR-like copper-responsive repressor. While comC expression is modulated by RpoE, CRP and ComR, it was found that ComR was the most effective in regulation. (note: Mermod, M., Magnani, D., Solioz, M. and Stoyanov, J.V. (2011). The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli. BioMetals, 25(1), pp.33–43. doi:https://doi.org/10.1007/s10534-011-9510-x. )

What are we using this for?

Since comR is a repressor of comC, upregulation of comR downregulates comC hence increasing the permeability of outer membrane to copper and increasing cofactor concentration in the periplasmic space - thus more cofactor would be available for the reductase to function. Studies have proven that the maturation of the enzyme, including the metallation of the CuA site occurs in the periplasm, meaning that increasing copper availability in the periplasm may increase the rate of maturation, or potentially the rate of enzymatic activity. (note: Wunsch, P., Herb, M., Wieland, H., Schiek, U.M. and Zumft, W.G. (2003). Requirements for Cu A and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier Pseudomonas putida. Journal of Bacteriology, 185(3), pp.887–896. doi:https://doi.org/10.1128/jb.185.3.887-896.2003. ) (note: Zumft, W.G. (2005). Biogenesis of the Bacterial Respiratory CuA, Cu-S Enzyme Nitrous Oxide Reductase. Microbial physiology, 10(2-4), pp.154–166. doi:https://doi.org/10.1159/000091562. ) Since studies have shown that copper is the limiting factor (see above), we hypothesise that by upregulating ComR and increasing the availability of copper in the periplasm, N2OR can function at a faster rate. Given the short nature of the protein and the simplicity of the system, introduction of ComR is a sensible and low-cost option for increasing periplasmic copper levels.

Chassis selection

Our team used DH5α competent cells derived from E. coli K-12 strain for our wet lab. DH5α is a commonly used strain due to its high transformation efficiency and high insert stability, which makes it ideal for transformations. (note: Chan WT, Verma CS, Lane DP, Gan SK. A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci Rep. 2013 Dec 12;33(6):e00086. doi: https://doi.org/10.1042/BSR20130098. PMID: 24229075; PMCID: PMC3860579. ) (note: Kostylev M, Otwell AE, Richardson RE, Suzuki Y. Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies. PLoS One. 2015 Sep 8;10(9):e0137466. doi: https://doi.org/10.1371/journal.pone.0137466. PMID: 26348330; PMCID: PMC4562628. )

Plasmid construction

In order to enable E. coli to break down nitrous oxide we designed two plasmid inserts each containing parts of the nos gene cluster. We also designed two inserts used in our wet lab: one to synthesise ComR and the other as a reporter to measure the efficacy of ComR in downregulating ComC. As a high school team without regular access to a lab, we only had one week to perform our wet lab work. We therefore decided to focus on the ComR/GFP inserts which we were able to successfully construct in the time we had. The nos inserts were not constructed, but we have published their designs for future iGEM teams to use. For further details on our parts please see the contribution page and the diagrams below.