Part:BBa_K4849023
E. coli NucA/NuiA kill switch
Description
We designed, built, and tested a kill switch in Escherichia coli based on NucA nuclease (toxin) and its inhibitor NuiA (antitoxin) from cyanobacterium Anabaena sp. PCC 7120. We synthesized the whole expression cassette containing the nuiA gene under control of the E. coli zinc-inducible promoter PzntA and TrrnB double terminator, and the nucA gene under control of constitutive Anderson promoter (BBa_J23114) and a TECK120029600 terminator, which is a strong E. coli terminator with termination efficiency (TE) values of >99.5% (Gale et al., 2021). We added overhangs to the synthesized composite part suited for CyanoGate (Vasudevan et al., 2019) assembly, and ligated it into a Level T acceptor vector pSEVA421-T (https://www.addgene.org/119555/) by BbsI assembly (Gale et al., 2019).
NucA is a non-specific DNA/RNA nuclease that can cut single-stranded and double-stranded DNA and RNA (Ghosh et al., 2005). Nucleases of this type are present in several bacterial species and are believed to have evolved to serve for nutritional purposes and sometimes as bacteriocides (Meiss et al., 1998). NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, the optimal concentration for these being around 5 mM (Meiss et al., 1998). NucA activity was shown to decrease with increasing concentration of monovalent salt (Meiss et al., 1998). NucA contains a ββα metal finger motif and a hydrated divalent metal ion at the active site (Ghosh et al., 2005). Ghosh et al. (2005) proposed that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state. Since our intention was to use the NucA nuclease as a toxin in the kill switch, we had to optimize it for this purpose. First, in order to achieve intracellular localization of the nuclease and to make sure it cuts cellular DNA upon kill switch induction, we shortened the coding sequence of nucA by 69 nucleotides to remove the signal peptide mediating the export of nuclease to the periplasm (Muro-Pastor et al., 1992). Second, it was previously observed that for the efficient cloning of the nuclease-based kill switch degradation tags had to be introduced to curtail the cellular half-life of the nuclease (Čelešnik et al., 2016). We added the T1 degradation tag (RPAANDENYAAAV) (Huang et al., 2010) to the C-terminal of the NucA nuclease (Huang et al., 2010).
NuiA is a specific inhibitor of the NucA nuclease (Ghosh et al., 2005). NucA and NuiA form a 1:1 complex leading to complete inhibition of NucA (Meiss et al., 1998) (Figure 1). The inhibition involves an unusual divalent metal ion bridge between the nuclease with its inhibitor (Ghosh et al., 2007). The C-terminal Thr-135 hydroxyl oxygen in NuiA interacts directly with the catalytic Mg2+ in the nuclease active site, while Glu-24 in NuiA extends into the active site, mimicking the charge of a scissile phosphate (Ghosh et al., 2007). NuiA residues Asp-75 and Trp-76 contribute to the strength and specificity of the interaction (Ghosh et al., 2007).
Figure 1. NucA-NuiA complex. Taken from Ghosh et al. (2007).
In our design, NuiA is expressed from a zinc-inducible E. coli promoter PzntA (Figure 2), which regulates the expression of the P-type ATPase ZntA, which is a major component of the zinc homeostasis system in E. coli and is associated with the translocation of zinc from the cytoplasm to the periplasm (Brocklehurst et al., 1999; Outten and O’Halloran, 2001). PzntA is bound by ZntR transcription factor dimer. The spacing between the -35 and -10 element of PzntA is suboptimal at 20 bp, but Zn2+ causes a conformational change in ZntR dimer, underwinding the DNA in the promoter which brings the spacing between the -35 and -10 hexamer to optimal spacing, allowing RNA Polymerase (RNAP) to bind, thus upregulating gene expression (Brocklehurst et al., 1999; Outten and O’Halloran, 2001) (Figure 3). Ingram (2021) has created a library of mutated PzntA promoters and found that PzntA(-11_-7>TGACA) mutant (Figure 4) showed expression with no significant difference to wild-type PzntA but only when induced by 400 µM ZnSO4. Since our intention was to express NuiA antitoxin in zinc-rich environment and minimize its expression in zinc-low environment, we decided to use this mutated PzntA promoter in our kill switch.
Figure 2. Sequence of PzntA promoter. Bold indicates either -35 or -10 element. Underline indicates ZntR binding site, space indicates separation of the 11-11 bp palindromic repeat. Green indicates +1 transcription start site. Taken from Ingram (2021).
Figure 3. Diagram of ZntR Regulation (a) ZntR binds to 11-11 bp palindromic repeats in PzntA, repressing RNAP binding. (b) Excess Zn2+ binds to ZntR and induces a conformation change in the transcription factor, leading to underwinding of the DNA in the promoter, which allows RNAP binding, upregulating gene expression. Taken from Ingram (2021).
Figure 4. Sequence of PzntA(-11_-7>TGACA) mutant promoter. Bold indicates either -35 or -10 element. Red indicates mutation. Underline indicates ZntR binding site. Green indicates +1 transcription start site. Taken from Ingram (2021).
References
Brocklehurst, K. R., Hobman, J. L., Lawley, B., Blank, L., Marshall, S. J., Brown, N. L. and Morby, A. P. (1999) ‘ZntR is a Zn(II)-responsive MerR-like transcriptional regulator of zntA in Escherichia coli’, Molecular Microbiology, 31(3), pp. 893–902.
Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A., Mustar, J., Vidmar, V. and Dolinar, M., 2016. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5(4), pp.519-528.
Gale, G.A., Osorio, A.A.S., Puzorjov, A., Wang, B. and McCormick, A.J., 2019. Genetic modification of cyanobacteria by conjugation using the CyanoGate modular cloning toolkit. JoVE (Journal of Visualized Experiments), (152), p.e60451.
Gale, G.A., Wang, B. and McCormick, A.J., 2021. Evaluation and comparison of the efficiency of transcription terminators in different cyanobacterial species. Frontiers in Microbiology, 11, p.624011.
Ghosh, M., Meiss, G., Pingoud, A., London, R.E. and Pedersen, L.C., 2005. Structural insights into the mechanism of nuclease A, a ββα metal nuclease from Anabaena. Journal of Biological Chemistry, 280(30), pp.27990-27997.
Ghosh, M., Meiss, G., Pingoud, A.M., London, R.E. and Pedersen, L.C., 2007. The nuclease a-inhibitor complex is characterized by a novel metal ion bridge. Journal of Biological Chemistry, 282(8), pp.5682-5690.
Huang, H.H., Camsund, D., Lindblad, P. and Heidorn, T., 2010. Design and characterization of molecular tools for a synthetic biology approach towards developing cyanobacterial biotechnology. Nucleic acids research, 38(8), pp.2577-2593.
Meiss, G., Franke, I., Gimadutdinow, O., Urbanke, C. and Pingoud, A., 1998. Biochemical characterization of Anabaena sp. strain PCC 7120 non‐specific nuclease NucA and its inhibitor NuiA. European journal of biochemistry, 251(3), pp.924-934.
Muro‐Pastor, A.M., Flores, E., Herrero, A. and Wolk, C.P., 1992. Identification, genetic analysis and characterization of a sugar‐non‐specific nuclease from the cyanobacterium Anabaena sp. PCC 7120. Molecular microbiology, 6(20), pp.3021-3030.
Outten, C. E. and O’Halloran, T. V (2001) ‘Femtomolar sensitivity of metalloregulatory proteins controlling zinc homeostasis.’, Science, 292, pp. 2488–2492.
Ingram, J., 2021. Utilizing the zinc homeostasis system of Escherichia coli as a novel inducible promoter system (Doctoral dissertation, University of Nottingham).
Vasudevan, R., Gale, G.A., Schiavon, A.A., Puzorjov, A., Malin, J., Gillespie, M.D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C.J. and Lea-Smith, D.J., 2019. CyanoGate: a modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), pp.39-55.
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