Utilizing the zinc homeostasis system of Escherichia coli as a novel inducible promoter system

Ingram, Joseph (2021) Utilizing the zinc homeostasis system of Escherichia coli as a novel inducible promoter system. PhD thesis, University of Nottingham.

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Abstract

Zn(ii) is an essential post-transition metal found in all life, however, at high concentrations Zn(ii) can become toxic, causing oxidative stress and inactivation of essential enzymes by replacing other metals in catalytic centres of proteins. Escherichia coli cells can control their internal zinc to a femtomolar concentration, equivalent to one to two free Zn(ii) ions per cell. This tightly controlled zinc homeostasis system is regulated by two transcription factors, ZntR and Zur, which regulated the expression of the major zinc export and zinc acquisition genes. This zinc homeostasis system of E. coli has the potential to be developed as a novel inducible promoter system.

The ZntR regulated promoter (PzntA) demonstrated a strong correlation between increasing zinc concentrations and promoter induction, as well as showing a comparable induction level to the IPTG inducible promoter, Ptrc. Six Zur regulated promoters showed a variation of induction level when induced with the zinc chelator TPEN (Pc1265>PykgM>PznuA>PznuCB>PpliG>PzinT). Promoters Pc1265 and PykgM demonstrated more desirable characteristics than the IPTG inducible Ptrc; showing lower basal expression, higher induced expression and higher fold induction. Both ZntR and Zur regulated promoters show strong potential to be utilized as either a zinc or TPEN inducible promoter for use in both research and biotechnology.

Flow cytometry data of E. coli zntA:rfp in combination with Bayesian analysis demonstrated that E. coli mounts a heterogenous gene expression of zntA. This analysis further showed that with increasing zinc concentration, E. coli shifts the heterogenous zntA expression, reducing low level gene expression and increasing high level gene expression.

In silico analysis of the uncharacterised Zur regulated C1265-7 suggested that C1265 is a TonB-dependent receptor which translocates zinc, C1266 may be involved in bacteria-host adhesion, and C1276 is likely a COG0523 protein. In vitro analysis did not suggest a phenotype and it is likely that the true phonotype of C1265-7 can only be observed in an infection model.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hobman, Jon
Hill, Phil
Stekel, Dov
Keywords: Zinc homeostasis system, Escherichia coli
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 65080
Depositing User: Ingram, Joseph
Date Deposited: 04 Aug 2021 04:41
Last Modified: 04 Aug 2021 04:41
URI: http://eprints.nottingham.ac.uk/id/eprint/65080

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