عنوان

Zinc on the move :

Number

TLets720499

Title Proper

Zinc on the move :

General Material Designation

[Thesis]

First Statement of Responsibility

Ksibe, Amira Z.

Title Proper by Another Author

insights towards understanding zinc homeostasis in the open ocean cyanobacterium Synechococcus sp. WH8102

Name of Publisher, Distributor, etc.

University of Warwick

Date of Publication, Distribution, etc.

2017

Dissertation or thesis details and type of degree

Ph.D.

Body granting the degree

University of Warwick

Text preceding or following the note

2017

Text of Note

Since the discovery of zinc as an essential element for living organisms including humans, animals, plants, fungi and bacteria, much has been learnt about zinc in biological systems, ranging from its effects at the whole organism level, including the uptake/efflux of zinc, identification of important zinc-regulator proteins, down to structural, thermodynamic, and kinetic details of zinc-protein interactions. One of the most exciting fields to work on, currently, is understanding the importance of zinc "on the move" - in particular the "acquisition and homeostasis" of this micronutrient element by the organisms. In this wider context, zinc homeostasis by open ocean cyanobacteria, which occupy variable ecosystems with an erratic nutrient supply, has become of recent interest. In an attempt to resolve how open ocean cyanobacteria persist in regions where the zinc concentration is thought to be limited, we hypothesised that the cyanobacterium Synechococcus sp. WH8102 might provide an extracellular zinc scavenger for the acquisition of this essential element. Therefore, the current study developed a method to isolate and purify the putative biogenic zinc-binding ligand (zincophore) using polystyrene-divinylbenzene resin and liquid chromatographies. However, it seems likely Synechococcus sp. WH8102 produce ligands that can bind to zinc only under zinc-depleted conditions. Then, we performed zinc limitation and repletion experiments on axenic cultures of marine Synechococcus sp. WH8102, and data determined that this strain mounted an adaptive response for zinc under depleted and replete conditions, resulting in the induction and/or repression of a number of proteins. As a homologue to the Fur family, Zur from Synechococcus sp. WH8102 was sub-cloned and purified in the absence of zinc ions in the cultures. Synechococcus sp. WH8102 Zur protein at neutral pH (~pH 7.8), in mass spectrometry, presented as a mixture of species including a monomer with two zinc ions bound Zn2Zur, a dimer with four zinc ions bound, and another well folded Zn4Zur2 dimer. The complementary technique, ICP-OES, confirmed that the zinc binding stoichiometry was in agreement with mass spectrometric findings, 2.1±0.2 Zn(II) ions per monomer. Mimicking a drop in available cellular zinc as may be expected in zinc-deplete conditions, it was found that only one of the two metal ions bound per monomer was removed by EDTA from Synechococcus sp. WH8102 Zur protein, giving 0.9 ± 0.3 Zn(II) atoms per monomer, with shifting in the monomer/dimer equilibrium toward Zn1Zur species. No large difference in the secondary structure between the metallo-species of the SynZur protein was found in CD spectroscopy. Moreover, Synechococcus sp. WH8102 Zur protein was found to behave in a similar manner to previously studied Fur family proteins, where zinc removal from the sensory site is fully reversible and has the ability to re-establish a (2:1) Zn(II):protein ratio in less than 15 min with 1.9 ± 0.4 Zn(II) atoms per monomer. Similarly, Synechococcus sp. WH8102 Zur was found to act as a transcriptional factor in the presence of zinc ions and bind specifically to 23 AT-rich DNA sequence. The "free" cytosolic zinc concentrations that Zur protein trigger transcription of znuABC and smtA found to be in femtomolar range ~ 1.78 ×10-15, with the dissociation constant ~ 62.65±1.47 nM and 61.76 ± 2.42 nM for Zur-PznuA promoter and SynZur-PsmtA promoter, respectively.

QD Chemistry

Ksibe, Amira Z.

University of Warwick

Electronic name

p

[Thesis]

276903

a

Y