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Zinc regulation in an open ocean cyanobacterium
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Mikhaylina, Alevtina (2019) Zinc regulation in an open ocean cyanobacterium. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3491556~S15
Abstract
Cyanobacteria of the genera Synechococcus and Prochlorococcus are key players in marine CO2 fixation contributing ~25% of the global total. Since oceanic zinc concentrations may directly affect the CO2 fixation process via the role of carbonic anhydrase in cyanobacterial photosynthesis, understanding cyanobacterial zinc metabolism is of critical importance.
The current study used the model open ocean cyanobacterium Synechococcus sp. WH8102. In this species zinc uptake is thought to be regulated by a predicted zinc uptake regulator (SynZur) which controls expression of at least four genes thought to be involved in zinc homeostasis: znuA, znuB, znuC and bmtA (encoding a predicted high affinity zinc uptake transporter ZnuABC and a metallothionein SynBmtA). These components and Zur are largely uncharacterized.
SynZur was over-expressed recombinantly in E. coli, purified, crystallized and its X-ray crystal structure resolved. This is the first member of the Fur family of proteins from cyanobacteria to have a known structure. Two zinc binding sites were identified: a conserved structural site 1 (Cys4), and a novel sensory site (His2AspCys). Both zinc binding sites reside within the dimerization domain. ESI-MS detected a decrease in the abundance of the SynZur dimer in the absence of zinc at the sensory site, hinting at a potentially novel zinc sensing mechanism that might be typical of cyanobacterial Zur proteins. The affinity of the sensory site for zinc was KD = 8.27×10-13 M. SynZur with two zinc ions per monomer bound also showed selective binding to pznuABC with a KD ~5 nM per dimer or ~10 nM per monomer. ESI-MS revealed that Cd2+ can replace Zn2+ in the structural site suggesting that it is not kinetically inert.
A Synechococcus sp. WH8102 zur mutant was also constructed and characterised. Growth rate comparisons showed that the mutant has a lower tolerance to high zinc levels compared to the WT, whilst ICP-MS analysis revealed different accumulation behaviour for trace metals and most significantly for zinc. This is consistent with SynZur repressing a predicted znuABC system. RNA-seq analysis of the WT and zur mutant facilitated experimental identification of new members of the Synechococcus sp. WH8102 Zur regulon. Phyre2 structural prediction suggested that two of the newly identified genes (SYNW0972 and SYNW0973) encode components of a novel outer membrane zinc uptake system. Significantly, these two genes are conserved in other marine cyanobacteria. Furthermore, the RNA-seq data (confirmed using RT-qPCR) showed that SynZur can activate a metallothionein (SYNW0359, SynBmtA) in Synechococcus sp. WH8102 previously thought to be repressed by SynZur.
SynZur binds to the operator-promoter region of SynBmtA (pbmtA) in a 4:1 stoichiometry, with an affinity of 26.7 nM per monomer. The SynBmtA protein was over-expressed and purified with four zinc ions bound. SynBmtA was de-metallated and its affinity to Zn2+ was determined (KD = 1.13×10-14 M). ESI-MS showed that apo-SynBmtA is able to remove zinc from metallated SynZurZn2 in agreement with the order of zinc affinities for SynZur and SynBmtA. Finally, SynBmtA was analysed by 1H,15N-NOESY-HSQC and 1H,15N-TOCSY-HSQC NMR spectroscopy. The spectra were partially assigned and the assignment indicated that the characteristic zinc-finger fold of bacterial MTs is indeed present in SynBmtA, but that the C-terminus is likely to differ significantly from that of SmtA from the Synechococcus elongatus strain PCC 7942.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Cyanobacteria, Carbon dioxide, Zinc, Transcription factors | ||||
Official Date: | September 2019 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Chemistry ; School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Blindauer, Claudia A. ; Scanlan, David J. | ||||
Format of File: | |||||
Extent: | xxi, 278 leaves : illustrations, charts | ||||
Language: | eng |
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