Overview

The micronutrients iron, zinc, copper, cobalt, and manganese play essential roles in the biogeochemical cycling of carbon, nitrogen, oxygen and phosphorus [1], and ensuring sufficiently high metal quotas is especially critical for photoautotrophs in oligotrophic environments. Marine cyanobacteria have the ability to bio-concentrate extremely scarce essential metal ions by several orders of magnitude [2]. Despite major advances in understanding bacterial metal homeostasis [3,4], our understanding of how marine cyanobacteria achieve this remarkable bio-concentration is far from complete: in contrast to other bacteria – including freshwater cyanobacteria such as Synechocystis sp. PCC 6803 [5] - that possess systems to energise active metal transport through the outer membrane, marine cyanobacterial genomes are devoid of genes for such systems [6]. Recent collaborative work by the Blindauer and Scanlan labs has led to the hypothesis that cyanobacterial porins play a critical role in promoting metal transport through the outer membrane at extremely low external metal concentrations (Figure 1).

Despite their ubiquitous distribution, little is known of the functioning of cyanobacterial porins (CBPs; Transporter Classification Database Identifier 1.B.23). The proposed project will focus on Synechococcus sp. WH8102 as model organism, and on one particular cyanobacterial porin encoded by the gene synw2224. This candidate protein has been identified in previous metalloproteomic screens through its metal-binding ability, and stood out by displaying higher abundance in metal-depleted conditions [7]. We hypothesise that this protein transports uncomplexed metal ions, but may also aid decomplexation and accumulation at the cell surface. Understanding metal transport in marine Synechococcus underpins explaining a key adaptive mechanism that cyanobacteria possess to occupy and proliferate in the oligotrophic oceans – organisms that are critical for global carbon cycling and whose abundance is expected to increase as a result of global warming.

Figure 1: Overview of known and suspected players in cyanobacterial zinc uptake. Transport through the inner membrane is active, requiring ATP to energise transport through the permease of the ABC system ZnuABC. Similar networks are thought to be at play for other metal ions such as iron, manganese, cobalt, and copper. The driving force for passage through the outer membrane is unknown.

Methodology

The function of the porin encoded by synw2224 will be assessed by the construction of a cyanobacterial mutant defective in this gene and subsequent characterisation of the mutant using transcriptomics, proteomics, metal uptake and metal quota analysis e.g. using ICP-MS. The SYNW2224 protein will also be over-expressed in E. coli and the protein characterised using biophysical (including metal binding and transport) and structural approaches e.g. Cryo-EM and crystallography.

Training and Skills

The Supervisory team has an excellent record in PhD supervision. Students that have graduated from the two groups have received training in a broad range of techniques and skills, and have typically had no trouble finding employment after graduating. For example, former students of the Blindauer lab now work for MedImmune, Malvern Instruments, Lonza, Immunocore and other biotech companies. Students have the opportunity to disseminate their work widely, through participation at national and international conferences, and by publishing their work in high profile journals such as The ISME Journal, Current Biology, and Scientific Reports. This exciting project will provide cutting-edge training on contemporary omics approaches, including transcriptomics and proteomics, as well as sophisticated analytical chemistry techniques including ICP-MS and biological mass spectrometry. It will also provide excellent training in wider aspects of marine microbiology, biogeochemistry and molecular biology using cutting edge techniques as well as in a variety of analytical and structural techniques currently available at Warwick, including mass spectrometry, cryo-EM and crystallography.

Timeline

Year 1: Define metal quotas in marine Synechococcus sp. WH8102; generate a synw2224 knockout mutant strain; clone and optimise recombinant expression of the SYNW2224 protein in E. coli.

Year 2: Comparative studies of wild type and synw2224 mutant Synechococcus sp. WH8102: growth rates at different concentrations of respective metals; Metal quotas; Crystallisation trials (and/or cryo-EM analysis). Proteomic/transcriptomic characterisation of the mutant.

Year 3: SYNW2224 structural studies; Metallo-proteomics analysis of mutant and wild type.

Partners and collaboration (including CASE)

The supervisors are world-leading experts in metal biogeochemistry and marine microbiology, as evidenced by regularly publishing in high profile interdisciplinary journals (e.g. Proc. Natl. Acad. Sci. USA) and field specific high impact journals (e.g. Metallomics, The ISME Journal, Chemical Science), and giving invited lectures at pertinent conferences. For example, Dr Blindauer gave a plenary lecture at the 2017 International Conference on the Biogeochemistry of Trace Elements. The supervisors have complementary expertise in bio-inorganic chemistry and marine molecular biology as it relates to biogeochemical cycles. Current research in the groups is well-funded by NERC, the Leverhulme Trust and The European Union. Further details on their research activities and their group members can be found via the links below.

Dr Blindauer’s group: https://warwick.ac.uk/fac/sci/chemistry/research/blindauer/blindauergroup

Prof Scanlan’s group http://www2.warwick.ac.uk/fac/sci/lifesci/people/dscanlan

Further Details

Applicants from the UK or the EU are eligible. Applicants should hold a BSc and/or MSc degree in relevant subjects. Informal enquires can be made to Dr Claudia Blindauer (c.blindauer@warwick.ac.uk). Details of how to apply can be found at https://warwick.ac.uk/fac/sci/lifesci/study/pgr/studentships/nerc-centa/