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How do metals cross the bacterial outer membrane?

Primary Supervisor: Professor Claudia Blindauer, Department of Chemistry

Secondary supervisor: Professor David Scanlan

PhD project title: How do metals cross the bacterial outer membrane?

University of Registration: University of Warwick

Project outline:

General Background

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 photosynthetic organisms. Marine cyanobacteria inhabiting the nutrient-poor open ocean have the ability to bio-concentrate extremely scarce essential metal ions by several orders of magnitude. Despite major advances in understanding bacterial metal homeostasis, our understanding of how marine cyanobacteria achieve this remarkable bio-concentration is far from complete: in contrast to other bacteria (including freshwater cyanobacteria) that possess systems to energise active metal transport through the outer membrane, marine cyanobacterial genomes are devoid of genes for such systems. Recent collaborative work by the Blindauer and Scanlan labs has identified two strong candidates predicted to play a critical role in promoting metal transport through the outer membrane at extremely low external metal concentrations. This project aims to elucidate structure and transport properties of proteins encoded by the synw0972 and synw2224 [2] genes from Synechococcus sp. WH8102.

Objectives

Understand structure and function of cyanobacterial outer-membrane proteins suspected to be involved in metal uptake, and their role in enabling the support of photosynthesis in oligotrophic environments

Methodology

Genes will be cloned from the native host (Synechococcus sp. WH8102) and recombinantly expressed in E. coli. This will enable biophysical characterisation of these proteins, including structure elucidation by either cryo-electron microscopy or X- Ray crystallography.

Transport properties will be assessed both in vitro and in vivo. For in vitro studies, the proteins will be reconstituted in proteoliposomes, and metal transport will be assessed by stopped-flow fluorescence measurements. For in vivo studies, knock-out mutants deficient in the gene of interest will be constructed. The mutants will be studied using transcriptomics, proteomics, metal uptake and metal quota analysis by ICP-MS (metallomics).

References:

  1. F. M. Morel (2008) The co-evolution of phytoplankton and trace element cycles in the oceans. Geobiology 6:318-324.

  2. J.P. Barnett, D.J. Scanlan, C.A. Blindauer (2014) Identification of major zinc-binding proteins from a marine cyanobacterium: insight into metal uptake in oligotrophic environments. Metallomics 6: 1254-1268.

  3. A. Mikhaylina, A. Z. Ksibe, R. C. Wilkinson, D. Smith, J. P. C. Coverdale, V. Fülöp, D. J. Scanlan, C. A. Blindauer (under revision at Nature Chemical Biology) A single sensor controls large variations in zinc quotas in a marine cyanobacterium.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Microbiology & Structural Biology & Systems Biology
Techniques that will be undertaken during the project:
  • Cyanobacterial cell culture
  • Bacterial genetics and molecular biology
  • RNA-sequencing
  • Mass-spectrometry based proteomics
  • Recombinant protein expression
  • Membrane protein purification and reconstitution
  • Cryo-EM (collaboratively with Monash)
  • Stopped-flow fluorescence spectrometry
  • Inductively-coupled-plasma mass spectrometry (ICP-MS) and ICP-optical emission spectroscopy (ICP-OES)

Contact: Professor Claudia Blindauer, University of Warwick