Organisation and mobility of respiratory electron transport complexes in Escherichia coli

Conrad Mullineaux
Professor of Microbiology
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road,
London E1 4NS
UK

This is the research project of Tchern Lenn, a PhD student funded by a QMUL College Studentship. It involves collaboration with Mark Leake (Clarendon Laboratory and Department of Biochemistry, Oxford) who has provided essential input with TIRF microscopy and data analysis. We also collaborate with Thorsten Friedrich (Freiburg, Germany) and his group. Some mutants and gene constructs used in the project come from the Keio collection and ASKA library.

In E. coli (as in many other bacteria) there is a respiratory electron transport chain, consisting of a number of protein complexes in the plasma membrane. These proteins play a crucial role in energy transduction in the cell. Most of the individual protein complexes are very well characterised (with X-ray crystallographic structures in some cases). However, we know much less about their organisation and interactions in the intact membrane. Do the different respiratory complexes cluster together in the membrane? Or do individual complexes diffuse randomly and interact only transiently?

Our approach is to use gene-fusions to tag respiratory complexes with Green Fluorescent Protein (GFP), and then use advanced fluorescence microscopy to study their organisation and dynamics in vivo. The example shown below is for the cytochrome bd terminal oxidase complex. The complex was GFP-tagged at the C-terminus of CydB. The gfp gene fusion is expressed from native chromosomal locus to avoid any artefacts due to overexpression. Growth and electron transport measurements show that the GFP-tagged complex is fully functional.

Frames taken from a TIRF video sequence showing fluorescence from GFP-tagged cytochrome bd complexes in the plasma membrane of an E.coli cell. Note that GFP fluorescence is concentrated into mobile spots: we estimate that each spot contains about 75 complexes on average and is about 100 nm in diameter. These spots may represent localised regions of the membrane dedicated to respiratory activity ("respirazones").

The video clip (16 MB) from which these frames are taken can be downloaded here. The clip plays in real time.

Key publications on this topic:

Lenn, T., Leake, M.C. and Mullineaux, C.W. (2008) Are Escherichia coli OXPHOS complexes concentrated in specialised zones within the plasma membrane? Biochem. Soc. Trans. 36, 1032-1036.

Lenn, T., Leake, M.C. and Mullineaux, C.W. (2008) Clustering and dynamics of cytochrome bd-I complexes in the Escherichia coli plasma membrane in vivo. Molecular Microbiology 70, 1397-1407.