POSTERS - BLAST X - University of Utah

BLAST X Wed. Morning Session
ELECTRON CRYOTOMOGRAPHY OF BACTERIAL CHEMOTAXIS ARRAYS
Ariane Briegel 1 , H. Jane Ding 1 , Zhuo Li 1 , John Werner 2 , Zemer Gitai 2 , D. Prabha Dias 1 ,
Rasmus B. Jensen 3 , Elitza Tocheva 1 and Grant Jensen 1
1 California Institute of Technology, CA
2 Princeton University, NJ
3 University of Roskilde, Denmark
Motile prokaryotes are able to sense and to respond to ambient conditions through a
process known as chemotaxis. Attractants and repellents bind to the sensing domain of methylaccepting
chemotaxis proteins (MCPs), thereby regulating the activity of the histidine kinase
CheA. Together with the linking protein CheW, CheA is located at the distal tip of the
cytoplasmic signaling domain of the MCPs. If activated, CheA phophorylates CheY (CheY-P),
which in turn controls the direction of flagellar rotation. Together with CheA and a linking protein
CheW, the MCPs form extended chemotaxis arrays at the cell poles.
Electron cryotomograhy (ECT) makes it possible to visualize chemoreceptor clusters in
prokaryotes in vivo at macromolecular resolution (4-8 nm). While high-resolution structures of
the individual chemotaxis proteins are available, their arrangement and position in the arrays
remain unclear. Understanding this "mesoscale" architecture of the clusters is critical, however,
since it is vital to the arrays' cooperative signal amplification and regulation. In order to
unambiguously identify the chemotaxis arrays inside cells, we have correlated ECT with
fluorescent light microscopy (FLM), using slightly fixed and immobilized Caulobacter crescentus
cells with a fusion of the red-fluorescent protein, mCherry, to the C-terminus of the
chemoreceptor (McpA). After plunge freezing, we imaged the same cells by ECT. In
combination with ECT of near-native wild-type and mutant cells, we used the correlated FLM
and ECT approach to identify the chemotactic array, its location and its in-vivo structure. We
demonstrate that in wild-type Caulobacter crescentus cells preserved in a near-native state, the
chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of
nanometers away from the flagellar motor that they control. The arrays were always found on
the concave side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral
as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of
receptor dimers at each vertex of the lattice accounts well for the density, supporting an array
composition unlike the published models for Escherichia coli [1] or Thermotoga maritima [2].
We are now in the process of comparing the chemotaxis arrays of a wide range of bacteria to
determine the similarities and differences of these macromolecular assemblies at the
‘mesoscale’ level.
1. Shimizu T. S. et al, Nat Cell Biol 11 (2000) 792.
2. Park, S.-Y. et al, Nat Struct Mol Biol 13 (2006) 400.
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