In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 23 ( 2012-06-05)
Abstract:
The high connectivity among MCP trimers in our model provides a basis for understanding the emergent properties of the receptor array, such as its high sensitivity, extensive dynamic range, and impressive signal amplification. We are now in a position to understand the detailed configuration of receptor arrays in various signaling states, such as immediately after the addition of attractants or repellents. Arrays formed by mutant MCPs that generate either exclusively CCW or CW flagellar rotation can also be examined. We anticipate that high-resolution images of receptor arrays in cells exposed to different sensory inputs and generating different signal outputs will help to elucidate the highly cooperative mechanisms required to achieve optimal chemotaxis ( 3 ). In addition, the combination of cryo-ET with the genetic manipulation of E. coli to make very small minicells will be of general interest for structural studies of molecular machines in living cells. In our model displayed in Fig. P1 , the membrane-distal tips of two MCP trimers are joined by a P3 dimerization domain of CheA flanked on either side by a receptor-interaction P5 domain of CheA and a CheW monomer. Each P5 domain and each CheW is bound to a different receptor dimer. The critical CheA P1 domain, which is involved in phosphotransfer signaling, lies below the plane of the trimer tip/CheW/P3/P5 layer. An unknown interaction, to our knowledge, between CheA and CheW, now revealed by our reconstruction, illustrates how multiple core signaling complexes may assemble into extended arrays ( Fig. P1 ). There are densities within our maps indicating that, at least under conditions of protein overproduction, regular hexameric rings of CheW proteins may occupy the otherwise unpaired third MCP dimer within each trimer of the extended array. This feature may explain the discrepancy between the relative numbers of associated receptor-CheA-CheW molecules evident in the core signaling complex and in living cells. By averaging the images from hundreds of minicells, we have achieved 3.2-nm resolution electron-density maps of the intact arrays in their native environment. The level of resolution is sufficient to allow us to fit the known atomic structures of the MCPs, CheA, and CheW accurately into our electron-density maps. This combined approach allowed us to construct a model for the core signaling complex ( Fig. P1 ), which consists of two trimers of chemoreceptor homodimers (hereafter called trimers), one CheA dimer, and two CheW molecules, as determined by biochemical studies ( 5 ). Here, we used a genetically engineered “skinny” E. coli strain that overproduces all flagellar and chemotaxis proteins and that generates chromosome-free cells (minicells) for cryo-ET. Minicells are produced by asymmetrical cell division that occurs near one pole of the rod-shaped cell rather than normally at its midpoint. This aberrant event is caused by mutations at a genetic locus called minCD . Minicells produced by our engineered skinny minCD mutant are smaller than those produced by typical minCD strains, but often display an overabundance of flagella and larger chemoreceptor arrays. The small diameter of these minicells is optimal for cryo-ET and allows arrays to be visualized at a high resolution. MCPs localize to the poles of rod-shaped E. coli in highly ordered arrays that also include the CheA kinase and the CheW coupling proteins. The precise architecture of these arrays is responsible for the sensitivity, high dynamic range, and strong amplification of chemotactic signaling ( 3 ). These arrays have been visualized in a number of bacterial species ( 4 ) by using cryoelectron tomography (cryo-ET), a technique that employs an electron microscope to acquire 3D reconstruction of an intact organism at cryogenic temperatures. However, until now, the structural details of the array have not been well defined. Spatial chemical gradients are sensed by a temporally controlled mechanism that compares concentrations of attractants and repellents over time ( 1 , 2 ). E. coli is able to “remember” the chemical concentrations over a period of a few seconds. Adaptation abolishes this memory, in essence making the cells forget so that they can adjust their sensitivity to sense further changes in concentration. The adaptation process involves the chemical modification of the chemoreceptors that recognize the attractants and repellents. Because these modifications involve the addition and removal of methyl groups (i.e., methylation, demethylation), the receptors are also known as methyl-accepting chemotaxis proteins (MCPs). Chemotaxis in Escherichia coli , the most abundant inhabitant of the human gut, is one of the best understood behaviors at the molecular level exhibited by any organism. The basic phenomenon is very simple. Propelled by long, thin flagellar filaments, swimming cells move toward higher concentrations of attractant chemicals and toward lower concentrations of repellent chemicals. These movements are accomplished by biasing a 3D random walk ( 1 ), such that periods of smooth swimming, known as “runs,” become longer as the cell moves in a favorable direction. These lengthened runs are achieved by suppressing “tumbling” events, in which the counterclockwise (CCW) flagellar rotation that produces smooth swimming is interrupted by brief periods of clockwise (CW) flagellar rotation that randomly reorient the cell ( 1 ).
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1200781109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
Permalink
