Abstract
Our aim was to isolate from bacteria a flagellated, subcellular system whose content could be changed at will. Because the control of bacterial chemotaxis resides in the direction of rotation of the flagella, such a system would be ideal for the study of this control mechanism. By incubating bacteria with penicillin and then lysing them osmotically, we were able to isolate cell envelopes from Escherichia coli and Salmonella typhimurium. These envelopes have the same sidedness and similar shape and dimensions as the original bacteria; they are practically free of cytoplasm; they are osmotically sensitive, having intact the cytoplasmic membrane and at least part of the cell wall; and they have flagella. This preparation was used to find out what is required to restore flagellar rotation, which had been lost during osmotic lysis. By visualizing the image of individual flagella with high intensity light microscopy or by tethering the cell envelopes, we found that adding artificial electron donors as an energy source is enough to restore rotation. This seems to indicate that no cytoplasmic components are required and that the proton electrochemical potential is indeed the driving force for flagellar rotation. However, the rotation was almost entirely counterclockwise, while in intact bacteria the flagella rotate in both directions. This may indicate that a cytoplasmic component is required to allow clockwise rotation. The significance of these results for the study of chemotaxis is discussed.
Highlights
By incubating bacteriawith penicillin and thelnysing them osmoticallyw, e were able tiosolate cell envelopes from Escherichia coli and Salmonella typhimurium
Known subcellular systems consist of either nonflagellated vesicles of the cell envelope [8,9,10,11,12,13,14], isolated, intact flagella [15,16,17], or flagella attached to vesicular fragments of cytoplasmic membrane [18],but the small size of these fragments makes such a system very difficult to study from the point of view of motility
This report describes the isolation of ering the cell envelopes,we found that adding artificial bacteria-sized cell envelopesof E. coli and S. typhimurium to electron donors as an energy source is enough to re- which functional flagella are still attached
Summary
From theDepartments of Biochemistry and Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706. By incubating bacteriawith penicillin and thelnysing them osmoticallyw, e were able tiosolate cell envelopes from Escherichia coli and Salmonella typhimurium These envelopes have the same sidedness and similar shape and dimensionas s the original bacteriat;hey are practically free of cytoplasm;they are osmoticallysensitive, having intact the cytoplasmic membrane andat least part of the cellwall; and they have flagella. Since chemotaxis results from controlling the direction of rotation of the flagella, a flagellated subcellular system whose content can be changed at will should be ideal for the study of the chemotactic mechanism. Store rotation.This seems to indicate thatno cytoplasm i c components are required and that the protoelnec-
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