Motile Escherichia coli placed at one end of a capillary tube containing an energy source and oxygen migrate out into the tube in one or two bands, which are clearly visible to the naked eye and can also be demonstrated by photography, microscopy, and densitometry and by assaying for bacteria throughout the tube. The formation of two bands is not due to heterogeneity among the bacteria, since the bacteria in each band, when reused, will form two more bands. If an anaerobically utilizable energy source such as galactose is present in excess over the oxygen, the first band consumes all the oxygen and a part of the sugar and the second band uses the residual sugar anaerobically. On the other hand, if oxygen is present in excess over the sugar, the first band oxidizes all the sugar and leaves behind unused oxygen, and the second band uses up the residual oxygen to oxidize an endogenous energy source. The essence of the matter is that the bacteria create a gradient of oxygen or of an energy source, and then they move preferentially in the direction of the higher concentration of the chemical. As a consequence, bands of bacteria (or rings of bacteria in the case of agar plates) form and move out. These results show that E. coli is chemotactic toward oxygen and energy sources such as galactose, glucose, aspartic acid, threonine, or serine. The full repertoire of chemotactic responses by E. coli is no doubt greater than this, and a more complete list remains to be compiled. The studies reported here demonstrate that chemotaxis allows bacteria to find that environment which provides them with the greatest supply of energy. It is clearly an advantage for bacteria to be able to carry out chemotaxis, since by this means they can avoid unfavorable conditions and seek optimum surroundings. Finally, it is necessary to acknowledge the pioneering work of Englemann, Pfeffer, and the other late-19thcentury biologists who discovered chemotaxis in bacteria, and to point out that the studies reported here fully confirm the earlier reports of Beijerinck (4) and Sherris and his collaborators (5,6) on a band of bacteria chemotactic toward oxygen. By using a chemically defined medium instead of a complex broth, it has been possible to study this band more closely and to demonstrate in addition the occurrence of a second band of bacteria chemotactic toward an energy source. Beijerinck (4) did, in fact, sometimes observe a second band, but he did not offer an explanation for it.

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherchia coli and have discovered pressure-activated ion channels. The channels have the following properties: (t) activation by slight positive or negative pressure; (it) voltage dependence; (ifi) large conductance; (iv) selectivity for anions over cations; (v) dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.Ion channels are gated protein pores found in biological membranes; these channels regulate many cellular interactions with the environment, including responses to hormonal, neuronal, and sensory stimuli (1). Ion channels have been studied in animals, plants, and microorganisms (2-4). In bacteria, in vivo channel activity has not been demonstrated, although the activity of isolated channel proteins has been measured in artificial membranes (5, 6).The patch-clamp technique allows recording of current through individual ion channels in the native membrane by sucking the membrane onto a recording pipette to form a tight (gigaohm) electrical seal (7). This method has been used to study single channels in vivo in many eukaryotic cells, and it has demonstrated that the large currents measured across the membranes of a whole cell are really composed ofmany small currents passing through individual channels.The lower limit to the diameter ofthe patch-pipette opening is about 1 ,um (1); this precludes measurement ofion channels in bacteria directly. Cells of Escherichia coli, however, can become giant spheroplasts when grown in the presence of chemicals such as mecillinam to prevent cell wall (peptidoglycan) synthesis, and membrane potential has been measured in such spheroplasts by conventional electrophysiology (8). Giant spheroplasts can also be formed by growth of cells in the presence of cephalexin to prevent cell division and form filamentous "snakes"; these snakes can then be treated with lysozyme and EDTA to dissolve the cell wall (the spheroplasts can revert to normal form when returned to growth medium in the absence of these chemicals) (9). We used this latter method to make spheroplasts with a diameter of -6 ,.m. We demonstrate here the application of in vivo patch-clamp recording to such giant spheroplasts. This method should be generally applicable to any bacterial species.We discovered that a low positive or negative pressure (tens of millimeters of mercury; 1 mm Hg = 133 Pa) applied to the spheroplast membrane activates ion channels. This pressure could be caused by an osmotic difference of as little as a few milliosmolar across the membrane. We believe that these channels may allow E. coli to detect and to respond to small osmotic changes in the surrounding medium. The preliminary work has been reported in abstract form (10). MATERIALS AND METHODSMaterials. Organic components of the growth medium were purchased from Difco. Tris was purchased from Boehringer Mannheim; other salts and chemicals for preparation of...

Chemotaxis of a bacterium such as Escherichia coli is assayed by measuring the number of organisms attracted into a capillary tube containing an attractant. Rate of bacterial accumulation in capillaries and a concentration-response curve for L-aspartate taxis are presented and interpreted, and the effect of bacterial concentration is reported. Other parameters of the assay were studied, such as the volume of fluid in the capillary and the size of the capillary opening. The concentration gradient of chemical was also described. Escherichia coli chemotaxis requires EDTA to allow motility, a buffer to maintain the pH at its optimum near neutrality, and L-methionine if it cannot be synthesized. Under certain conditions there is stimulation by inorganic ions, either by K+ or, less effectively, by Na+. Chemotaxis is dependent on temperature, there being a 20-fold increase in the number of bacteria accumulating in a capillary when the temperature is raised from 20 to 30 "C.

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Mechanosensitive channels have been found in more than 30 cell types, including bacterial, yeast, plant and animal cells. Whether tension is transferred to the channel through the lipid bilayer and/or underlying cytoskeleton is not clear. Using the patch-clamp method, we found that amphipathic compounds, which are molecules having hydrophobic and hydrophilic character with positive, negative or no net electric charge at pH 7, could slowly activate the mechanosensitive channels of giant Escherichia coli spheroplasts, with effectiveness proportional to their lipid solubility. The cationic or anionic amphipaths were able to compensate for each other's effect. After a channel was activated by an amphipath of one charge, if that amphipath was gradually replaced by one with the opposite charge, the channel first inactivated before reactivating. These findings support the view that the mechanical gating force can come from the surrounding lipids.