Effects of Organic Antagonists of Ca²⁺, Na⁺, and K⁺ on Chemotaxis and Motility of Escherichia coli

Abstract

Various Ca²⁺ antagonists used in animal research, many of them known to be Ca²⁺ channel blockers, inhibited Escherichia coli chemotaxis (measured as entry of cells into a capillary containing attractant). The most effective of these, acting in the nanomolar range, was ω-conotoxin GVIA. The next most effective were gallopamil and verapamil. At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant). Various other Ca²⁺ antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility. Cells treated with each of these Ca²⁺ antagonists swam with a running bias, i.e., tumbling was inhibited. Similarly, some Na⁺ antagonists used in animal research inhibited bacterial chemotaxis. E. coli chemotaxis was inhibited by saxitoxin at concentrations above 10⁻⁷ M, while more than 10⁻⁴ M was needed to inhibit motility. Cells treated with saxitoxin swam with a tumbling bias. In the case of other Na⁺ antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration. In the case of K⁺ antagonists used in animal research, 4-aminopyridine blocked E. coli chemotaxis between 10⁻³ M and, totally, 10⁻² M, while motility was not affected at 10⁻² M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10⁻² M.

Ion channels are membrane components which function to allow rapid entry or exit of ions into or out of cells. In eukaryotic cells, a wide variety of pharmacological agents are known to block ion channel proteins (23). Several of these agents have been shown to inhibit specifically and to bind with high affinity to either Ca²⁺, Na⁺, K⁺, or Cl⁻ ion channels, while in other cases the inhibition is not specific (23). Besides their use in the characterization of ion channels, organic channel blockers have been employed to isolate and purify channel proteins, such as the acetylcholine receptor (34) and the voltage-gated Na⁺ channel (10, 11).

Ion channels have been demonstrated also in bacteria (for a review, see reference 5). Ion channels have been located in both the cytoplasmic (inner) and outer membranes of gram-negative bacteria such as Escherichia coli. Porins, the first class of bacterial ion channels to be discovered, are located in the outer membrane of gram-negative bacteria and function as molecular sieves to allow passage of small compounds (41). Mechanosensitive channels from the cytoplasmic membrane of E. coli and other species have been studied (4, 12, 14, 15, 22, 28, 31, 54, 55, 66). K⁺ transporters and K⁺ channels are known to occur in the cytoplasmic membrane of E. coli (14, 25, 38, 66). A K⁺ channel occurs in Streptomyces lividans (17, 29, 49), and a glutamate receptor which is a K⁺ channel has been discovered in cyanobacteria (13). The K⁺ channels found in bacteria closely resemble those of animals (17, 25, 29, 38, 49, 67). Ca²⁺ channels have been reported for the cytoplasmic membrane of E. coli (16) and Bacillus subtilis (33). The Ca²⁺ channel of E. coli consists of inorganic polyphosphate bound together with poly-β-hydroxybutyrate (16); it appears that animal-like Ca²⁺ channels are not coded for by the E. coli genome (3). Na⁺ channels have not been reported for bacteria to our knowledge and appear not to be coded for by the E. coli genome (3).

Inorganic cations play a role in bacterial taxis. (For reviews of chemotaxis, see references 19 and 51). In B. subtilis, Ca²⁺ has been shown to be involved in chemotaxis (44). Several organic Ca²⁺ channel blockers in animals have been reported to inhibit chemotaxis of B. subtilis: ω-conotoxin, verapamil, nitrendipine, and diltiazem (32). In Spirochaeta aurantia, chemotaxis was blocked by inhibitors affecting animal Ca²⁺ channels (botulinum toxin A), Na⁺ channels (aconitine, tetrodotoxin, and sea anemone venom), and K⁺ channels (scorpion venom and tetraethylammonium) (20). In Spirillum volutans various inorganic and organic agents, some of them neurotoxic, produce uncoordination of flagella (9, 27). Ca²⁺ produces constant tumbling of E. coli in the presence of a Ca²⁺ ionophore (43). (See also the discussion about Ca²⁺ and E. coli taxis in the paper by Brey and Rosen [7].) Ca²⁺ has been implicated in photophobic responses of Phormidium uncinatum, a cyanobacterium (or blue-green alga), by the use of ruthenium red, lanthanum, and a calcium-conducting ionophore (21, 39, 40). Ca²⁺ is also involved in the gliding motility of myxobacteria (65). Ca²⁺ plays a role in taxis by Halobacterium salinarum, an archaeon (2, 39).

More recent work documents further that Ca²⁺ is involved in E. coli chemotaxis (56–59, 62), but how the Ca²⁺ acts remains to be determined. The Ca²⁺ antagonist ω-conotoxin inhibits E. coli chemotaxis (59). We (56, 58) and Watkins et al. (62) have reported that the cytoplasmic concentration of Ca²⁺ rises when bacteria encounter repellents (which make them tumble) and falls with attractants (which make them run). In addition, mutants having a high concentration of Ca²⁺ are tumbly (57). (For recent reviews on the role of calcium ions in bacteria, including bacterial chemotaxis, see references 24, 42, and 52).

This communication reports an investigation on the effect of organic Ca²⁺ blockers, organic Na⁺ blockers, and organic K⁺ blockers used in animal research on chemotaxis and motility by E. coli. The aim of this survey is to identify ions involved in E. coli chemotaxis and to find pharmacological agents which could potentially be used to characterize, and ultimately to purify, ion channel components or other ion-binding components involved in the chemotaxis mechanism.

MATERIALS AND METHODS

Bacterial strains.

AW405 (36), AW574 (60), and RP487 (45) were used as chemotactically wild-type strains of E. coli.

Growth conditions.

Cells were grown in tryptone broth and then in Vogel-Bonner medium (61) containing 50 mM glycerol (except that 25 mM dl-lactate was used for the experiment for Fig. 1) plus the required amino acids at 1 mM.

FIG. 1.

Effect of ω-conotoxin GVIA on bacterial chemotaxis and motility by chemotactically wild-type E. coli (strain AW405). Bacteria were treated as described in Materials and Methods. Cells were preincubated for 45 min at 30°C in chemotaxis medium with different concentrations of ω-conotoxin. They were then presented with capillaries containing the same concentrations of ω-conotoxin and the attractant l-serine (10⁻³ M) or no attractant (“motility”), all in chemotaxis medium. Thus, in each case, the concentration of ω-conotoxin in the pond of bacteria was the same as that in the capillary. Circles (A) represent l-serine chemotaxis, and triangles (B) represent motility in the same medium but lacking added attractant. Points in panel A are the averages of two measurements indicated by the bars; points in panel B are single measurements.

Chemotaxis assay.

Cells were grown in the Vogel-Bonner growth medium by shaking at 35°C to an optical density of 0.4 to 0.6 at 590 nm. Then they were harvested by centrifugation at 6,000 × g for 3 min. The supernatant fluid was discarded, the pellet was resuspended, and chemotaxis medium was added (10 mM K⁺ phosphate [pH 7.0], 0.1 mM K⁺ EDTA, and, for RP487, 0.1 mM l-methionine). This was followed by two more such washes in chemotaxis medium, and finally the cells were resuspended in chemotaxis medium to an optical density of 0.005 at 590 nm (about 4 × 10⁶ bacteria/ml). Chemotaxis was assayed in chemotaxis medium by the capillary method (1) for 30 min (unless otherwise indicated) at 30°C. l-Serine (1 mM) or l-aspartate (10 mM) was used as an attractant in the capillary tubes (36). The cells in the capillary were plated on tryptone broth agar, and colonies were counted after incubation at 37°C overnight. Motility was assayed in the same way, but attractant was omitted. Antagonists to be tested for their effects on chemotaxis and motility were added to both the cell suspension and the capillary at equal concentrations to eliminate a gradient of the antagonists. Each experiment was repeated several times, and typical results are presented.

Viability assay.

To measure survival, cells were suspended in chemotaxis medium containing varying concentrations of the test antagonists and were incubated at room temperature for 30 min. The cells were then plated on tryptone broth agar, and colonies were counted after incubation overnight at 37°C.

Analysis of swimming behavior.

Swimming behavior was observed at 30°C by phase-contrast microscopy at a magnification of ×400. The cells were suspended in chemotaxis medium to an optical density of 0.1 at 590 nm. The microscopic behavior was videotaped, digitized, and analyzed by motion analysis (48).

Chemicals.

All chemicals are available from commercial sources, except that tiapamil (18) was a gift from R. Eigenmann at Hoffman-La Roche.

RESULTS

We have tested the effects of a number of organic ion antagonists known to be active in animals on E. coli chemotaxis and motility. By use of the capillary assay, the dose required to inhibit chemotaxis by 50% was measured for each antagonist tested and the dose required to inhibit motility by 50% was also measured; in addition, the dose required to reduce survival by 50% was determined (Table 1).

TABLE 1.

Drug concentrations required to inhibit E. coli chemotaxis, motility, and survival^a

Drug	Concn producing 50% inhibition of:		Ratio of concn producing 50% inhibition of motility/concn producing 50% inhibition of chemotaxis	Concn producing 50% loss of survival
	Chemotaxis	Motility
Calcium antagonists
ω-Conotoxin GVIAb	0.2 μM (0.01 μM)b	2.5 μM (1 μM)	13 (100)	No effect at 1 μM
Gallopamilcd	5 μM	500 μM	100	No effect at 1 mM
Verapamilcd	10 μM	650 μM	65	No effect at 1 mM
Diltiazemd	50 μM	800 μM	16	No effect at 1 mM
Tiapamilc	50 μM	800 μM	16	No effect at 1 mM
Chlorpromazinee	1 μM	10 μM	10	50 μM
Tetracainef	10 μM	100 μM	10	70 μM
Nifedipinedg	30 μM	300 μM	10	5 mM
Nisoldipinedg	80 μM	300 μM	4	5 mM
Dibucainef	37 μM	100 μM	3	50 μM
Nitrendipinedg	100 μM	300 μM	3	5 mM
Prenylaminedg	7 μM	10 μM	2	1 mM
Bepridild	5 μM	5 μM	1	100 μM
Sodium antagonists
Saxitoxinh	10 μM	1 mM	100	No effect at 1 mM
Aconitinei	10 μM	100 μM	10	No effect at 1 mM
Lidocainej	10 μM	10 μM	1	1 mM
Procainej	5 mM	5 mM	1	50 mM
Potassium antagonists
4-Aminopyridinek	2 mM	>10 mM	>5	No effect at 10 mM
Tetraethylammonium chloridek	>10 mM	>10 mM	1	No effect at 10 mM

^aBoth chemotaxis and motility were measured by the capillary assay in chemotaxis medium as described in Materials and Methods. Bacteria were treated as described in Materials and Methods. Cells were incubated for 30 min at 30°C in chemotaxis medium with different concentrations of each antagonist and were then presented with capillaries containing the antagonist and 10 mM l-aspartate or no attractant, all in chemotaxis medium. In each case, the concentration of the antagonist in the capillary was the same as that in the pond of bacteria. The viability assay for measuring survival is described in Materials and Methods. 

^bSome of these results with ω-conotoxin GVIA come from the work of Tisa, Olivera, and Adler (59). The concentrations stated in the parentheses represent the results obtained with Tris-EDTA-treated cells. This treatment allows the free passage of ω-conotoxin (molecular weight of 3,043) to the cytoplasmic membrane. 

^cGallopamil, also called D-600, is methoxyverapamil. Tiapamil is another verapamil derivative; it is a calcium channel blocker with cardiovascular effects (18). 

^dIn animals, a calcium channel blocker with cardiovascular effects (8). 

^eIn animals, a tranquilizer with effects on dopamine receptors (8). 

^fIn animals, a local anesthetic (8). 

^gDrug soluble in organic solvents; ethanol was used as a solvent in these experiments. At high concentrations, ethanol acts as an inhibitor of chemotaxis and motility. To correct for this effect and to test the effects of the drug on chemotaxis and motility, the results were compared with control cells in the presence of ethanol. 

^hIn animals, a Na⁺ channel blocker (8, 23, 26, 53, 64). 

ⁱIn animals, it produces heart arrhythmia (8). 

^jIn animals, a local anesthetic (8). 

^kIn animals, a K⁺ channel blocker (8, 23). 

Calcium blockers.

A variety of Ca²⁺ channel blockers in animals was tested (Table 1). ω-Conotoxin GVIA (here called ω-conotoxin) is a peptide in the venom of cone shells which in animals specifically blocks Ca²⁺ channels at picomolar concentrations (35). Figure 1 shows that ω-conotoxin was highly effective in inhibiting chemotaxis as previously reported (59); chemotaxis was up to 100 times more sensitive to ω-conotoxin than was motility (Fig. 1; Table 1, see column 3). Gallopamil (called also D-600), verapamil, diltiazem, tiapamil, nifedipine, and nitrendipine block Ca²⁺ channels of animals at 20 nM to 50 μM (23), but they are not perfectly selective for Ca²⁺ channels, and so high concentrations can depress Na⁺ and K⁺ channel currents as well (23); all those compounds are modeled after papavarine, a muscle relaxant found in opium (23). Gallopamil inhibited chemotaxis 100 times more effectively than it inhibited motility, though higher concentrations were needed for gallopamil than for ω-conotoxin. Verapamil was a close third, and diltiazem and tiapamil ranked next. None of these listed above harmed viability at the highest concentrations tested. Then ranked chlorpromazine, tetracaine, and nifedipine, each inhibiting chemotaxis 10 times more effectively than motility. In the remainder of the list (Table 1, see column 3) are Ca²⁺ antagonists which inhibited chemotaxis not much better than they inhibited motility.

The effect of these drugs on swimming behavior was tested. Normally, E. coli alternately runs and tumbles. Cells treated with each of these drugs swam with a running bias, i.e., running was predominant and tumbling was inhibited. To provide an objective analysis of these effects, the observations were subjected to computer analysis. Microscopic observations of free-swimming bacteria were videotaped, digitized, and analyzed (48). This technique measures both the average angular speed (the rate of change in direction) and the average linear speed (the rate of movement in a straight line) of a population of motile bacteria. The angular and linear speeds are directly and inversely proportional, respectively, to the tumbling frequency: an increase in the angular speed reflects an increase in the time spent tumbling, while a decrease corresponds to reduction in tumbling. The results are presented in Fig. 2. All of the Ca²⁺ blockers tested as reported in Table 1 inhibited tumbling and thus promoted a running bias which lasted as long as the blocker was present (unlike in the case of attractants, where pure running stops due to adaptation). There was no reduction in running speed, and so there should be no problem with reduced proton motive force.

FIG. 2.

The effect of Ca²⁺ antagonists on the average angular and linear speeds of chemotactically wild-type E. coli (strain AW574). The swimming behavior was videotaped, digitized, and analyzed by motion analysis (48) as described in Materials and Methods. This experiment has been repeated a number of times with similar results. Averages for three to six measurements of different fields in several experiments are plotted, and the range is indicated by the error bars. l-aspartate at 200 μM is a known attractant (36), while 4 mM NiSO₄ is a known repellent (60). (A) Average angular speeds; (B) average linear speeds.

The inhibition of chemotaxis by verapamil (Table 1) can be completely overcome by addition of Ca²⁺ (Fig. 3). Mg²⁺, even more effectively than Ca²⁺, overcame the inhibition by verapamil (Fig. 3): inhibition by 100 μM verapamil was completely overcome by 30 μM MgCl₂ compared to 300 μM CaCl₂. (The cause of the decline at high concentrations of Mg²⁺ and Ca²⁺ shown in Fig. 3 is not known.) Verapamil is used in humans as a coronary vasodilator because it blocks Ca²⁺ flux, but it is known to block Mg²⁺ flux, too (47). The ability of inorganic ions to overcome inhibition by organic antagonists has been tested also for ω-conotoxin—Ca²⁺ was nearly 100 times more effective than was Mg²⁺ (59)—but not for any of the other antagonists.

FIG. 3.

Ca²⁺ or Mg²⁺ overcomes inhibition of chemotaxis by verapamil (strain AW574). In the capillary assays, l-aspartate (10⁻² M) was present as an attractant in the capillary and verapamil (10⁻⁴ M), together with various concentrations of CaCl₂ (closed squares) or MgCl₂ (open squares), was present in both the capillary and the cell suspension. Incubation was at 30°C for 1 h. The uninhibited response (10 mM K⁺ phosphate [pH 7.0] and 0.1 mM K⁺ EDTA, no verapamil, no CaCl₂, and no MgCl₂) was 47,800 (±7%) bacteria in the experiment for CaCl₂ and 230,000 (±8%) bacteria in the experiment for MgCl₂; the difference between the two is due to variation in results that was obtained between the two different days when experiments were performed.

Sodium blockers.

Saxitoxin is a specific Na⁺ channel blocker of action potentials in nerve and muscle of animals, although it is actually produced by certain bacteria residing in a poisonous dinoflagellate; saxitoxin blocks Na⁺ current at nanomolar concentrations in animals (23, 26, 53, 64). About 1 μM saxitoxin began the inhibition of E. coli chemotaxis, and 10 μM (in some experiments, 30 μM) inhibited chemotaxis by 50% (Fig. 4 and Table 1). Chemotaxis was inhibited 100 times more effectively than was motility (Table 1).

FIG. 4.

Effect of saxitoxin on bacterial chemotaxis by chemotactically wild-type E. coli (strain RP487). The procedure was as described for Fig. 1, except that there was no preincubation. The capillaries contained 10⁻² M K⁺ l-aspartate. (To avoid K⁺, we tried l-serine instead of K⁺ l-aspartate as the attractant; similar results were obtained.) The pond of bacteria contained the same chemotaxis medium as did the capillary. In each case, the concentration of saxitoxin in the pond of bacteria was the same as that in the capillary.

The effect of saxitoxin on swimming behavior was studied. The cells were observed to tumble in 1 mM saxitoxin, and to a lesser degree in 100 μM, as long as the saxitoxin remained present (unlike in the case of repellents, where tumbling stops due to adaptation). Saxitoxin at 1 mM had no effect on cell viability (Table 1).

Among the other Na⁺ channel blockers of animals tested, aconitine inhibited chemotaxis at a micromolar level and motility at a 10-fold-higher concentration, while lidocaine and procaine inhibited chemotaxis at about the same concentrations that affected motility (Table 1).

Potassium blockers.

In animals, 4-aminopyridine and tetraethylammonium chloride are classical, specific K⁺ antagonists acting at about 1 to 10 mM on action potentials of nerve and muscle (23), but 4-aminopyridine and tetraethylammonium chloride are known sometimes to act differently from each other (see Table 2 on p. 131 of reference 23). In E. coli, 4-aminopyridine completely eliminated chemotaxis at 10 mM and had no significant effect at 1 mM or less, while the motility assay was not influenced at 10 mM (Fig. 5). Tetraethylammonium chloride had no effect on either chemotaxis or motility at 10 mM.

FIG. 5.

Effect of 4-aminopyridine on bacterial chemotaxis and motility by chemotactically wild-type bacteria (strain AW574). The procedure was as described for Fig. 4. Circles (A) represent the effect of 4-aminopyridine on chemotaxis to l-aspartate (10⁻² M in the capillary), and triangles (B) represent motility in the same chemotaxis medium as that for panel A but lacking l-aspartate. The pond of bacteria contained the same chemotaxis medium as did the capillary. In each case, the concentration of 4-aminopyridine in the pond of bacteria was the same as that in the capillary.

DISCUSSION

This study has shown that organic antagonists for the action of calcium ions, sodium ions, or potassium ions in animals have inhibitory effects on E. coli chemotaxis. The most effective Ca²⁺ antagonist that we have found for bacterial chemotaxis is ω-conotoxin GVIA, the most effective Na⁺ antagonist is saxitoxin, and the K⁺ antagonist is 4-aminopyridine. See Results for a discussion of how these antagonists act in animals.

While animal-like K⁺ channels do occur in bacteria (17, 25, 29, 38, 49, 67), animal-like Ca²⁺ channels and animal-like Na⁺ channels do not appear to be coded for by the E. coli genome (3) (see the introduction). Therefore, it is surprising that the Ca²⁺ antagonists and the Na⁺ antagonists studied here should inhibit E. coli chemotaxis. The mechanism of inhibition by the various antagonists needs to be determined.

It is clear that the Ca²⁺ antagonists all block tumbling and promote running, while the Na⁺ antagonist saxitoxin does the opposite. How this is accomplished is not known. One possibility is that the methyl-accepting chemotaxis proteins are themselves ion channels, or else the methyl-accepting chemotaxis proteins could signal to a separate ion channel (or channels). A second possibility is that the complex of methyl-accepting chemotaxis proteins-CheW-CheA-CheY or -CheY-phosphate could signal to an ion channel (or channels). A third possibility is that the antagonists do not block ion channels at all in bacteria but rather act in some different ways.

The ion channel hypothesized above could allow all three ions, Ca²⁺, Na⁺, and K⁺, to move through it, much as is done in animals by the acetylcholine receptor channel, the cGMP-gated visual receptor channel, or the cyclic AMP-gated olfaction receptor channel (23). Alternatively, there could be separate channels for each of these ions: a Na⁺ channel and a K⁺ channel perhaps to allow a change in membrane potential, similar to the typical eukaryotic action potential, and then a Ca²⁺ flux to signal the flagella to bring about tumbling, much as there is signalling by Ca²⁺ at the end of a nerve cell. There is evidence that CheY-phosphate interacts with the Fli proteins at the base of the flagellum to bring about clockwise rotation and thus tumbling (6, 46, 63); a change in membrane potential could be used to introduce Ca²⁺ at that site.

It is interesting in this regard that Metzner wrote in 1920 (translated), “The transmission [in Spirillum volutans] is usually so fast that both flagellar bundles [one bundle at each end of the cell] are reversed practically simultaneously” (p. 409 in reference 37). Krieg et al., working with flagellar coordination in S. volutans, have stated: “The most remarkable aspect, however, was that in these spirilla both flagellar fascicles [bundles of flagella at each end of the cell] were reoriented simultaneously, and it seemed almost inescapable that some kind of coordination mechanism existed between the cell poles, despite the large distance (ca. 50 μm) that intervened. . . . . By analogy to animal nerve cells, the spirillar cell membrane is a likely candidate for the location of this coordination mechanism, and the generation of an action potential in this membrane by a wave of depolarization, with resulting transmission of an electrical impulse, is not inconsistent with the rapidity with which simultaneous reorientation of flagellar fascicles occurs in normal cells of S. volutans. . . . . Certainly a variety of information will be required to illuminate further the fascinating frontier of bacterial ‘nervous systems’ ” (27). However, Caraway and Krieg have strongly suggested that action potentials are not a factor in the flagellar coordination mechanism of S. volutans but that the cell membrane may still be the site of the mechanism (9).

For E. coli, the idea of an action potential for the flagella has not received support; there is evidence for only a short-range signalling system for the flagella (50). Our recent work on filaments (“snakes”) of E. coli is in agreement with this conclusion: an action potential seems unnecessary because all along the filament there are clusters of chemoreceptors able to communicate with the nearby flagella by means of CheY-phosphate (30).

Since S. volutans (9, 27, 37) and S. aurantia (20) are 20 to 50 μm long, they may require an action potential while smaller bacteria do not, but for smaller bacteria (about 2 μm long) such as E. coli (16) and B. subtilis (33) calcium channels have been found, and for E. coli (25, 38), S. lividans (17, 29, 49), and the cyanobacterium Synechocystis spp. (13), K⁺ channels have been reported and characterized. Ion channels may be important for motility and chemotaxis, even when an action potential does not occur.