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Electric field effects on bacterial motility and chemotaxis
Bioelectrochemistry and Bioenergetics, 10 (1983) 499-5 10 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
571-ELECTRIC CHEMOTAXIS
MICHAEL HENRYK
EISENBACH *, JERALD R. ZIMMERMAN FISCHLER and RAF1 KORENSTEIN
Department (Revised
FIELD EFFECTS ON BACTERIAL
499
MOTILITY AND
**, ADINA
CIOBOTARIU,
of Membrane Research, The Weizmann Institute of Science, 76100 Rehooot (Israel)
manuscript
received
April 29th 1983)
SUMMARY The molecular nature of signal transduction in bacterial chemotaxis is virtually unknown. If the signal transduction is electrical in nature, an externally applied electric field should affect chemotactic behavior. We therefore studied the effect of an electromagnetically induced electric field on macroscopic assays of chemotaxis and motility of Escherichia coli. The electric field had opposing effects on these phenomena: it doubled motility, but inhibited chemotaxis by 70%. Controls for viability, for electrophoretic effects, and for other parameters that may affect chemotaxis, showed that this inhibition was specific for chemotaxis. These observations suggest that an electrical process may be involved in the chemotaxis machinery of E. coli. However, other interactions of the electric field with one or more of the membrane components of the chemotaxis machinery cannot be excluded.
INTRODUCTION
The swimming behavior of bacteria is not random. It is oriented towards favorable chemicals (attractants) or away from unfavorable ones (repellents). This behavior is called chemotaxis (for reviews see Refs. l-4). Bacteria detect chemicals by means of chemoreceptors, and the sensed information is transmitted to the flagella [5]. (For general considerations of sensory signals see Ref. 6). The nature of the transmitted signal is not known. It might be transmitted by means of a protein-protein interaction [7], a diffusible substance (e.g. cGMP [8], or neurotransmitters [9]), or by an electrical signal [lo-121. An electrical signal is appealing because of the analogy to eucaryotic systems [ 13- 151. Indirect evidence in favor of an electrical signal in bacteria was obtained for Spirillum volutans [ 161, Thiospirillum jenense [ 171, Rhodospirillum rubrum [ 17,181 and Spirochaeta aurantia [ 1I]. The finding of Krieg et al. that flagellar reversals in S. * To whom correspondence should be addressed. ** Present address: Dept. of Orthopedic Surgery, MN 55455, U.S.A. 0302-4598/83/$03.00
Q 1983 Elsevier Sequoia
University
S.A.
of Minnesota
Medical
School, Minneapolis,
500
volutuns are synchronous when filmed at 48 frames/s [16], led Berg to calculate that one end of the cell can signal the other in an interval of approximately 10e2 s [19]. By showing that diffusion of a substance of low molecular weight from one end of the cell to the other would take at least 1 s, Berg suggested that the signal in S. volutuns was possibly electrical in nature. The early finding of Caraway and Krieg, that S. volutans can be made to back up when an electric field is switched on or off [20], has been used as supporting evidence for this hypothesis. Similar calculations and conclusions have been published by Lee and Fitzsimons for Rhodospirillum rubrum [ 181. The situation may be different in small bacteria like Escherichia coli: it has been calculated that the response delay time of E. coli (0.2 s-Ref. 21) is sufficient to allow a small molecule to diffuse from one end of the bacterium to the other and thus to be the signal transducer [22]. If signal transduction in E. coli is of an electrical nature, an externally applied electric field should perturb the chemotactic behavior of the bacteria, as is the case with the photophobic behavior of blue-green algae [23]. Since the absence of such an effect would argue against an electrical nature for signal transduction, it is the purpose of this study to examine the effect of electric field on chemotaxis of E. coli. EXPERIMENTAL
Bacteria The strains used in this study were obtained from Dr. Julius Adler (University of Wisconsin at Madison). They are E. coli K12 derivatives. AW546 [24] and A W546edu+ are wild type for chemotaxis. A W546edu +cheAlOl is a generally non-chemotactic mutant. Allele 101 of cheA (previously used in a different background by Kort et al. [25]; it appears there as elOg1) was transferred into A W546edu+ by J. Pierce in J. Adler’s laboratory. M524 is a paralyzed mutant, having a defect in the motB gene [26]. The bacteria were precultured to stationary phase (overnight growth) at 35°C in tryptone broth (Difco). Before the capillary-assay experiment the bacteria were diluted 1 : 100 with tryptone broth and grown for 3-4 h until they gained vigorous motility (OD,,, = 0.4) and were then harvested and washed twice with wash medium consisting of KPi (10 mM, pH7), EDTA (0.1 mM), and methionine (0.1 mM). The bacteria were then diluted with wash medium to OD,,, of 0.05 (- 4 X 10’ cells per cm3) and kept on ice until used. Bacteria for swarm assays were used from fresh tryptone-broth cultures without washing. Chemotaxis and motility assays Capillary assays, measuring the accumulation of bacteria in attractant-filled (for chemotaxis) or attractant-free (for motility) capillaries, were performed as described by Adler [27]. A U-shaped capillary tube with 14 mm distance between the two arms was placed on a glass microscope slide as far to the edge of a 9 cm plastic Petri dish
501 Glass
Glass slide
U -Shaped tube Fig. 1. Schematic description text for details.
of the measuring system. The electric field is in the plane of the plate. See
as possible, and was covered by a glass cover slip. The plate and its contents were warmed in an incubator at 30°C. Two Petri dishes were removed at a time from the incubator, and the well formed by the U-shaped tube and the cover slip was filled with pre-warmed, washed bacteria (3O’C; OD,,, = 0.05). A straight capillary tube filled to a height of 5 mm with cY-methyl-DL-aspartate (cY-MeAsp) at the given concentration in wash medium, or wash medium only (for motility assays), was then inserted into the pool of bacteria in each Petri dish (Fig. 1). The experiment was carried out in couples: one of the dishes was placed in a 30°C incubator in the center of magnetic coils producing the electric field, while the other dish (the control) was placed in the incubator outside the electric field. At a given time the electric field was switched off (or not switched off at all), but the assay was continued for a total period of 30 min. The capillaries were immediately removed, and their contents were expelled into tryptone broth. Dilutions of this harvest were made and plated onto tryptone broth agar. The plates were incubated overnight at 35°C and the number of colonies grown on each plate were counted. For each condition at least eight such pairs of determinations were carried out (to give one point in a graph), and Student’s t-distribution test was applied to determine the probability for a significant difference between the measurements in and out of the electric field. Tryptone-agar swarm assays were carried out at 30°C as described by Adler [28] in the same type of Petri plates as those used for the capillary assays and under identical conditions. Each assay was carried out for 4-6 h. Those plates which were inside the coils producing the electric field were exposed to the electric field (same as in Fig. 3) for 15 mm periods with 15 min intervals.
502 Matching impedance
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Fig. 2. Schematic diagram of experimental details.
set-up of externally applied electric field to E. co/i. See text for
Electric field stimulation The experimental set-up for the electric stimulation is shown in Fig. 2. Negative rectangular voltage pulses (U’) from a high-power pulse generator were applied through an impedance matching device to two flat rectangular coils connected in series. The surfaces of the two coils were positioned parallel to one another. The magnetic fields generated by each coil enhance each other, creating between them a uniform, time-dependent magnetic field (B in Fig. 2). The field was uniform within the space enclosed by the coil up to a distance of 1 cm from the edge of the coil. The
Fig. 3. (a) Display of pulse form of the generator output Us applied to magnetic field coils. (b) Display of voltage UP induced in the search probe (for coil, see text) located within the magnetic field.
503
rate of the field strength variation was proportional to the output amplitude of the driving generator (U,). Calibration of the magnetic flux change was performed by a search probe (five turns of a single-layer air-cored coil of 16 mm diameter) yielding a typical wave form of the induced voltage shown in Fig. 3b (when applying a generator’s output voltage pulse of a form given in Fig. 3a). The applied voltage output (25 ps pulse length, 3 Hz frequency) yielded a rate of flux change in the range of 0.054 G */ps (at 20 V) to 1.21 G/ps (at 450 V). The medium under the cover slip (Fig. 1) can be considered as a cylindric conductor with a radius of 7 mm. The induced electromotive force in a conductor has a radial symmmetry, being maximal at the periphery and decreasing towards the center of the medium. The calculated maximal electric field in the periphery of such a conductor by a flux change of 0.95 G/ps is - 3.3 mV/cm and the induced current is - 0.3 PA/cm2 (the conductivity of the medium being 2.1 mmho.cm-i). RESULTS
We used the capillary assay as a macroscopic measure of chemotaxis and motility of bacteria [27, 291. In this assay the attractant diffuses out of a capillary and forms a concentration gradient in a drop of bacteria. The bacteria follow the gradient, accumulate in the capillary and are then counted by plating. To avoid metabolic perturbations we used a non-metabolizable attractant, cY-methyl-DL-aspartate ((YMeAsp). The measuring system is described in the Experimental section and is schematically presented in Fig. 1. An external electric field was applied to this system in the sample plane in the experimental set-up shown in Fig. 2. The field was induced by a pulsed, time-varying magnetic field (normal to the sample plane) created between two coils surrounding the sample. We applied low electric fields to avoid electrolytic products and lethal effects on the bacteria [30]. To examine the effect of an electric field on the motility of the bacteria we used capillaries that contained no attractant [31]. In the absence of an electric field, 760 f 100 bacteria ( f S.d., 16 determinations) accumulated in the capillary within 30 min at 30°C. When a time-dependent magnetic field of 0.95 G/hs (resulting from an output of 350 V, 25 /JS pulse length, 3 Hz frequency of the pulse generator) was applied for the first 15 min, 1590 f 270 bacteria (+s.d., 12 determinations) accumulated in the capillary within 30 min. Thus, the electric field enhanced motility to 209 -+ 35% of the value obtained in the absence of an electric field. The Student’s t-distribution test indicates a probability larger than 99.6% that the values obtained in the presence and the absence of an electric field were different. The electric field had no effect on the viability of the bacteria. We repeated the capillary assays with sealed capillaries (to avoid swimming of bacteria into them), and assayed the concentrations of the bacteria under the cover-glass slips (Fig. 1). These concentrations were (5.6 + 0.8) X 10’ and (5.3 _+ 1.4) X 10’ cells/cm (fs.d.) * The symbol G denotes the unit Gauss.
b 200
100
[a-MeAsp] C 0
10-4
10-3
lo-'
F
I
’
10-4
(M)
I
I
10-3
1o-2
Fig. 4. Capillary assays in the presence and absence of electric field as a function of the attractant concentration. The experiment was carried out as detailed in the text. The numbers of bacteria accumulated in the capillary tubes in different experiments were normalized according to an identical control assay performed in the absence of an electric field. (a) (0) Electric field pulses at a frequency of 3 Hz and 25 ps pulse length, corresponding to a fhtx change of 0.95 G/es (350 V) were applied (- - -); ). (b) (+) Relative accumulation of bacteria in the capillary; (m) electric field was not applied (( . . . . . .) presence over absence of electric field. The probability that the results in an electric field were different from those in the absence of an electric field was larger than 99.6%, 95% and 99.9% for a-MeAsp concentrations of 0, 10m4 and 10m2 M respectively, as determined by Student’s t-distribution test. At 10K3 M a-MeAsp the results were not significantly different (probability = 90% for 23 determinations).
for samples respectively exposed or not exposed to the electric field (at the same pulse frequency and intensity as before). Similarly, the electric field had no effect on the passive accumulation of bacteria in the capillary. For this control we used the paralyzed motB mutant, M524, and compared its accumulation in capillary tubes (without attractant) exposed or not exposed to an electric field. In the absence of an electric field 1030 1fl360 paralyzed bacteria ( f s.d., 9 determinations) accumulated in the capillary within 30 min under the conditions of Fig. 4, as compared to 800 f 360 bacteria ( f s-d., 9 determinations) in the presence of an electric field. This apparent difference was not significant, as determined by Student’s t-distribution test. The effect of an electric field on a chemotactic response is shown in Fig. 4. At lo-* M a-MeAsp, the electric field decreased the number of bacteria accumulated in the capillary from 128000 f 7000 to 87000 f 5000 bacteria. When the simultaneous enhancement of motility is taken into account, chemotaxis appears to be inhibited by approximately 70%. Thus, it seems that an electric field has opposing effects: it enhances motility but inhibits chemotaxis. Between 10m4 and 10e3 M a-MeAsp, the enhancement of motility just balanced the inhibition of chemotaxis, and the electric field had no apparent effect on the number of bacteria accumulating in the capillary. The relative inhibition of chemotaxis is shown in Fig. 5 as a function of the electric-field intensity (proportional to the magnetic flux change or to the output
505
I
0
I
I 100 0.27
I
I 300 081
I
I 500 v 135 Gauss/p
Fig. 5. Inhibition of chemotaxis by externally applied electric field. The output of the pulse generator was calibrated by a search coil and the corresponding flux changes (G/ps) are given with the corresponding voltage output (V). The experiment was carried out as described in the text with capillary tubes containing lo-* M o-MeAsp. The relative accumulation is the ratio between the number of bacteria accumulated in the capillary in the presence and the absence of an electric field. For each voltage a separate control in the absence of an electric field was carried out and considered as 100%. The enhancement of motility was not considered in this calculation. (Taking the increased motility into account, the percentage of bacteria accumulated in the capillary would be approximately half of the written values.) The number of bacteria accumulated in the capillary in these controls were 25 000 f 5000 (6) for 20 V (0.054 G/gs) ( f s. d. in parentheses are the number of control experiments); 31000~2000 (12) for 100 V (0.27 G/ps); 43000~2000 (12) for 250 V (0.67 G/ps); 48000~3000 (26) for 350 V (0.95 G/ps); 14OOOf 1000 (27) for 450 V (1.21 G/ps). The number of experiments in an electric field were 5, 14, 12, 28, 26 for 20, 100, 250, 350,450 V respectively. The probability that the results in an electric field were different from those in the absence of an electric field was less than 50% (not significant) for 20 V, but greater than 99.7% for 100 and 250 V, greater than 99.9% for 350 V and greater than 75% (not significant) for 450 V, as determined by Student’s r-distribution test.
voltage of the pulse generator). A wide peak was observed, with maximal inhibition of chemotaxis in the range 0.27-0.95 G/ps (NO-350 V). The lowest frequency (3 Hz) and the longest pulse (25 ps) of our pulse generator gave the largest effect of the electric field. Perhaps lower frequencies and longer pulses would have stronger effects on motility and chemotaxis (cf. the low frequency of electric field required to inhibit photophobic reactions in blue-green algae [23]). To test the above observations we used a quite different technique to measure chemotaxis, the tryptone-agar swarm assay. Bacteria are put in the center of a plate containing a concentration of agar in tryptone broth (0.35%) that allows migration [28]. The amino acids of casein are the major constituents of tryptone, and some are potent attractants of E. coli. As the bacteria grow they use up the local supply of attractant amino acid, and then they follow the gradient they themselves have
506
Fig. 6. Tryptone-swarm assays in the presence and absence of an electric field. Freshly grown bacteria in tryptone broth were inoculated from a sterile loop (2 mm in diam.) in the middle of a Petri dish containing soft tryptone agar (0.35% w/w). The experiment was carried out at 3O’C as detailed in the text. The bacteria are in the white concentric rings or at the white area in the center of the plate. The large external ring in each photograph is the edge of the plate. The photographs at the left (A, C, E) are in the absence of an electric field, and those at the right (B, D, F) are in its presence (same magnitude and pulse frequency as in Fig. 3). The bar at the bottom represents 1.0 cm. (A, B) Wild-type strain, AW546. Bacteria
507
produced. At the same time they multiply. The result is an expanding ring of bacteria for each metabolizable attractant, primarily serine and aspartate [28]. These rings were clearly observable in the control experiments, carried out in the absence of an electric field (Figs. 6A, C). To make sure that every generation of bacteria experienced the electric field, we applied it (at the same pulse frequency and intensity as before) for 15 min periods with 15 min intervals throughout the assay (4-6 h). The effect of the electric field on a swarm assay that was initiated from a dense inoculum of bacteria at their stationary growth phase is shown in Fig. 6A, B. Three rings were formed during 4 h in the absence of an electric field, as compared to one diffused ring in its presence. When the assay was initiated from a more dilute inoculum of bacteria at their exponential growth phase, three rings were formed in the absence of an electric field within 5.75 h (Fig. 6C). The bacteria that were exposed to the electric field under the same conditions formed two smaller rings (Fig. 6D). We carried out 10 such assays with results varying between a strong inhibition of ring formation (65% inhibition as measured according to the external ring diameter in Fig. 6A, B) to weak inhibition (20% inhibition as measured from Fig. 6C, D). In all these assays inhibition by the electric field was observed. Motile but non-chemotactic mutants cannot form rings in the swarm assay. Instead, they randomly distribute around the inoculation site [32]. This is shown in Fig. 6E for a non-chemotactic mutant derivative of the wild-type strain used above. The distribution of the cells around the inoculation site was of similar size when the electric field was applied (Fig. 6F). This observation once more indicates that the motility was not harmed. Also, the viability of the eells was not harmed by the 6 h alternate exposure to the electric field. This was confirmed in a separate experiment by counting the number of colonies formed from a given inoculum that had either been exposed or unexposed to the electric field. DISCUSSION
This study shows that an externally applied electric field affects both motility and chemotaxis of bacteria, though in an antagonistic manner: it enhances motility and inhibits chemotaxis. Factors, not directly related to motility or chemotaxis, that could, in principle, be affected by the electric field and perturb the observations are as follows: (1) Sample heating: the induced electric field and current were extremely low in this study (see Experimental) and could not account for any heating effects.
at their stationary growth phase (5 x lo9 cells/cm3) were used for the inoculum. The time length of the assay was 4 h. (C, D) Wild-type strain, AW546edu+. Bacteria at their exponential growth phase (1 x lo9 cells/cm3) were used for the inoculum. The time length of the assay was 5.75 h. Identical results were obtained with AW546 under the same conditions. (E, F) Motile but non-chemotactic mutant, A W546educc/zeA101. The same pattern was observed with other the mutants tested. Experimental conditions as in C and D.
508
(2) Viability: separate measurements did not reveal any harmful effects. (3) Electrophoresis: at the low electric field applied the electrophoretic motion of either the bacteria (see Results with the paralyzed mutant) or the chemicals was negligible. The chemical gradient formed by the attractant diffusing from the capillary was not, therefore, perturbed. In fact, any factor, not related to the attractant, should have the same effect on the bacteria accumulating in the attractant-filled or attractant-free capillaries. Since opposing effects were observed, the decrease in the number of bacteria accumulating in the attractant-filled capillary should be either specific to chemotaxis or to perturbations related to the attractant. Processes related to the latter are its transport, metabolism and chemotaxis. Metabolism of the attractant was eliminated in the capillary assay because we used a non-metabolizable attractant in non-growth medium. The only way in which altered transport of the attractant could affect the number of bacteria accumulating in the capillary would be by perturbing the gradient of the attractant diffusing from the capillary (transport of the attractant is not required for chemotaxis [5]). If this were the case, such effects on the gradient should be at low attractant concentrations. The contrary was observed: inhibition by electric fields was observed at the highest attractant concentration examined (lop2 M, the peak response in the control assay). At this concentration, it is hard to envisage how any altered transport could significantly affect the gradient. Thus, it seems that the electric field affects the chemotaxis machinery directly. This conclusion is strongly supported by the independent technique used to measure chemotaxis, the tryptone-swarm assay (Fig. 6). Furthermore, this assay indicates that the effect of the electric field was not specific for a single attractant, but rather was a general phenomenon in chemotaxis. What is the mode of interaction between the electric field and the bacterial cell? Since the extracellular and the intracellular conductances are much higher than the membrane conductance, the effect of the electric field is on the membrane only. The voltage drop in the cytoplasm would then be orders of magnitude lower than that across the membrane. At a magnetic flux change of 0.95 G/ps the calculated external electric field for the capillary assay is - 3.3 mV/cm (see Experimental). The polarization of the cytoplasmic membrane induced by such an electric field can be calculated [33] to be in the order of 1 pV. (Electric fields of few millivolts per cm were used clinically in orthopedic treatment of bone non-unions [34,35]. Furthermore, modulation of cellular activity of bone cells in culture by similar low electromagnetic fields has been recently demonstrated [36].) The molecular mechanism of interaction of such a low external electric field with a cellular system at the membrane level is still unknown. An example for a plausible mechanism to account for the observed interaction could involve electrically induced perturbation of surface charge changes that might be involved in signalling or in inducing the activity of an enzyme. The inhibitory effect of the external electric field on the chemotaxis machinery may be attributed to either one of the following mechanisms: (a), perturbation of the hypothesized electrical signalling;
509
(b), modulation of one or more proteins that are involved in chemotaxis; (c), modulation of a trans-membrane ion flux involved in chemotaxis [37-391. The scope of this study does not allow us to distinguish between these possibilities. For this purpose, the effect of an electric field on the behavior of individual bacteria is currently under study. In these studies prominent effects of the electric field on tethered E. co/i and Salmonella typhimurium cells have been clearly observable under the microscope (R. Perl-Treves and M. Eisenbach, unpublished observations). ACKNOWLEDGEMENTS
We thank Dr. Julius Adler for his suggestion to perform a viability test, and to Dr. Robert M. Macnab for his constructive comments on this manuscript. This study was supported by research grants to M.E. from the U.S. National Institute of Allergy and Infectious Diseases and from the U.S.-Israel Binational Science Foundation (B.S.F.), Jerusalem, Israel; and by a research grant to R.K. from Stiftung Volkswagenwerk. M.E. holds the Barecha Foundation Career Development Chair. R.K. is an incumbent of the Association of Friends of the Weizmann Institute of Science in Israel Career Development Chair. REFERENCES 1 J. Adler in the Harvey Lectures, Academic Press, Series 72, 1978, p. 195. ? D.E. Koshland, Jr. Bacterial Chemotaxis as a Model Behavioral System. Raven Press, New York, 1980. 3 B.L. Taylor and D.J. Laszlo in the Perception of Behavioral Chemicals, D.M. Norris, (Editor), Else.vier/North-Holland, Amsterdam, 1981 p. 1. 4 R.M. Macnab, Sot. Exp. Biol. Symp. (Cambridge University Press), 35 (1982) 77. 5 J. Adler, Science, 166 (1969) 1588. 6 D.E. Koshland Jr., A. Goldbeter and J.B. Stock, Science, 217 (1982) 220. 7 J.S. Parkinson, Annu. Rev. Genet., 11 (1977) 397. 8 R.A. Black, A.C. Hobson and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 3879. 9 I. Chet, Y. Henis, and R. Mitchell, J. Bacterial., 115 (1973) 1215. 10 S. Szmelcman and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 4387. 11 E.A. Goulboume, Jr. and E.P. Greenberg, J. Bacterial., 148 (1981) 837. 12 G.V. Murvanidze and A.N. Glagolev, J. Bactehol., 150 (1982) 239. 13 R. Eckert, Science, 176 (1972) 473. 14 C. Kung, S.-Y. Chang, Y. Satow, J. Van Houten and H. Hansma, Science, 188 (1975) 898. 15 W. Ulbricht, Annu. Rev. Biophys. Bioeng., 6 (1977) 7. 16 N.R. Krieg, J.P. Tomelty and J.S. Wells, Jr., J. Bacterial., 94 (1967) 1431. 17 M.A. Faust and R.N. Doetsch, Can. J. Microbial., 17 (1971) 191. 18 A.G. Lee and J.T.R. Fitzsimons, J. Gen. Microbial, 93 (1976) 346. 19 H.C. Berg, Annu. Rev: Biophys. Bioeng., 4 (1975) 119. 20 B.H. Caraway and N.R. Krieg, Can. J. Microbial, 18 (1972) 1749. 21 J.E. Segall, M.D. Manson and H.C. Berg. Nature (London), 296 (1982) 855. 22 M.A. Snyder, J.B. Stock and D.E. Koshland, Jr., J. Mol. Biol., 149 (1981) 241. 23 D.P. HBder, Arch. Microbial., 114 (1977) 83. 24 G.W. Ordal and J. Adler, J. Bacterial., 117 (1974) 517.
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25 26 27 28 29 30 31 32 33 34 35 36
E.N. Kort, M.F. Goy, S.H. Larsen and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 3939. J.B. Armstrong and J. Adler, Genetics, 56 (1967) 363. J. Adler, J. Gen. Microbial., 74 (1973) 77. J. Adler, Science, 153 (1966) 708. J. Adler and M.M. Dahl, J. Gen. Microbial., 46 (1967) 161. H. Htilsheger and E.-G. Niemann, Radiat. Environ. Biophys., 18 (1980) 281. J. Adler and B. Templeton, J. Gen. Microbial., 46 (1967) 175. J.B. Armstrong and J. Adler, J. Bacterial., 97 (1969) 156. D. Farkas, R. Korenstein and S. Malkin, FEBS Lett., 120 (1980) 236. C.A.L. Bassett, R.J. Pawluk and A.A. Pilla, Science, 184 (1974) 575. C.A.L. Bassett, A.A. Pilla and R.J. Pawluk, Clin. Orthop., 124 (1977) 128. R.A. Luben, CD. Cain, C.-Y. Chen, D.M. Rosen and W.R. Adey, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4180. 37 M. Eisenbach, Biochemistry, 21 (1982) 6818. 38 M. Eisenbach in Biological Structures and Coupled Flows, A. Oplatka and M. Balaban (Editors), Academic Press, New York and Balaban I.S.S., Philadelphia, 1983, p. 349. 39 M. Eisenbach, T. Raz and A. Ciobotariu, Biochemistry, 22 (1983) 3293.
571-ELECTRIC CHEMOTAXIS
MICHAEL HENRYK
EISENBACH *, JERALD R. ZIMMERMAN FISCHLER and RAF1 KORENSTEIN
Department (Revised
FIELD EFFECTS ON BACTERIAL
499
MOTILITY AND
**, ADINA
CIOBOTARIU,
of Membrane Research, The Weizmann Institute of Science, 76100 Rehooot (Israel)
manuscript
received
April 29th 1983)
SUMMARY The molecular nature of signal transduction in bacterial chemotaxis is virtually unknown. If the signal transduction is electrical in nature, an externally applied electric field should affect chemotactic behavior. We therefore studied the effect of an electromagnetically induced electric field on macroscopic assays of chemotaxis and motility of Escherichia coli. The electric field had opposing effects on these phenomena: it doubled motility, but inhibited chemotaxis by 70%. Controls for viability, for electrophoretic effects, and for other parameters that may affect chemotaxis, showed that this inhibition was specific for chemotaxis. These observations suggest that an electrical process may be involved in the chemotaxis machinery of E. coli. However, other interactions of the electric field with one or more of the membrane components of the chemotaxis machinery cannot be excluded.
INTRODUCTION
The swimming behavior of bacteria is not random. It is oriented towards favorable chemicals (attractants) or away from unfavorable ones (repellents). This behavior is called chemotaxis (for reviews see Refs. l-4). Bacteria detect chemicals by means of chemoreceptors, and the sensed information is transmitted to the flagella [5]. (For general considerations of sensory signals see Ref. 6). The nature of the transmitted signal is not known. It might be transmitted by means of a protein-protein interaction [7], a diffusible substance (e.g. cGMP [8], or neurotransmitters [9]), or by an electrical signal [lo-121. An electrical signal is appealing because of the analogy to eucaryotic systems [ 13- 151. Indirect evidence in favor of an electrical signal in bacteria was obtained for Spirillum volutans [ 161, Thiospirillum jenense [ 171, Rhodospirillum rubrum [ 17,181 and Spirochaeta aurantia [ 1I]. The finding of Krieg et al. that flagellar reversals in S. * To whom correspondence should be addressed. ** Present address: Dept. of Orthopedic Surgery, MN 55455, U.S.A. 0302-4598/83/$03.00
Q 1983 Elsevier Sequoia
University
S.A.
of Minnesota
Medical
School, Minneapolis,
500
volutuns are synchronous when filmed at 48 frames/s [16], led Berg to calculate that one end of the cell can signal the other in an interval of approximately 10e2 s [19]. By showing that diffusion of a substance of low molecular weight from one end of the cell to the other would take at least 1 s, Berg suggested that the signal in S. volutuns was possibly electrical in nature. The early finding of Caraway and Krieg, that S. volutans can be made to back up when an electric field is switched on or off [20], has been used as supporting evidence for this hypothesis. Similar calculations and conclusions have been published by Lee and Fitzsimons for Rhodospirillum rubrum [ 181. The situation may be different in small bacteria like Escherichia coli: it has been calculated that the response delay time of E. coli (0.2 s-Ref. 21) is sufficient to allow a small molecule to diffuse from one end of the bacterium to the other and thus to be the signal transducer [22]. If signal transduction in E. coli is of an electrical nature, an externally applied electric field should perturb the chemotactic behavior of the bacteria, as is the case with the photophobic behavior of blue-green algae [23]. Since the absence of such an effect would argue against an electrical nature for signal transduction, it is the purpose of this study to examine the effect of electric field on chemotaxis of E. coli. EXPERIMENTAL
Bacteria The strains used in this study were obtained from Dr. Julius Adler (University of Wisconsin at Madison). They are E. coli K12 derivatives. AW546 [24] and A W546edu+ are wild type for chemotaxis. A W546edu +cheAlOl is a generally non-chemotactic mutant. Allele 101 of cheA (previously used in a different background by Kort et al. [25]; it appears there as elOg1) was transferred into A W546edu+ by J. Pierce in J. Adler’s laboratory. M524 is a paralyzed mutant, having a defect in the motB gene [26]. The bacteria were precultured to stationary phase (overnight growth) at 35°C in tryptone broth (Difco). Before the capillary-assay experiment the bacteria were diluted 1 : 100 with tryptone broth and grown for 3-4 h until they gained vigorous motility (OD,,, = 0.4) and were then harvested and washed twice with wash medium consisting of KPi (10 mM, pH7), EDTA (0.1 mM), and methionine (0.1 mM). The bacteria were then diluted with wash medium to OD,,, of 0.05 (- 4 X 10’ cells per cm3) and kept on ice until used. Bacteria for swarm assays were used from fresh tryptone-broth cultures without washing. Chemotaxis and motility assays Capillary assays, measuring the accumulation of bacteria in attractant-filled (for chemotaxis) or attractant-free (for motility) capillaries, were performed as described by Adler [27]. A U-shaped capillary tube with 14 mm distance between the two arms was placed on a glass microscope slide as far to the edge of a 9 cm plastic Petri dish
501 Glass
Glass slide
U -Shaped tube Fig. 1. Schematic description text for details.
of the measuring system. The electric field is in the plane of the plate. See
as possible, and was covered by a glass cover slip. The plate and its contents were warmed in an incubator at 30°C. Two Petri dishes were removed at a time from the incubator, and the well formed by the U-shaped tube and the cover slip was filled with pre-warmed, washed bacteria (3O’C; OD,,, = 0.05). A straight capillary tube filled to a height of 5 mm with cY-methyl-DL-aspartate (cY-MeAsp) at the given concentration in wash medium, or wash medium only (for motility assays), was then inserted into the pool of bacteria in each Petri dish (Fig. 1). The experiment was carried out in couples: one of the dishes was placed in a 30°C incubator in the center of magnetic coils producing the electric field, while the other dish (the control) was placed in the incubator outside the electric field. At a given time the electric field was switched off (or not switched off at all), but the assay was continued for a total period of 30 min. The capillaries were immediately removed, and their contents were expelled into tryptone broth. Dilutions of this harvest were made and plated onto tryptone broth agar. The plates were incubated overnight at 35°C and the number of colonies grown on each plate were counted. For each condition at least eight such pairs of determinations were carried out (to give one point in a graph), and Student’s t-distribution test was applied to determine the probability for a significant difference between the measurements in and out of the electric field. Tryptone-agar swarm assays were carried out at 30°C as described by Adler [28] in the same type of Petri plates as those used for the capillary assays and under identical conditions. Each assay was carried out for 4-6 h. Those plates which were inside the coils producing the electric field were exposed to the electric field (same as in Fig. 3) for 15 mm periods with 15 min intervals.
502 Matching impedance
Hi h power pu9 se generator r--___--__~
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Fig. 2. Schematic diagram of experimental details.
set-up of externally applied electric field to E. co/i. See text for
Electric field stimulation The experimental set-up for the electric stimulation is shown in Fig. 2. Negative rectangular voltage pulses (U’) from a high-power pulse generator were applied through an impedance matching device to two flat rectangular coils connected in series. The surfaces of the two coils were positioned parallel to one another. The magnetic fields generated by each coil enhance each other, creating between them a uniform, time-dependent magnetic field (B in Fig. 2). The field was uniform within the space enclosed by the coil up to a distance of 1 cm from the edge of the coil. The
Fig. 3. (a) Display of pulse form of the generator output Us applied to magnetic field coils. (b) Display of voltage UP induced in the search probe (for coil, see text) located within the magnetic field.
503
rate of the field strength variation was proportional to the output amplitude of the driving generator (U,). Calibration of the magnetic flux change was performed by a search probe (five turns of a single-layer air-cored coil of 16 mm diameter) yielding a typical wave form of the induced voltage shown in Fig. 3b (when applying a generator’s output voltage pulse of a form given in Fig. 3a). The applied voltage output (25 ps pulse length, 3 Hz frequency) yielded a rate of flux change in the range of 0.054 G */ps (at 20 V) to 1.21 G/ps (at 450 V). The medium under the cover slip (Fig. 1) can be considered as a cylindric conductor with a radius of 7 mm. The induced electromotive force in a conductor has a radial symmmetry, being maximal at the periphery and decreasing towards the center of the medium. The calculated maximal electric field in the periphery of such a conductor by a flux change of 0.95 G/ps is - 3.3 mV/cm and the induced current is - 0.3 PA/cm2 (the conductivity of the medium being 2.1 mmho.cm-i). RESULTS
We used the capillary assay as a macroscopic measure of chemotaxis and motility of bacteria [27, 291. In this assay the attractant diffuses out of a capillary and forms a concentration gradient in a drop of bacteria. The bacteria follow the gradient, accumulate in the capillary and are then counted by plating. To avoid metabolic perturbations we used a non-metabolizable attractant, cY-methyl-DL-aspartate ((YMeAsp). The measuring system is described in the Experimental section and is schematically presented in Fig. 1. An external electric field was applied to this system in the sample plane in the experimental set-up shown in Fig. 2. The field was induced by a pulsed, time-varying magnetic field (normal to the sample plane) created between two coils surrounding the sample. We applied low electric fields to avoid electrolytic products and lethal effects on the bacteria [30]. To examine the effect of an electric field on the motility of the bacteria we used capillaries that contained no attractant [31]. In the absence of an electric field, 760 f 100 bacteria ( f S.d., 16 determinations) accumulated in the capillary within 30 min at 30°C. When a time-dependent magnetic field of 0.95 G/hs (resulting from an output of 350 V, 25 /JS pulse length, 3 Hz frequency of the pulse generator) was applied for the first 15 min, 1590 f 270 bacteria (+s.d., 12 determinations) accumulated in the capillary within 30 min. Thus, the electric field enhanced motility to 209 -+ 35% of the value obtained in the absence of an electric field. The Student’s t-distribution test indicates a probability larger than 99.6% that the values obtained in the presence and the absence of an electric field were different. The electric field had no effect on the viability of the bacteria. We repeated the capillary assays with sealed capillaries (to avoid swimming of bacteria into them), and assayed the concentrations of the bacteria under the cover-glass slips (Fig. 1). These concentrations were (5.6 + 0.8) X 10’ and (5.3 _+ 1.4) X 10’ cells/cm (fs.d.) * The symbol G denotes the unit Gauss.
b 200
100
[a-MeAsp] C 0
10-4
10-3
lo-'
F
I
’
10-4
(M)
I
I
10-3
1o-2
Fig. 4. Capillary assays in the presence and absence of electric field as a function of the attractant concentration. The experiment was carried out as detailed in the text. The numbers of bacteria accumulated in the capillary tubes in different experiments were normalized according to an identical control assay performed in the absence of an electric field. (a) (0) Electric field pulses at a frequency of 3 Hz and 25 ps pulse length, corresponding to a fhtx change of 0.95 G/es (350 V) were applied (- - -); ). (b) (+) Relative accumulation of bacteria in the capillary; (m) electric field was not applied (( . . . . . .) presence over absence of electric field. The probability that the results in an electric field were different from those in the absence of an electric field was larger than 99.6%, 95% and 99.9% for a-MeAsp concentrations of 0, 10m4 and 10m2 M respectively, as determined by Student’s t-distribution test. At 10K3 M a-MeAsp the results were not significantly different (probability = 90% for 23 determinations).
for samples respectively exposed or not exposed to the electric field (at the same pulse frequency and intensity as before). Similarly, the electric field had no effect on the passive accumulation of bacteria in the capillary. For this control we used the paralyzed motB mutant, M524, and compared its accumulation in capillary tubes (without attractant) exposed or not exposed to an electric field. In the absence of an electric field 1030 1fl360 paralyzed bacteria ( f s.d., 9 determinations) accumulated in the capillary within 30 min under the conditions of Fig. 4, as compared to 800 f 360 bacteria ( f s-d., 9 determinations) in the presence of an electric field. This apparent difference was not significant, as determined by Student’s t-distribution test. The effect of an electric field on a chemotactic response is shown in Fig. 4. At lo-* M a-MeAsp, the electric field decreased the number of bacteria accumulated in the capillary from 128000 f 7000 to 87000 f 5000 bacteria. When the simultaneous enhancement of motility is taken into account, chemotaxis appears to be inhibited by approximately 70%. Thus, it seems that an electric field has opposing effects: it enhances motility but inhibits chemotaxis. Between 10m4 and 10e3 M a-MeAsp, the enhancement of motility just balanced the inhibition of chemotaxis, and the electric field had no apparent effect on the number of bacteria accumulating in the capillary. The relative inhibition of chemotaxis is shown in Fig. 5 as a function of the electric-field intensity (proportional to the magnetic flux change or to the output
505
I
0
I
I 100 0.27
I
I 300 081
I
I 500 v 135 Gauss/p
Fig. 5. Inhibition of chemotaxis by externally applied electric field. The output of the pulse generator was calibrated by a search coil and the corresponding flux changes (G/ps) are given with the corresponding voltage output (V). The experiment was carried out as described in the text with capillary tubes containing lo-* M o-MeAsp. The relative accumulation is the ratio between the number of bacteria accumulated in the capillary in the presence and the absence of an electric field. For each voltage a separate control in the absence of an electric field was carried out and considered as 100%. The enhancement of motility was not considered in this calculation. (Taking the increased motility into account, the percentage of bacteria accumulated in the capillary would be approximately half of the written values.) The number of bacteria accumulated in the capillary in these controls were 25 000 f 5000 (6) for 20 V (0.054 G/gs) ( f s. d. in parentheses are the number of control experiments); 31000~2000 (12) for 100 V (0.27 G/ps); 43000~2000 (12) for 250 V (0.67 G/ps); 48000~3000 (26) for 350 V (0.95 G/ps); 14OOOf 1000 (27) for 450 V (1.21 G/ps). The number of experiments in an electric field were 5, 14, 12, 28, 26 for 20, 100, 250, 350,450 V respectively. The probability that the results in an electric field were different from those in the absence of an electric field was less than 50% (not significant) for 20 V, but greater than 99.7% for 100 and 250 V, greater than 99.9% for 350 V and greater than 75% (not significant) for 450 V, as determined by Student’s r-distribution test.
voltage of the pulse generator). A wide peak was observed, with maximal inhibition of chemotaxis in the range 0.27-0.95 G/ps (NO-350 V). The lowest frequency (3 Hz) and the longest pulse (25 ps) of our pulse generator gave the largest effect of the electric field. Perhaps lower frequencies and longer pulses would have stronger effects on motility and chemotaxis (cf. the low frequency of electric field required to inhibit photophobic reactions in blue-green algae [23]). To test the above observations we used a quite different technique to measure chemotaxis, the tryptone-agar swarm assay. Bacteria are put in the center of a plate containing a concentration of agar in tryptone broth (0.35%) that allows migration [28]. The amino acids of casein are the major constituents of tryptone, and some are potent attractants of E. coli. As the bacteria grow they use up the local supply of attractant amino acid, and then they follow the gradient they themselves have
506
Fig. 6. Tryptone-swarm assays in the presence and absence of an electric field. Freshly grown bacteria in tryptone broth were inoculated from a sterile loop (2 mm in diam.) in the middle of a Petri dish containing soft tryptone agar (0.35% w/w). The experiment was carried out at 3O’C as detailed in the text. The bacteria are in the white concentric rings or at the white area in the center of the plate. The large external ring in each photograph is the edge of the plate. The photographs at the left (A, C, E) are in the absence of an electric field, and those at the right (B, D, F) are in its presence (same magnitude and pulse frequency as in Fig. 3). The bar at the bottom represents 1.0 cm. (A, B) Wild-type strain, AW546. Bacteria
507
produced. At the same time they multiply. The result is an expanding ring of bacteria for each metabolizable attractant, primarily serine and aspartate [28]. These rings were clearly observable in the control experiments, carried out in the absence of an electric field (Figs. 6A, C). To make sure that every generation of bacteria experienced the electric field, we applied it (at the same pulse frequency and intensity as before) for 15 min periods with 15 min intervals throughout the assay (4-6 h). The effect of the electric field on a swarm assay that was initiated from a dense inoculum of bacteria at their stationary growth phase is shown in Fig. 6A, B. Three rings were formed during 4 h in the absence of an electric field, as compared to one diffused ring in its presence. When the assay was initiated from a more dilute inoculum of bacteria at their exponential growth phase, three rings were formed in the absence of an electric field within 5.75 h (Fig. 6C). The bacteria that were exposed to the electric field under the same conditions formed two smaller rings (Fig. 6D). We carried out 10 such assays with results varying between a strong inhibition of ring formation (65% inhibition as measured according to the external ring diameter in Fig. 6A, B) to weak inhibition (20% inhibition as measured from Fig. 6C, D). In all these assays inhibition by the electric field was observed. Motile but non-chemotactic mutants cannot form rings in the swarm assay. Instead, they randomly distribute around the inoculation site [32]. This is shown in Fig. 6E for a non-chemotactic mutant derivative of the wild-type strain used above. The distribution of the cells around the inoculation site was of similar size when the electric field was applied (Fig. 6F). This observation once more indicates that the motility was not harmed. Also, the viability of the eells was not harmed by the 6 h alternate exposure to the electric field. This was confirmed in a separate experiment by counting the number of colonies formed from a given inoculum that had either been exposed or unexposed to the electric field. DISCUSSION
This study shows that an externally applied electric field affects both motility and chemotaxis of bacteria, though in an antagonistic manner: it enhances motility and inhibits chemotaxis. Factors, not directly related to motility or chemotaxis, that could, in principle, be affected by the electric field and perturb the observations are as follows: (1) Sample heating: the induced electric field and current were extremely low in this study (see Experimental) and could not account for any heating effects.
at their stationary growth phase (5 x lo9 cells/cm3) were used for the inoculum. The time length of the assay was 4 h. (C, D) Wild-type strain, AW546edu+. Bacteria at their exponential growth phase (1 x lo9 cells/cm3) were used for the inoculum. The time length of the assay was 5.75 h. Identical results were obtained with AW546 under the same conditions. (E, F) Motile but non-chemotactic mutant, A W546educc/zeA101. The same pattern was observed with other the mutants tested. Experimental conditions as in C and D.
508
(2) Viability: separate measurements did not reveal any harmful effects. (3) Electrophoresis: at the low electric field applied the electrophoretic motion of either the bacteria (see Results with the paralyzed mutant) or the chemicals was negligible. The chemical gradient formed by the attractant diffusing from the capillary was not, therefore, perturbed. In fact, any factor, not related to the attractant, should have the same effect on the bacteria accumulating in the attractant-filled or attractant-free capillaries. Since opposing effects were observed, the decrease in the number of bacteria accumulating in the attractant-filled capillary should be either specific to chemotaxis or to perturbations related to the attractant. Processes related to the latter are its transport, metabolism and chemotaxis. Metabolism of the attractant was eliminated in the capillary assay because we used a non-metabolizable attractant in non-growth medium. The only way in which altered transport of the attractant could affect the number of bacteria accumulating in the capillary would be by perturbing the gradient of the attractant diffusing from the capillary (transport of the attractant is not required for chemotaxis [5]). If this were the case, such effects on the gradient should be at low attractant concentrations. The contrary was observed: inhibition by electric fields was observed at the highest attractant concentration examined (lop2 M, the peak response in the control assay). At this concentration, it is hard to envisage how any altered transport could significantly affect the gradient. Thus, it seems that the electric field affects the chemotaxis machinery directly. This conclusion is strongly supported by the independent technique used to measure chemotaxis, the tryptone-swarm assay (Fig. 6). Furthermore, this assay indicates that the effect of the electric field was not specific for a single attractant, but rather was a general phenomenon in chemotaxis. What is the mode of interaction between the electric field and the bacterial cell? Since the extracellular and the intracellular conductances are much higher than the membrane conductance, the effect of the electric field is on the membrane only. The voltage drop in the cytoplasm would then be orders of magnitude lower than that across the membrane. At a magnetic flux change of 0.95 G/ps the calculated external electric field for the capillary assay is - 3.3 mV/cm (see Experimental). The polarization of the cytoplasmic membrane induced by such an electric field can be calculated [33] to be in the order of 1 pV. (Electric fields of few millivolts per cm were used clinically in orthopedic treatment of bone non-unions [34,35]. Furthermore, modulation of cellular activity of bone cells in culture by similar low electromagnetic fields has been recently demonstrated [36].) The molecular mechanism of interaction of such a low external electric field with a cellular system at the membrane level is still unknown. An example for a plausible mechanism to account for the observed interaction could involve electrically induced perturbation of surface charge changes that might be involved in signalling or in inducing the activity of an enzyme. The inhibitory effect of the external electric field on the chemotaxis machinery may be attributed to either one of the following mechanisms: (a), perturbation of the hypothesized electrical signalling;
509
(b), modulation of one or more proteins that are involved in chemotaxis; (c), modulation of a trans-membrane ion flux involved in chemotaxis [37-391. The scope of this study does not allow us to distinguish between these possibilities. For this purpose, the effect of an electric field on the behavior of individual bacteria is currently under study. In these studies prominent effects of the electric field on tethered E. co/i and Salmonella typhimurium cells have been clearly observable under the microscope (R. Perl-Treves and M. Eisenbach, unpublished observations). ACKNOWLEDGEMENTS
We thank Dr. Julius Adler for his suggestion to perform a viability test, and to Dr. Robert M. Macnab for his constructive comments on this manuscript. This study was supported by research grants to M.E. from the U.S. National Institute of Allergy and Infectious Diseases and from the U.S.-Israel Binational Science Foundation (B.S.F.), Jerusalem, Israel; and by a research grant to R.K. from Stiftung Volkswagenwerk. M.E. holds the Barecha Foundation Career Development Chair. R.K. is an incumbent of the Association of Friends of the Weizmann Institute of Science in Israel Career Development Chair. REFERENCES 1 J. Adler in the Harvey Lectures, Academic Press, Series 72, 1978, p. 195. ? D.E. Koshland, Jr. Bacterial Chemotaxis as a Model Behavioral System. Raven Press, New York, 1980. 3 B.L. Taylor and D.J. Laszlo in the Perception of Behavioral Chemicals, D.M. Norris, (Editor), Else.vier/North-Holland, Amsterdam, 1981 p. 1. 4 R.M. Macnab, Sot. Exp. Biol. Symp. (Cambridge University Press), 35 (1982) 77. 5 J. Adler, Science, 166 (1969) 1588. 6 D.E. Koshland Jr., A. Goldbeter and J.B. Stock, Science, 217 (1982) 220. 7 J.S. Parkinson, Annu. Rev. Genet., 11 (1977) 397. 8 R.A. Black, A.C. Hobson and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 3879. 9 I. Chet, Y. Henis, and R. Mitchell, J. Bacterial., 115 (1973) 1215. 10 S. Szmelcman and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 4387. 11 E.A. Goulboume, Jr. and E.P. Greenberg, J. Bacterial., 148 (1981) 837. 12 G.V. Murvanidze and A.N. Glagolev, J. Bactehol., 150 (1982) 239. 13 R. Eckert, Science, 176 (1972) 473. 14 C. Kung, S.-Y. Chang, Y. Satow, J. Van Houten and H. Hansma, Science, 188 (1975) 898. 15 W. Ulbricht, Annu. Rev. Biophys. Bioeng., 6 (1977) 7. 16 N.R. Krieg, J.P. Tomelty and J.S. Wells, Jr., J. Bacterial., 94 (1967) 1431. 17 M.A. Faust and R.N. Doetsch, Can. J. Microbial., 17 (1971) 191. 18 A.G. Lee and J.T.R. Fitzsimons, J. Gen. Microbial, 93 (1976) 346. 19 H.C. Berg, Annu. Rev: Biophys. Bioeng., 4 (1975) 119. 20 B.H. Caraway and N.R. Krieg, Can. J. Microbial, 18 (1972) 1749. 21 J.E. Segall, M.D. Manson and H.C. Berg. Nature (London), 296 (1982) 855. 22 M.A. Snyder, J.B. Stock and D.E. Koshland, Jr., J. Mol. Biol., 149 (1981) 241. 23 D.P. HBder, Arch. Microbial., 114 (1977) 83. 24 G.W. Ordal and J. Adler, J. Bacterial., 117 (1974) 517.
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E.N. Kort, M.F. Goy, S.H. Larsen and J. Adler, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 3939. J.B. Armstrong and J. Adler, Genetics, 56 (1967) 363. J. Adler, J. Gen. Microbial., 74 (1973) 77. J. Adler, Science, 153 (1966) 708. J. Adler and M.M. Dahl, J. Gen. Microbial., 46 (1967) 161. H. Htilsheger and E.-G. Niemann, Radiat. Environ. Biophys., 18 (1980) 281. J. Adler and B. Templeton, J. Gen. Microbial., 46 (1967) 175. J.B. Armstrong and J. Adler, J. Bacterial., 97 (1969) 156. D. Farkas, R. Korenstein and S. Malkin, FEBS Lett., 120 (1980) 236. C.A.L. Bassett, R.J. Pawluk and A.A. Pilla, Science, 184 (1974) 575. C.A.L. Bassett, A.A. Pilla and R.J. Pawluk, Clin. Orthop., 124 (1977) 128. R.A. Luben, CD. Cain, C.-Y. Chen, D.M. Rosen and W.R. Adey, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 4180. 37 M. Eisenbach, Biochemistry, 21 (1982) 6818. 38 M. Eisenbach in Biological Structures and Coupled Flows, A. Oplatka and M. Balaban (Editors), Academic Press, New York and Balaban I.S.S., Philadelphia, 1983, p. 349. 39 M. Eisenbach, T. Raz and A. Ciobotariu, Biochemistry, 22 (1983) 3293.