JOURNAL OF BACTERIOLOGY, Sept. 1996, p. 5199–5204 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 178, No. 17
Oxygen Taxis and Proton Motive Force in Azospirillum brasilense IGOR B. ZHULIN,1* VYACHESLAV A. BESPALOV,1† MARK S. JOHNSON,1
AND
BARRY L. TAYLOR1,2
Department of Microbiology and Molecular Genetics1 and Center for Molecular Biology and Gene Therapy,2 School of Medicine, Loma Linda University, Loma Linda, California 92350 Received 6 May 1996/Accepted 26 June 1996
The microaerophilic nitrogen-fixing bacterium Azospirillum brasilense formed a sharply defined band in a spatial gradient of oxygen. As a result of aerotaxis, the bacteria were attracted to a specific low concentration of oxygen (3 to 5 mM). Bacteria swimming away from the aerotactic band were repelled by the higher or lower concentration of oxygen that they encountered and returned to the band. This behavior was confirmed by using temporal gradients of oxygen. The cellular energy level in A. brasilense, monitored by measuring the proton motive force, was maximal at 3 to 5 mM oxygen. The proton motive force was lower at oxygen concentrations that were higher or lower than the preferred oxygen concentration. Bacteria swimming toward the aerotactic band would experience an increase in the proton motive force, and bacteria swimming away from the band would experience a decrease in the proton motive force. It is proposed that the change in the proton motive force is the signal that regulates positive and negative aerotaxis. The preferred oxygen concentration for aerotaxis was similar to the preferred oxygen concentration for nitrogen fixation. Aerotaxis is an important adaptive behavioral response that can guide these free-living diazotrophs to the optimal niche for nitrogen fixation in the rhizosphere. distinct aerotactic bands were described for microaerophilic bacteria, such as Spirochaeta aurantia (14), Spirillum volutans (8), and Azospirillum brasilense (3, 15). In contrast to most other chemoeffectors, oxygen can act as both an attractant and a repellent (34). Positive (attractant) aerotaxis in E. coli and S. typhimurium involves the transport of reducing equivalents through the respiratory chain (28). The concentration of oxygen that elicits a half-maximal response (K0.5) for this response is similar to the Km of oxygen uptake by cytochrome o (26); however, the oxygen-bound terminal oxidases are not the transducers of the aerotactic signal in E. coli (36). The positive aerotactic signal in E. coli, S. typhimurium, and B. subtilis is generated by the changes in the redox state of the components of the respiratory chain or by consequent changes in the proton motive force (Dp) (27, 28, 35, 36). An unsolved problem in elucidating the mechanism of bacterial aerotaxis is how bacteria avoid high oxygen concentrations, i.e., what is the signal for negative (repellent) aerotaxis? A. brasilense (a former member of the genus Spirillum) is a typical microaerophilic organism. This nitrogen-fixing, plantassociated bacterium has an aerobic type of metabolism; however, nitrogen fixation in A. brasilense occurs only under low (,8 mM) concentrations of dissolved oxygen (18, 32). This allows energy generation with the most efficient electron acceptor (oxygen) while protecting the oxygen-sensitive nitrogenase. Vigorous respiration of A. brasilense at such low oxygen concentrations was demonstrated previously (6) and is thought to be made possible by a high-affinity terminal oxidase. The physiology of motile behavior, chemokinesis, chemotaxis, and redox taxis in this microorganism (15, 43) and the possible role of behavioral responses in its ecology (42, 48) have been described previously. We now present evidence that the microaerophilic bacterium A. brasilense generates maximum energy (the proton motive force) at a specific low oxygen concentration which appears to be the optimal oxygen concentration for both chemotaxis to oxygen and nitrogen fixation. (A preliminary report of this work was presented at the 1st European Nitrogen Fixation Conference [47] and the 95th General Meeting of the American Society for Microbiology [44].)
Aerotaxis (chemotaxis to oxygen) enables bacteria to find concentrations of dissolved oxygen that are favorable for their metabolic lifestyle (reviewed in references 39 and 40). Aerotaxis was the first behavioral response reported for microorganisms: in 1881, Engelmann described accumulation of Bacterium termo around plant cells that were producing oxygen (10). Among the bacteria described by Engelmann, the most sensitive to oxygen was Spirillum tenue, which accumulated at a low oxygen concentration (11). In 1893, Beijerinck described the formation of bacterial “Atmungsfiguren” around the source of oxygen. The bacteria, most likely spirilla, formed a band at some distance from the oxygen source; the band descended if air was replaced by oxygen or ascended if air was replaced by hydrogen (4). In 1901, Jennings and Crosby investigated the aggregation of Spirillum species in the region of illuminated algae that were evolving oxygen. The bacteria that were distant from the algae swam randomly. However, the cells became trapped if they entered a zone around the algae that had an optimal concentration of oxygen. Whenever the bacteria reached the edge of the zone, the direction of their movement was reversed (20). This aerotactic response, originally termed phobo-chemotaxis (33), resulted in the accumulation of bacteria in the zone of optimal oxygen concentration. Formation of aerotactic bands is a characteristic of motile bacterial species. Aerotaxis in the aerobe Bacillus subtilis (41), in the facultative anaerobes Escherichia coli, Salmonella typhimurium (1, 34, 36), and Halobacterium salinarium (7, 29), and in the anaerobe Desulfovibrio vulgaris (22) has been extensively investigated. At least one motile bacterial species, Rhizobium meliloti, does not have a true chemotactic response to oxygen and responds to oxygen only by chemokinesis (45). The most
* Corresponding author. Phone: (909) 824-4480. Fax: (909) 8244035. Electronic mail address:
[email protected] at internetmail. † Permanent address: Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Saratov 410015, Russian Federation. 5199
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MATERIALS AND METHODS Bacterial strains and culture conditions. A. brasilense Sp7 (ATCC 29145) is wild type for chemotaxis. A. brasilense strains IZ15 (frequently reversing) and IZ21 (smooth swimming) are mutants with a defect in chemotaxis that were isolated after N-methyl-N9-nitro-N-nitrosoguanidine mutagenesis of strain Sp7. The isolation procedure and genetic description of the mutants (in progress) will be presented elsewhere. The cells were grown aerobically at 308C on defined malate medium as described previously (15, 43). Assays for aerotaxis. A spatial gradient assay for aerotaxis was performed in an optically flat capillary (inner dimensions, 0.1 by 2 by 50 mm; Vitro Dynamics Inc., Rockaway, N.J.) that was placed in a microchamber ventilated with a humidified O2-N2 gas mixture. The concentration of oxygen in the gas mixture was determined with a gas proportioner as described previously (28). The formation of a band of bacteria near the air-liquid interface was observed and video recorded (28, 29). The response to a temporal gradient of oxygen was determined by spreading 2 ml of diluted cell suspension (optical density at 600 nm 5 0.1; 2 3 107 cells ml21) on a microscope slide in the microchamber (28). The cell suspension was equilibrated with an N2-O2 gas mixture for 30 to 60 s, then the oxygen concentration of the gas mixture was altered, and the behavior of the bacteria was videotaped for motion analysis. To minimize evaporative loss, the gas mixture was humidified and the time of exposure was kept to a minimum. A novel assay that utilized spatial oxygen gradients in a drop of bacterial suspension was developed to measure aerotaxis. A 10-ml drop of diluted bacterial suspension was placed, without spreading, on a microscope slide in the microchamber. Nitrogen or oxygen gas flowing over the drop rapidly created a spatial gradient of oxygen from the top to the bottom of the drop. The microscope was focused at the bottom of the drop, at the glass surface. A steep spatial gradient of oxygen resulted in bacteria migrating toward the bottom of the drop. The change in the number of motile bacteria at the glass surface was recorded after introducing oxygen into the ventilating gas. Motion analysis. Manual frame-by-frame analysis involved tracing individual tracks of free-swimming cells on transparent acetate sheets directly from the monitor screen as previously described (43). Twenty cells were tracked for each assay, typically for 5 to 8 s each. A minimum of three replicates was performed. A computerized motion analysis system was used to measure cell speed and reversal frequency as previously described (29). Video records were digitized at 30 frames per s. Measurement of membrane potential. Membrane potential was determined by using a tetraphenylphosphonium ion (TPP1)-selective electrode constructed as described by Kamo et al. (23). The TPP1-selective electrode and a semi-micro calomel reference electrode (Orion, Boston, Mass.) were connected to an ion meter (Corning, Wilmington, Del.) and a MacLab MKIII datum recording system (Analog Digital Instruments, Milford, Mass.). All measurements were performed in a 10-ml closed vessel at 308C and pH 7.1. The vessel was ventilated with either nitrogen or oxygen gas. The cells were permeabilized to TPP1 by treatment with EDTA (38). Membrane potential was calculated by using the Nernst equation after correction for nonspecific TPP1 binding (9) and estimation of the cell volume. The intracellular cell volume was determined by the centrifugation method (37). [3H]water and inulin-[14C]carboxyl were used as the probes. Measurement of DpH. The chemical potential difference of protons across the membrane (DpH) was determined by measuring the accumulation of [14C]benzoic acid (final concentration, 45 mM) by flow dialysis as described previously (35). A high concentration of protonophores (carbonyl cyanide m-chlorophenylhydrazone, 200 mM; pentachlorophenol, 2 mM) was used to collapse DpH. Measurements of oxygen concentrations. The oxygen concentration in a bacterial suspension in a closed vessel ventilated with oxygen or nitrogen was measured with a needle oxygen electrode and a chemical microsensor (Transidyne General, Ann Arbor, Mich.). The spatial oxygen gradient in a bacterial suspension in a capillary was quantified by using a needle oxygen electrode which had a 5-mm-diameter sensing tip (no. 723; Diamond General, Ann Arbor, Mich.). The electrode was inserted into a capillary with a micromanipulator (12). The oxygen concentration was recorded at intervals of 20 mm from the meniscus. Respiration rates of bacterial suspensions were measured with a Clark-type electrode and an oxygen monitor (Yellow Springs Instruments, Yellow Springs, Ohio). The outputs of the chemical microsensor and the oxygen monitor were collected in separate channels of the MacLab datum recording system. The data collected were analyzed and stored in a Macintosh computer using Chart v3.3 software (Analog Digital Instruments).
RESULTS Aerotaxis in a spatial oxygen gradient. Aerotaxis studies were performed with exponentially growing cells (optical density at 600 nm 5 0.4 to 0.6) which had maximum respiration rates. After the bacteria were introduced into a flat glass capillary, they formed a distinct band 400 to 900 mm from the air-suspension interface (meniscus) (Fig. 1b). The band
FIG. 1. Aerotactic band formation in suspensions of A. brasilense Sp7 in spatial gradients of oxygen in a capillary (see Materials and Methods). The meniscus is at the left of each panel. Oxygen concentrations (percentages) in headspace: 100 (a), 21 (b), 1 (c), 0 (d), 0.5 (e), 0.35 (f), 0.22 (g), and 0.02 (h). Magnification, 3200 (a to d) or 31,500 (e to h).
formed in 0.5 to 3 min, depending on the cell density and respiration rate. If oxygen replaced air in the headspace, the aerotactic band moved further away from the meniscus (Fig. 1a). If nitrogen replaced air or oxygen in the headspace, the band moved to the meniscus and then disappeared (Fig. 1d). When air was reintroduced, the band reformed and the cycle of band movement could be repeated indefinitely, indicating that the bacteria moved toward a specific low concentration of oxygen. The behavior of individual cells in the capillary was observed at high magnification using dark-field illumination. A spatial gradient of oxygen was formed in a capillary as described in the legend to Fig. 1, with a gas phase of air. The oxygen concentration was determined with a needle electrode (see Fig. 2). Average cell speed was determined by manual frame-by-frame analysis of video recordings (see Materials and Methods). To determine the speed of the cells inside the aerotactic band, one cell was traced only for 1 or 2 s because of the cell crowding. Each frame of the video recording was time-tagged, facilitating an accurate calculation of swimming speed. Essentially all the motile cells between the band and the meniscus eventually reached the band and stayed in the band. Bacteria both proximal and distal to the band were motile, as were the cells in the band. However, the swimming speed of the cells in the band appeared to be much higher than the speed of cells outside the band. The average speeds, in micrometers per second, were as follows (means 6 standard deviations) for the indicated oxygen concentrations: ,0.2% oxygen, 19 6 5; 0.3 to 0.5% oxygen, 49 6 6; 1 to 5% oxygen, 22 6 6; and 10 to 20% oxygen, 14 6 4. The cells swimming in either direction away from the
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FIG. 3. Rapid spatial assay for aerotaxis. See Materials and Methods for details. A drop of A. brasilense Sp7 suspension was placed in a microchamber and equilibrated with 100% nitrogen for 2 min until no motile cells were seen on the bottom of the slide. After the field of view was observed for an additional 5 s, pure oxygen (100%) was introduced into the chamber (shaded area) for 25 s and then nitrogen was reintroduced. The number of motile cells in the microscopic field of view was determined.
FIG. 2. Oxygen profile in a suspension of A. brasilense Sp7. The oxygen gradient was formed by oxygen diffusion through the air-liquid interface and oxygen consumption by the respiring cells. (A) Oxygen concentration determined as described in Materials and Methods. (B) The position of the aerotactic band in the gradient. The linear scale is the same in panels A and B.
band reversed immediately and returned to the band. The cells swimming into the band from either direction did not reverse until they reached the opposite side of the band. Bacteria in the region where the oxygen concentration in the gradient was between 5 and 21% oxygen (Fig. 2) had a random motility pattern, and any bias in the reversal frequency was not obvious. We confirmed that formation of the aerotactic band in suspensions was due to chemotaxis to oxygen, using the generally nonchemotactic mutants IZ15 and IZ21. IZ15 cells reversed direction three times more frequently than the wild-type cells, and IZ21 cells did not reverse direction, analogous to the constantly tumbling and smooth-swimming chemotaxis mutants, respectively, of E. coli (2). The average cell speed and respiration rate of the mutant cells were similar to those of the wild type. Cells of the IZ15 or IZ21 strain did not form an aerotactic band, even after 48 h, but remained fully motile. The profile of oxygen concentration in the Azospirillum suspension in a flat capillary was measured with microelectrodes (Fig. 2). The oxygen concentration linearly decreased from the meniscus to near the band, reaching the limit of electrode sensitivity (approximately 1% oxygen) at the boundary of the band. This indicated that the preferred concentration of oxygen for A. brasilense is ,1% (,10 mM dissolved oxygen). We estimated the preferred concentration of oxygen by using a novel strategy. A capillary containing A. brasilense cells was inserted into a microchamber in which the oxygen concentration of the ventilating gas could be controlled by a gas proportioner. The aerotactic band of bacteria moved toward the meniscus as the oxygen concentration in the chamber was reduced, and the front of the band was adjacent to the meniscus when the oxygen concentration was 0.5% (Fig. 1e). A further decrease in oxygen concentration resulted in a decrease of the size of the band (Fig. 1f and g), and the band completely disappeared when the oxygen concentration in the gas phase was lowered to 0.05%. We conclude that the preferred (at-
tractant) concentration of oxygen for A. brasilense cells is in the range of 0.3 to 0.5% in the gas phase, which corresponds to 2.8 to 4.8 mM dissolved oxygen. These values are approximate, since we were unable to determine the effect of bacterial respiration on the oxygen concentration in the band. We observed both positive and negative aerotaxis of A. brasilense when a spatial oxygen gradient was rapidly created in a 10-ml drop of bacterial suspension (see Materials and Methods). When oxygen was flowing over the drop, bacteria quickly concentrated on the bottom of the drop, avoiding high oxygen concentrations. When nitrogen was substituted for oxygen, the cells immediately left the zone at the glass surface and moved down the oxygen gradient (Fig. 3). Multiple drops can be placed on a microscope slide, making this a convenient and rapid method for screening isolates for aerotaxis in a spatial gradient of oxygen. Aerotaxis in a temporal oxygen gradient. To investigate the aerotactic response in a temporal gradient, a small drop (1 to 2 ml) of bacterial suspension was spread on a microscope slide and the cell suspension was ventilated with a controlled oxygen concentration (28). There were no significant changes in swimming behavior when the ventilating gas was changed from nitrogen to air (21% oxygen) or from air to 100% oxygen (Table 1). In E. coli, similar changes in oxygen concentration result in smooth-swimming and constantly tumbling responses,
TABLE 1. Reversal frequency of free-swimming A. brasilense cells after stimulation with temporal oxygen gradientsa Change in oxygen concn (%)
21 to 100 ........................................................................ 0 to 21 .......................................................................... 100 to 21 .......................................................................... 21 to 0 ............................................................................ 0 to 0.5 ......................................................................... 0.5 to 5 ............................................................................
Reversal frequencyb (s21)
0.32 6 0.04 0.27 6 0.04 0.24 6 0.06 0.22 6 0.05 0.09 6 0.03 0.49 6 0.09
a Temporal aerotaxis assays were performed as described in Materials and Methods. b Reversal frequency was determined by computerized motion analysis as described in Materials and Methods. Measurements were made 5 s after the change in oxygen concentration. Means and standard deviations are given.
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of factors which are described in a recent review by Harold and Maloney (16). DISCUSSION
FIG. 4. Temporal assay for aerotaxis. A 2-ml drop of A. brasilense Sp7 suspension was placed in a microchamber and equilibrated with 100% nitrogen for 30 s. After the motility of the bacteria in 100% nitrogen was observed for an additional 30 s, the chamber was ventilated with 0.5% oxygen in nitrogen (shaded area) for 60 s and then air (21% oxygen) was introduced. Reversal frequency was determined by computerized motion analysis (see Materials and Methods for details).
respectively. The A. brasilense cells decreased their reversal frequency after ventilation of the microchamber with nitrogen gas for 1 min and then exposure of the cells to 0.5% oxygen (Fig. 4). This indicates that the cells are attracted to 0.5% oxygen. Cells that were equilibrated with 0.5% oxygen and then exposed to nitrogen or air increased their reversal frequency, indicating a negative chemotaxis to both increased oxygen concentration and anaerobiosis (Table 1). A change in oxygen concentration from 5 to 21% did not cause a significant change in cell behavior. Relationship between oxygen concentration and proton motive force. The intracellular pH was 7.1 in A. brasilense cells at a neutral external pH (data not shown). Therefore, at an external pH of 7.1, the DpH component of the proton motive force in A. brasilense cells is 0, and membrane potential is the sole component of the proton motive force. We determined changes in the proton motive force upon stimulation of the cells with oxygen by monitoring changes in membrane potential at pH 7.1, using a TPP1-selective electrode. Membrane potential was calculated by using the Nernst equation with the experimentally determined value of 2 3 10215 liters for the cell volume (see Materials and Methods). The change in potential of a TPP1-selective electrode when A. brasilense cells were exposed to different oxygen concentrations is shown in Fig. 5. Dp increased from 2103 6 4 to 2115 6 2 mV (means 6 standard deviations) when A. brasilense cells that had been depleted of oxygen were exposed to 0.5% oxygen, the preferred oxygen concentration. Dp decreased to 253 6 4 mV when the oxygen concentration was increased from 0.5 to 5%. The decrease in the proton motive force could be reversed when the oxygen concentration was returned to 0.5%. The cells had a maximum proton motive force at the preferred oxygen concentration. Changes in the proton motive force under different oxygen concentrations correlated with changes in cell speed. This was expected, since the proton motive force is the source of energy for the bacterial flagellar motors (13, 25, 31). The calculated value of the proton motive force in A. brasilense is lower than that of E. coli but comparable with that of some other bacterial species (24). It is substantially lower than the thermodynamically expected value. The difference between calculated and expected values can be attributed to a number
In a spatial oxygen gradient, A. brasilense organisms always accumulated at an oxygen concentration of 3 to 5 mM, forming a distinct aerotactic band. The band is maintained in the zone of preferred oxygen concentration because the bacteria avoid swimming into regions of higher or lower oxygen concentrations. Positive aerotaxis in E. coli and S. typhimurium is dependent on changes in the functioning electron transport system, i.e., an increase in the flow of reducing equivalents and a consequent increase in the proton motive force (28, 35, 36). The mechanism for the aerophobic response, in which high concentrations of oxygen repel bacteria, has not been established. A hypothesis that the bacteria were repelled by reactive oxygen species, such as the superoxide ion (34), has been shown recently to be incorrect in studies of E. coli and S. typhimurium in our laboratory (21). The discovery that the proton motive force in A. brasilense substantially decreases when the oxygen concentration increases from 0.5 to 1% is important because it is consistent with a unified hypothesis for the mechanism of the aerophilic and aerophobic responses to oxygen (15). We propose that A. brasilense accumulates at the oxygen concentration that supports the maximum proton motive force in the cells. When bacteria leave the zone and move to a region of higher or lower oxygen concentration, they experience a decrease in the proton motive force. The bacteria sense the change in the proton motive force (or electron transport), and the probability of reversal of the direction of swimming is increased. In spatial oxygen gradients, such as those encountered in the present study, the oxygen concentration increases from 0.5 to 1% in about 40 mm (Fig. 2). A bacterial cell swimming away from the aerotactic band at an average speed of 50 mm/s experiences such a change and a corresponding decrease in the proton motive force within 1 s. A change in the
FIG. 5. Changes in membrane potential upon exposure of A. brasilense cells to oxygen. Membrane potential was monitored by a TPP1selective electrode as described in Materials and Methods. A 9-ml aliquot of 100 mM potassium phosphate buffer (pH 7.1), containing 20 mM malate as an energy source and 4 mM tetraphenylphosphonium, was placed in a vessel, equilibrated at 308C with stirring, and sparged with 100% nitrogen through a needle line. A trace of the electrode is shown. Cells (1 ml; 7.4 3 1010 cells per ml) were introduced at time zero. The times at which the oxygen concentration in the sparging gas was changed are indicated (arrows).
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direction of flagellar rotation would occur immediately (5, 30), causing the cell to return to the band. The hypothesis that the aerophilic and aerophobic responses to oxygen by a microaerophilic bacterium are regulated by changes in the proton motive force can be generalized for species of bacteria that have different affinities for oxygen. Preliminary studies of E. coli and B. subtilis in this laboratory suggest that in each species the proton motive force is maximal at the specific concentration of oxygen to which the bacteria are attracted. The proton motive force and the electron transport system are closely related. The present studies do not address the question of which of these parameters is the signal for aerotaxis. Two techniques that were developed for this study should be useful in investigations of other bacteria. The monitoring of cell density at the bottom of the drop of cell suspension is a rapid and convenient spatial gradient assay for screening bacteria for aerotaxis. Multiple samples can be observed per microscope slide, in less time than is required for the air bubble or capillary assay (29, 41). The use of the capillary to determine the concentrations of oxygen to which bacteria are attracted (Fig. 1) extends the range of oxygen concentrations that can be investigated. Needle electrodes that are currently available have a sensitivity limit of approximately 1% oxygen. Using the capillary method, we have extended our investigations to include oxygen concentrations as low as 0.01%. The observation that microaerophilic bacteria generate more energy at low than at high oxygen concentrations was unexpected. Increased respiration rates at low oxygen concentrations, which apparently provided more energy for effective nitrogen fixation, were recently reported for another plantassociated diazotroph, an Azoarcus sp. (19). Effective generation of the proton motive force at a low oxygen concentration by A. brasilense may be explained by the presence in its electron transport system of a high-affinity terminal oxidase which is coupled with proton translocation (6, 46). There is an obvious advantage of such a mechanism for A. brasilense. This nitrogenfixing, free-living bacterium can fix nitrogen only under conditions in which the oxygen concentration is below 1%, and it is able to maintain a vigorous aerobic metabolism at low oxygen concentrations (17, 32). The preferred oxygen concentration for aerotaxis (;3 to 5 mM) reported in this study coincides with a preferred oxygen concentration for nitrogen fixation (2 to 8 mM) reported for the same strain of A. brasilense (17). Thus, our results provide quantitative support for the hypothesis that aerotaxis is an adaptive response to optimize the efficiency of nitrogen fixation (3, 18). In contrast to rhizobia, which are also plant-associated diazotrophs, azospirilla are not provided with a low-oxygen-concentration environment by the plant host. Consequently, the active search for appropriate concentrations of oxygen is extremely important for these microorganisms. ACKNOWLEDGMENTS We are grateful to Christina Bennett and Swapnesh Patel for technical assistance. This work was supported by Public Health Service grant GM29481 from the National Institutes of Health (to B.L.T.). REFERENCES 1. Adler, J. 1966. Chemotaxis in bacteria. Science 153:708–716. 2. Armstrong, J. B., and J. Adler. 1969. Complementation of nonchemotactic mutants of Escherichia coli. Genetics 61:61–66. 3. Barak, R., I. Nur, Y. Okon, and Y. Henis. 1982. Aerotactic response of Azospirillum brasilense. J. Bacteriol. 152:643–649. 4. Beijerinck, M. W. 1893. Ueber Atmungsfiguren beweglicher Bakterien. Zentrabl. Bakteriol. Parasitenkd. 14:827–845. 5. Berg, H. C., and D. A. Brown. 1972. Chemotaxis in Escherichia coli analysed
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