Chemotaxis. after incubation at 40C (19), Schneider and Doetsch have subsequently reported that a. number of different bacteria, including a strain

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1 JOURNAL OF BACTERIOLOGY, July 1980, p /80/ /05$02.00/0 Vol. 143, No. 1 Effect of Temperature on Pseudomonas fluorescens Chemotaxis WILLIAM H. LYNCH Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 5A3 The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 500) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but not the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 50C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 00C and appeared to be functional over a broad temperature range of 3 to 350C. Motile bacteria respond to a wide variety of chemical stimuli by a process known as chemotaxis. The capillary assay technique, described by Adler (1), has frequently been used to measure chemotaxis in different bacteria. In Pseudomonas species, including P. aeruginosa (14, 15, 18), P. fluorescens (4, 18, 19), and P. lachrymans (5), attraction to a number of organic compounds has been demonstrated. More recently, some of these taxes in P. aeruginosa have been shown to be inducible (15). In this case, attraction to malate, succinate, and serine appeared to be constitutive, whereas attraction to glucose and citrate was dependent on prior exposure of the organism to the attractant. Several inducible taxes have also been reported for Escherichia coli (1, 2) and Salmonella typhimurium (6). The effect of temperature on chemotaxis has been examined in some mesophilic bacteria. With E. coli (1, 11) and S. typhimurium (12), chemotaxis, measured by the capillary assay technique, was severely curtailed at temperatures below 300C. In Bacillus subtilis, chemotaxis was shown to be independent of temperature from 29 to 42 C, but declined rapidly below 29 C (16). In both E. coli (1, 11) and B. subtilis (16), motility appeared to be less dependent on temperature than chemotaxis and was observed at temperatures below those at which chemotactic responses were detected. Although a strain of P. fluorescens was reported to be nonmotile 338 after incubation at 40C (19), Schneider and Doetsch have subsequently reported that a number of different bacteria, including a strain of P. fluorescens, demonstrated motility at low temperatures (17). In their report, P. fluorescens, incubated at 4 to 370C, was shown to be motile over the temperature range of 7.5 to 50 C. The purpose of this report was to examine the effects of temperature and attractants on functional chemotactic responses in psychrotrophic P. fluorescens. MATERIALS AND METHODS Organism, media, and cell preparation. P. fluorescens E-20 and conditions of cultivation have been described previously (10). Growth was performed in 250-ml Erlenmeyer flasks containing 50 ml of minimal medium consisting of: NH4H2PO4, 0.3%; K2HPO4, 0.2%; MgSO4. 7H20, 0.05%; FeSO4. 7H20, 0.5 ug/ml; and filter-sterilized carbon source as indicated, 0.2%. Cultures, grown to the exponential phase (absorbance at 660 nm of approximately 0.25), were harvested by centrifugation at 3,000 x g for 10 min. Absorbances were determined using a Zeiss PMQ II spectrophotometer in quartz cuvettes with a 1-cm light path. Cells grown at 30 C were harvested at room temperature, and cells grown at 5 C were harvested at 5 C. Cell pellets were gently washed twice and resuspended in chemotaxis medium (CM) consisting of 0.01 M potassium phosphate buffer at ph 7.4, 1 mm MgSO4. 7H20, and 0.1 mm disodium EDTA. The cells were suspended to give a final concentration of approximately 7 x 107 bacteria per ml. Chemotaxis assay. The capillary tube assay was

2 VOL. 143, 1980 performed basically as described by Adler (1). A small chamber was formed by placing a U-shaped piece of 1.7-mm-diameter capillary pipette glass with sealed ends between a glass plate (25 by 25 by 0.5 cm) and a cover slip (18 mm2). The chamber was filled with 0.45 ml of the above cell suspension in CM. The capillaries (1-pl Drummond type) were half-filled with CM, with and without attractant, and the open end of one was inserted into each chamber. Capillaries were removed after the specified times and rinsed with a stream of 0.01 M potassium phosphate buffer (ph 7.4) from a Pasteur pipette. The plugged end was broken off, and the contents of the open end were squirted into buffer. Serial dilutions were made in buffer, and 0.1-ml samples were spread on Trypticase soy agar plates. Each capillary test was performed in duplicate, and the results were averaged. Temperature on the glass plates was maintained by use of Psychrotherm model G-25 and G-26 controlled-environment incubator shakers (New Brunswick Scientific Co. Inc., Edison, N.J.), Lauda model R-2/KD circulating water pumps (Brinkmann Instruments Inc., Westbury, N.Y.), and refrigerators. All components of the assay system were maintained at the appropriate temperature before being used in the assay. Chemicals. Disodium EDTA and potassium citrate were obtained from J. T. Baker Chemical Co., Phillipsburg, N.J. The hemicalcium salt of 2-ketogluconate was obtained from Sigma Chemicfl Co., St. Louis, Mo., and was treated with Dowex 5CW-X8, H+ form, and neutralized with KOH before use. Other attractants and substrates were purchased from Sigma Chemical Co. as potassium salts or free acids and were neutralized with KOH. RESULTS Conditions for chemotaxis in P. fluorescens. The rate of accumulation of malate-grown bacteria in capillaries containing malate as attractant was linear up to approximately 30 min at 30 C and 60 min at 50C (Fig. 1). These two time periods were adopted for subsequent assays at the two respective temperatures. Cells grown with malate at 300C or 5 C demonstrated attraction to malate, showing a peak response at approximately 5 x 1i-O M malate and a threshold of approximately i0-4 M malate (Fig. 2). The chemotactic response to malate was proportional to the cell density over a range of 3.5 x 106 to 3 x 107 bacteria per ml. In one experiment, the peak accumulation to 5 x 10-3 M malate (mean of n = 10) was 9.16 (+1.40) x 104 cells per capillary compared to a background accumulation of 0.88 (±0.34) x 104 cells per capillary. In experiments on 20 different days, the average peak response to malate was 8.6 (±3.1) x 104 cells per capillary. The standard deviation between replicates was about 20%. Malate taxis was dependent on EDTA, with a maximum response at 10-4 M (Fig. 3). At 10-2 M EDTA, motility was inhibited. In contrast to CHEMOTAXIS IN P. FLUORESCENS 339 P. aeruginosa, where chemotaxis was shown to be dependent on EDTA and magnesium increased bacterial accumulation more than threefold (15), chemotaxis in P. fluorescens was not significantly enhanced by magnesium (Fig. 3). Based on these results, a CM containing 10'- M EDTA and 10-3 M MgSO4 was used for all assays. Effect of temperature. Chemotaxis in P. fluorescens to attractants such as malate, succinate (Fig. 2), and arginine (data not shown) 8- IL 4-I c27t INCUBATION TIME (MIN) FIG. 1. Rates of accumulation of P. fluorescens cells in capillaries containing CM with 5 x 10-3 M malate assayed at 30 C (0), 17 C (0), 10 C (A), and 5 C (A), or containing CM alone at 30 C (0). Means of n = 6 ± standard deviation. Cells were grown at 5 C with malate. SUCCINATE MOLARITY MALATE MOLARITY FIG. 2. Concentration response curves for P. fluorescens toward succinate and malate. Attraction to succinate at 300C (30-min assay) by 300C succinategrown cells (0). Attraction to succinate at 50C (60- min assay) by 30 C succinate-grown cells (0) and 50C succinate-grown cells (A)). Symbols for attraction to malate are as described above except that malategrown cells were used. Each point is the average of triplicate assays, and backgrounds were subtracted.

3 340 LYNCH was not affected by the growth temperature of the cells used for assay. Cells grown at 30 or 5 C responded equally well to the attractants. The concentration response curve for each attractant tested was very similar whether the cells used were grown at 30 or 5 C, or subsequently assayed at 30 or 5 C. The only detectable difference was in the rate of accumulation of bacteria into capillaries at different assay temperatures. The effect of assay temperature on chemotaxis to malate was examined over the range of 3 to 35 C with cells grown at both 5 and 300C (Fig. 4). The assay temperature response profile was similar in cells grown at both temperatures. The rate of accumulation of cells in capillaries containing malate showed a four- to fivefold change over the temperature range examined, and a maximum rate of accumulation was observed at 17 C. Similar results were obtained with serine as the attractant. These results suggested an 12 -_ c 44 II0 uj mr co 2 i A 0 10 id t A RI EDTA MOLARITY 4-4 l2 1C IL <.a :r mu 0 O10" 60 1 Ii66 1 I MAGNESIUM SULFATE MOLARITY FIG. 3. Effect of EDTA and MgSO4on chemotaxis of P. fluorescens to malate. Cells were grown with malate at 30'C and assayed for 30 min at 30 C. The CM contained 0.01 M potassium phosphate buffer with (A) 10-3 M MgSO4 and varying concentrations of EDTA, or (B) 10' M EDTA and varying concentrations of MgSO4. Capillaries contained CM with 5 x 10-3 M malate (0) or CM alone (0). A i le I, I-j II -L-r J. BACTERIOL. optimum temperature for chemotaxis to malate of approximately 170C. The rate of accumulation ofbacteria into capillaries containing malate at 170C was approximately two times faster than at 300C and four times faster than at 5 C (Fig. 1). Inducible taxes. Attraction to a number of organic compounds was examined in P. fluorescens E-20 (Fig. 5, Table 1). Each compound tested could serve as the sole carbon and energy source for growth of the microorganism in a minimal salts medium. In contrast to P. aeruginosa, which was reported to have an inducible taxis toward glucose (15, 20), no attraction to glucose or its oxidation products, gluconate and 2-ketogluconate, was detected with P. fluorescens. In these cases, the cells were grown in the presence of the respective substrates, which were subsequently tested as attractants over the concentration range of 10-6 to 10' M. All other compounds tested caused significant chemotactic responses with cells previously cultivated in medium containing the respective test substrate (Fig. 5). Threshold and peak response attractant concentrations varied considerably with the attractant used, as did the maximum accumulation of cells to each attractant. For example, observed peak response concentrations varied from 10-' M for leucine to 5 x 102 M for citrate and arginine. These are apparently widely observed phenomena (2, 12, 14-16). Tactic responses to citrate, to the amino acids leucine, arginine, and glutamic acid, and to the uronic acids, glucuronic and galacturonic, appeared to be inducible (Table 1). Strong responses to these attractants were observed only with cells previously cultivated in the presence of the respective attractant. Chemotaxis toward e6 Wa u a3; 4 V ax -C c] - 10 otu TEMPERATURE (C) FIG. 4. Effect of temperature on the rate of accumulation ofp. fluorescens to 5 x 10-3M malate. Cells, grown with malate at 30 C (0) and 5 C (0), were assayed as described for Fig. 1. o CO z 10 ATTRACTANT MOLARITY ATTRACTANT MOLARITY FIG. 5. Concentration response curves for chemotaxis of P. fluorescens to leucine (0), serine (0), arginine (A), glutamate (A), citrate (0), glucuronate (V), and galacturonate (O). Cells were grown with the substrate subsequently used as the attractant and were assayed for 30 min at 30'C. Backgrounds have been subtracted.

4 VOL. 143, 1980 TABLE 1. Growth substrate" CHEMOTAXIS IN P. FLUORESCENS 341 Effect ofgrowth substrate on chemotaxis to attractants in P. fluorescens Response to attractantb Malate Succinate Citrate Serine Arginine Leucine Glute- Galactumate ronate Malate Succinate Citrate Serine Arginine Argininec NP + Leucine Glutamate Galacturonate Glucose acells were grown at 30 C in minimal medium containing the carbon source indicated. b Attractants were tested at concentrations producing peak responses. Assay was at 30 C for 30 min. +, Accumulation to attractant was greater than threefold higher than to CM alone. -, Accumulation to attractant was less than twofold higher than to CM alone. NP, Not performed. c Cells were grown at 5 C. the organic acids, malate and succinate, appeared to be constitutive. Strong responses to these attractants were observed with cells grown with each substrate tested. Serine taxis was seen in cells cultivated with each substrate tested, with the exception of citrate-grown cells. The temperature at which the cells were grown did not appear to influence these results. Cells grown with arginine at 5 or 300C showed the same response to the attractants tested (Table 1). DISCUSSION The effect of temperature on chemotaxis in psychrotrophic P. fluorescens E-20 was examined. Cells grown at 5 or 300C, when assayed at the same temperature, demonstrated very similar rates and extents of accumulation to individual attractants and concentration response curves. Changes in the assay temperature for chemotaxis altered the rate but not the extent of accumulation of cells to attractants, nor the concentration response curves. Strong chemotactic responses were observed over a broad assay temperature range of 3 to 350C. The optimum temperature for attraction to malate appeared to be close to 17 C, well below the optimum growth temperature of 30 C for this microorganism (10). In contrast, chemotactic responses, as measured by the capillary assay, in mesophilic bacteria such as E. coli (1, 11), S. typhimurium (12), and B. subtilis (16) have been shown to be functional over a higher and narrower temperature range and to decline rapidly below 30 C. In E. coli, chemotaxis was not observed below 200C, although motility was still detected at lower temperatures. Low temperatures appeared to decrease the tumbling frequency with E. coli, and it has been suggested that this may be related to effects on membrane fluidity (8, 11). However, evidence presented by Miller and Koshland (13) strongly suggested that temperature effects on membrane fluidity have no significant effect on velocity of movement, tumbling frequency, and temporal gradient response time for E. coli and S. typhimurium. Although velocity was slower, tumbling frequency was decreased, and temporal gradient response time was increased at low temperatures, they were functional. Miller and Koshland suggested that the lack of observed accumulation of these mesophilic bacteria into capillaries containing attractants at low temperatures was due to a loss of cell viability (13). This would agree with the data presented here that chemotaxis with P. fluorescens E-20, using the capillary assay, was demonstrated with cells grown at 5 or 300C over the assay temperature range of 3 to 350C. P. fluorescens E-20 has a temperature range for growth of <0 to 350C (10). P. fluorescens E-20 differed from P. aeruginosa (15) in its lack of detectable attraction to glucose and the small stimulatory effect of magnesium ions on chemotaxis. It was similar to P. aeruginosa in the apparent requirement for EDTA for strong chemotactic responses, and in the apparent constitutive nature of taxes to malate and succinate and the inducible nature of taxis to citrate. P. aeruginosa exhibited attraction to serine whether the cells assayed were previously grown with succinate, citrate, malate, or glucose (15). Attraction to serine was observed with cells of P. fluorescens E-20 grown with eight of the nine carbon sources tested. The lack of detectable attraction to serine by citrategrown cells is not understood at the present

5 342 LYNCH time. Other taxes in P. fluorescens E-20 to three amino acids and two uronic acids appeared to be inducible. The apparent constitutive taxes to malate and succinate in both P. aeruginosa and P. fluorescens E-20 may reflect the aerobic metabolism of these microorganisms. Organic acids, such as malate and succinate, have been shown to be preferred substrates in P. aeruginosa (3, 7, 21) and P. fluorescens E-20 (9). The results presented here demonstrated that P. fluorescens E-20 exhibited chemotaxis to many but not all organic compounds which can serve as sole carbon and energy sources for growth. Many of these taxes were inducible, and the constitutive taxes may be related to the aerobic metabolism of this microorganism. Chemotaxis was functional over a wide temperature range, and strong responses were observed at low temperatures approaching 0 C. It might be relevant that P. fluorescens was shown to be a predominant species in an aquatic habitat (from which the microorganism under study was isolated) at low temperatures (M. Chalifour, M.Sc. thesis, University of New Brunswick, Fredericton, New Brunswick, Canada, 1974). ACKNOWLEDGMENTS This work was supported by grant A6754 from the Natural Sciences and Engineering Council Canada and by the University of New Brunswick Research Fund. LITERATURE CITED 1. Adler, J A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. Gen. Microbiol. 74: Adler, J., G. L. Hazelbauer, and M. M. Dahl Chemotaxis toward sugars in Escherichia coli. J. Bacteriol. 115: Belvins, W. T., T. W. Feary, and P. V. Phibbs, Jr Phosphogluconate dehydratase deficiency in pleiotrophic carbohydrate-negative mutant strains of Pseudomonas aeruginosa. J. Bacteriol. 121: Chet, I., Y. Henis, and R. Mitchell Effect of biogenic amines and cannabinoids on bacterial chemotaxis. J. Bacteriol. 115: Chet, I., Y. Zilberstein, and Y. Henis Chemotaxis of Pseudomonas lachrymans to plant extracts and to water droplets collected from the leaf surfaces of resis- J. BACTERIOL. tant and susceptible plants. Physiol. Plant Pathol. 3: Fahnestock, M., and D. E. Koshland, Jr Control of the receptor for galactose taxis in Salmonella typhimurium. J. Bacteriol. 137: Hylemon, P. B., and P. V. Phibbs, Jr Independent regulation of hexose-catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem. Biophys. Res. Commun. 48: Lofgren, K. W., and C. F. Fox Attractant-directed motility in Escherichia coli: requirement for a fluid lipid phase. J. Bacteriol. 118: Lynch, W. H., and M. Franklin Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens. Arch. Microbiol. 118: Lynch, W. H., J. MacLeod, and M. Franklin Effect of growth temperature on the accumulation of glucose-oxidation products in Pseudomonas fhuorescens. Can. J. Microbiol. 21: Maeda, K., Y. Imae, J. Shioi, and F. Oosawa Effect of temperature on motility and chemotaxis of Escherichia coli. J. Bacteriol. 127: Melton, T., P. E. Hartman, J. P. Stratis, T. L Lee, and A. T. Davis Chemotaxis of Salnonella typhimurium to amino acids and some sugars. J. Bacteriol. 133: Miller, J. B., and D. E. Koshland, Jr Membrane fluidity and chemotaxis: effects of temperature and membrane lipid composition on the swimming behavior of Salmonella typhimurium and Escherichia coli. J. Mol. Biol. 111: Moench, T. T., and W. A. Konetzka Chemotaxis in Pseudomonas aeruginosa. J. Bacteriol. 133: Moulton, R. C., and T. C. Montie Chemotaxis by Pseudomonas aeruginosa. J. Bacteriol. 137: Ordal, G. W., and K. J. Gibson Chemotaxis toward amino acids by Bacillus subtilis. J. Bacteriol. 129: Schneider, W. R., Jr., and R. N. Doetsch Temperature effects on bacterial movement. Appl. Environ. Microbiol. 34: Seymour, F. W. K., and R. N. Doetsch Chemotactic responses by motile bacteria. J. Gen. Microbiol. 78: Smith, J. L., and R. N. Doetsch Studies on negative chemotaxis and the survival value of motility in Pseudomonas fluorescens. 55: Stinson, M. W., M. A. Cohen, and J. M. Merrick Purification and properties of the periplasmic glucosebinding protein of Pseudomonas aeruginosa. J. Bacteriol. 131: Tiwari, N. P., and J. J. R. Campbell Enzymatic control of the metabolic activity of Pseudomonas aeruginosa grown in glucose or succinate media. Biochim. Biophys. Acta 192: