JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 798-804 0021-9193/79/12-0798/07$02.00/0
Vol. 140, No. 3
Response to a Metal Ion-Citrate Complex in Bacterial Sensing THOMAS D. INGOLIA AND D. E. KOSHLAND, JR.* Department of Biochemistry, University of California, Berkeley, California 94720 Received for publication 24 September 1979
Salmonella typhimurium responds chemotactically to gradients of divalent cations in the presence of citrate ions. The actual chemoeffector is the citratemetal ion complex, which acts as an attractant. Citrate (which is also a chemoeffector for Salmonella) and the citrate-metal ion complex are recognized by different receptors. The response of Salmonella, which can transport citrate through its membrane, is quite different than that of Escherichia coli, which cannot.
Bacteria respond to gradients of chemicals in their environment by modifying their swimming behavior (1, 7, 12, 19, 20, 22). They swim smoothly when they encounter an increase in concentration of attractant molecules (or a decrease in repellent molecules), but they tumble and randomize direction when they encounter increases in repellents (or decreases in attractants). The net migration, called chemotaxis, enables bacteria to move up gradients of certain sugars, amino acids, and metabolites and down unfavorable gradients of compounds like phenol and indole. Generally, bacteria move toward molecules that promote survival (such as carbon sources) and away from molecules detrimental to survival (such as uncouplers of oxidative
genes (adenosine triphosphatase [ATPase] protein) of
Salmonella, TT1039 and TT1042, were provided by
John Roth, University of Utah, and Jen-Shiang Hong, Brandeis University. Chemotaxis assay. Cultures (3 ml) of bacteria were grown to the midlog phase at 30°C with shaking. After the cells were collected by centrifugation at 3,000 x g for 10 min, the bacteria were resuspended at a density of about i08 cells per ml in 50 mM morpholinepropanesulfonic acid (MOPS)-10 jM EDTA and were then immediately diluted to 106 cells per ml in the desired assay medium. The cells were then incubated at 30°C with shaking for 15 to 45 min before chemotactic responses were tested. The temporal assay was performed by the general procedure of Tsang et al. (29). To 9 drops of bacteria in a test tube at 30°C, 1 drop of lOx attractant was added, and the contents of the tube were mixed rapphosphorylation). idly. From this mixture a sample was removed and Divalent cations, however, do not fit neatly added to a microscope slide. The behavior of the into this general pattern. Although beneficial population was recorded by either of two methods. metals such as Mg+ and Ca2" can act as attract- The first involved measuring the time after chemoefants at high concentrations, toxic cations such fector addition before an individual bacterium rea tumbling mode and averaging the values as cobalt and nickel are also attractants for turned to from obtained individual trials. The second method Salmonella typhimurium (32). The same diva- involved the time required for 80% of the lent cations can act as repellents for Escherichia populationestimating to return to the unstimulated swimming coli (30). Because of this anomaly and the im- pattern. The methods correlated well with each other. portance of cations in cellular metabolism and Capillary assays were performed by the procedure signalling systems, a study of metal ions in chem- of Adler and Templeton (3). Enzyme assays. Membranes were prepared from otaxis was made and has led to partial characterization of the divalent cation receptor for bacteria grown to midlog phase at 30°C and collected by centrifugation. The cells were washed in 50 mM chemotaxis. MOPS-10 mM MgCl2 (pH 7.0), suspended in the same buffer containing 10,ug of DNase per ml, and disrupted MATERIALS AND METHODS by two passages through a French pressure cell at Materials. L-Malic acid, D-(+)-malic acid, cis-aco- 20,000 lb/in2. After the cell debris was removed by nitic acid, trans-aconitic acid, and DL-isocitric acid centrifugation at 10,000 x g for 10 min, the membranes were obtained from Sigma Chemical Co. Citric acid were collected by centrifugation at 100,000 x g for 90 and sodium citrate were from Mallinckrodt. Nutrient min. The membranes were washed in 50 mM MOPSbroth was from Difco Laboratories. Bacterial strains 10 mM MgCl2, pH 7.0, and then resuspended in 50 ST1, ST171, ST356, and ST382 have been described mM Tris-hydrochloride-10 mM MgCl2, pH 8.0. ATP hydrolytic activity was measured on these previously (5, 6, 9). Strains AN1, M72, and M272, S. typhimurium strains mutant in the tricarboxylic acid membranes by a modification of the method of Evans transport system, were provided by Ko Imai, Institute (10). Buffer and cofactor (30 pl of 0.33 M Tris [pH 8.0] for Fermentation, Osaka, Japan. Mutants in the unc -0.33 M MgCl2) and various amounts of enzyme were 798
VOL. 140, 1979
METAL ION-CITRATE COMPLEX IN BACTERIAL SENSING
diluted to 480 pl with water. ATP (20 ,ul of 0.1 M ATP, pH 7.0) was added to initiate the reaction, which was allowed to proceed for 10 min at 37°C. The reaction was terminated by the addition of 500 jil of 14% trichloroacetic acid. If a precipitate formed in any tube, 0.1 ml of 10% sodium dodecyl sulfate was added to dissolve it. The inorganic phosphate released was immediately measured by the Fiske-SubbaRow colorimetric method (11). Succinate dehydrogenase activity was assayed spectrophotometrically following the reduction of dichlorophenolindophenol at 600 nm after the addition of enzyme, succinate, and phenazine methosulfate (17, 18). Protein concentration was determined by the method of Lowry et al. after trichloroacetic acid precipitation (21).
RESULTS Role of citrate in divalent cation chemotaxis. Citrate is required for S. typhimurium to respond to divalent cations. If the bacteria are suspended in media without citrate, such as 50 mM MOPS, no change in swimming behavior is observed when 0 to 10 mM temporal gradients (23) of either MgCl2 or CaCl2 are applied. The bacteria respond normally to amino acids and sugars in this buffer. If the buffer contained 10 mM citrate, a smooth swimming response of about 1 min was observed (Table 1). This smooth swimming response in the presence of citrate was observed with a variety of metal ions (Table 2). The original finding that citrate was a chemoattractant was made a number of years ago (R. M. Macnab and D. E. Koshland, Jr., unpublished data) and has now been expanded in a recent study (M. Kihara and R. M. Macnab, personal communication) and in this paper. L-Malate also facilitated the divalent cation response, but a number of other compounds tested were inactive (Table 3). Although L-malate was less effective than citrate, it was a strong attractant when added in the absence of divalent cations (24; M. Kihara and R. M. Macnab, manuscript in preparation). This chemotactic response was shown in a conventional capillary assay. Bacteria moved up a spatial gradient of divalent cations when citrate was present in uniform concentration. They accumulated inside a capillary containing either
799
TABLE 2. Effects of divalent cations on Salmonella in citratea Smooth swim-
ming response
Stimulus (0-1 mM)
time (min)
MgC2 .0.9 1.2 CaC12 ....
CoCl2 .0.7 0.6 ... NiCl2 . CuCl2 .. 0.6 .... 0.6 FeSO4 ..... 0.7 ZnCl2 a The experimental procedure was as discussed in the text except that 10 mM citrate was added to MOPS buffer. Tumbly mutant ST171 was used for the assay. TABLE 3. Specificity of the cofactor requirement in divalent cation chemotaxisa Smooth swimSmooth swimming response
Compound
to 0-10 mM compound
(mm)
ming response to 0-10 mMI MgCl2 for bacteria suspended in 10 mM compound (min)
0.8 1.1 Citrate 0.9 0.5 L-Malate 0.6
Vol. 140, No. 3
Response to a Metal Ion-Citrate Complex in Bacterial Sensing THOMAS D. INGOLIA AND D. E. KOSHLAND, JR.* Department of Biochemistry, University of California, Berkeley, California 94720 Received for publication 24 September 1979
Salmonella typhimurium responds chemotactically to gradients of divalent cations in the presence of citrate ions. The actual chemoeffector is the citratemetal ion complex, which acts as an attractant. Citrate (which is also a chemoeffector for Salmonella) and the citrate-metal ion complex are recognized by different receptors. The response of Salmonella, which can transport citrate through its membrane, is quite different than that of Escherichia coli, which cannot.
Bacteria respond to gradients of chemicals in their environment by modifying their swimming behavior (1, 7, 12, 19, 20, 22). They swim smoothly when they encounter an increase in concentration of attractant molecules (or a decrease in repellent molecules), but they tumble and randomize direction when they encounter increases in repellents (or decreases in attractants). The net migration, called chemotaxis, enables bacteria to move up gradients of certain sugars, amino acids, and metabolites and down unfavorable gradients of compounds like phenol and indole. Generally, bacteria move toward molecules that promote survival (such as carbon sources) and away from molecules detrimental to survival (such as uncouplers of oxidative
genes (adenosine triphosphatase [ATPase] protein) of
Salmonella, TT1039 and TT1042, were provided by
John Roth, University of Utah, and Jen-Shiang Hong, Brandeis University. Chemotaxis assay. Cultures (3 ml) of bacteria were grown to the midlog phase at 30°C with shaking. After the cells were collected by centrifugation at 3,000 x g for 10 min, the bacteria were resuspended at a density of about i08 cells per ml in 50 mM morpholinepropanesulfonic acid (MOPS)-10 jM EDTA and were then immediately diluted to 106 cells per ml in the desired assay medium. The cells were then incubated at 30°C with shaking for 15 to 45 min before chemotactic responses were tested. The temporal assay was performed by the general procedure of Tsang et al. (29). To 9 drops of bacteria in a test tube at 30°C, 1 drop of lOx attractant was added, and the contents of the tube were mixed rapphosphorylation). idly. From this mixture a sample was removed and Divalent cations, however, do not fit neatly added to a microscope slide. The behavior of the into this general pattern. Although beneficial population was recorded by either of two methods. metals such as Mg+ and Ca2" can act as attract- The first involved measuring the time after chemoefants at high concentrations, toxic cations such fector addition before an individual bacterium rea tumbling mode and averaging the values as cobalt and nickel are also attractants for turned to from obtained individual trials. The second method Salmonella typhimurium (32). The same diva- involved the time required for 80% of the lent cations can act as repellents for Escherichia populationestimating to return to the unstimulated swimming coli (30). Because of this anomaly and the im- pattern. The methods correlated well with each other. portance of cations in cellular metabolism and Capillary assays were performed by the procedure signalling systems, a study of metal ions in chem- of Adler and Templeton (3). Enzyme assays. Membranes were prepared from otaxis was made and has led to partial characterization of the divalent cation receptor for bacteria grown to midlog phase at 30°C and collected by centrifugation. The cells were washed in 50 mM chemotaxis. MOPS-10 mM MgCl2 (pH 7.0), suspended in the same buffer containing 10,ug of DNase per ml, and disrupted MATERIALS AND METHODS by two passages through a French pressure cell at Materials. L-Malic acid, D-(+)-malic acid, cis-aco- 20,000 lb/in2. After the cell debris was removed by nitic acid, trans-aconitic acid, and DL-isocitric acid centrifugation at 10,000 x g for 10 min, the membranes were obtained from Sigma Chemical Co. Citric acid were collected by centrifugation at 100,000 x g for 90 and sodium citrate were from Mallinckrodt. Nutrient min. The membranes were washed in 50 mM MOPSbroth was from Difco Laboratories. Bacterial strains 10 mM MgCl2, pH 7.0, and then resuspended in 50 ST1, ST171, ST356, and ST382 have been described mM Tris-hydrochloride-10 mM MgCl2, pH 8.0. ATP hydrolytic activity was measured on these previously (5, 6, 9). Strains AN1, M72, and M272, S. typhimurium strains mutant in the tricarboxylic acid membranes by a modification of the method of Evans transport system, were provided by Ko Imai, Institute (10). Buffer and cofactor (30 pl of 0.33 M Tris [pH 8.0] for Fermentation, Osaka, Japan. Mutants in the unc -0.33 M MgCl2) and various amounts of enzyme were 798
VOL. 140, 1979
METAL ION-CITRATE COMPLEX IN BACTERIAL SENSING
diluted to 480 pl with water. ATP (20 ,ul of 0.1 M ATP, pH 7.0) was added to initiate the reaction, which was allowed to proceed for 10 min at 37°C. The reaction was terminated by the addition of 500 jil of 14% trichloroacetic acid. If a precipitate formed in any tube, 0.1 ml of 10% sodium dodecyl sulfate was added to dissolve it. The inorganic phosphate released was immediately measured by the Fiske-SubbaRow colorimetric method (11). Succinate dehydrogenase activity was assayed spectrophotometrically following the reduction of dichlorophenolindophenol at 600 nm after the addition of enzyme, succinate, and phenazine methosulfate (17, 18). Protein concentration was determined by the method of Lowry et al. after trichloroacetic acid precipitation (21).
RESULTS Role of citrate in divalent cation chemotaxis. Citrate is required for S. typhimurium to respond to divalent cations. If the bacteria are suspended in media without citrate, such as 50 mM MOPS, no change in swimming behavior is observed when 0 to 10 mM temporal gradients (23) of either MgCl2 or CaCl2 are applied. The bacteria respond normally to amino acids and sugars in this buffer. If the buffer contained 10 mM citrate, a smooth swimming response of about 1 min was observed (Table 1). This smooth swimming response in the presence of citrate was observed with a variety of metal ions (Table 2). The original finding that citrate was a chemoattractant was made a number of years ago (R. M. Macnab and D. E. Koshland, Jr., unpublished data) and has now been expanded in a recent study (M. Kihara and R. M. Macnab, personal communication) and in this paper. L-Malate also facilitated the divalent cation response, but a number of other compounds tested were inactive (Table 3). Although L-malate was less effective than citrate, it was a strong attractant when added in the absence of divalent cations (24; M. Kihara and R. M. Macnab, manuscript in preparation). This chemotactic response was shown in a conventional capillary assay. Bacteria moved up a spatial gradient of divalent cations when citrate was present in uniform concentration. They accumulated inside a capillary containing either
799
TABLE 2. Effects of divalent cations on Salmonella in citratea Smooth swim-
ming response
Stimulus (0-1 mM)
time (min)
MgC2 .0.9 1.2 CaC12 ....
CoCl2 .0.7 0.6 ... NiCl2 . CuCl2 .. 0.6 .... 0.6 FeSO4 ..... 0.7 ZnCl2 a The experimental procedure was as discussed in the text except that 10 mM citrate was added to MOPS buffer. Tumbly mutant ST171 was used for the assay. TABLE 3. Specificity of the cofactor requirement in divalent cation chemotaxisa Smooth swimSmooth swimming response
Compound
to 0-10 mM compound
(mm)
ming response to 0-10 mMI MgCl2 for bacteria suspended in 10 mM compound (min)
0.8 1.1 Citrate 0.9 0.5 L-Malate 0.6
