enase than to that ofproline transport, but chemotaxis to proline was eliminated by mutations eliminating either or both of these activities. No responseto proline.
JOURNAL OF BACTFUOLOGY, June 1981, p. 902-906
Vol. 146, No. 3
0021-9193/81/060902-05$02.00/0
Genetic and Biochemical Requirements for Chemotaxis to LProline in Escherichia coli MAUREEN CLANCY, KAREN A. MADILL, AND JANET M. WOOD* Guelph- Waterloo Centre for Graduate Work in Chemistry, University of Guelph, Guelph, Ontario, Canada NIG 2W1
Received 20 November 1980/Accepted 23 March 1981
Chemotaxis to L-proline was examined by the capillary assay, using a set of Escherichia coli strains bearing well-defined defects in the enzymes of proline transport and utilization. Aspartate taxis was measured as a constitutive, control activity whose receptor and transducer requirements are known. Proline chemotaxis showed a pattern of induction more analogous to that of proline dehydrogenase than to that of proline transport, but chemotaxis to proline was eliminated by mutations eliminating either or both of these activities. No response to proline was observed in the absence of a proline concentration gradient or when succinate was provided as an oxidizable carbon source. These data suggest that the chemotactic response to proline results from a direct impact of proline oxidation on the energy metabolism of the cell. Bacterial chemotaxis is a model sensory system that is amenable to examination at the molecular level. Considerable progress has been made towards understanding the biophysical, biochemical, and genetic bases for sugar and amino acid taxis in the enteric bacteria Escherichia coli K-12 and Salmonella typhimurium (7, 11). In those cases that have been examined in detail, a temporal change in attractant or repellant concentration is sensed as a change in fractional occupancy of a specific receptor protein (14, 19). The signals from groups of receptors are communicated to the flagellar machinery by a mechanism whose details are unclear. This communication system is known to involve one of several methyl-accepting chemotaxis proteins or transducers (7, 11). The response to a chemotactic signal is a change in frequency of flagellar reversal, resulting in an increase or decrease in the frequency with which the bacteria change their swimming direction or tumble (4, 12). Strong evidence supports the view that several of the receptors for sugar taxis are periplasmic or plasma membrane-associated proteins that are also components of specific active transport systems (7, 11). Neither transport nor metabolism of these attractants is required to yield a chemotactic response, however (7, 11). Attempts to identify the receptors mediating amino acid taxis have been less fruitful, but recent experiments from two laboratories suggest that the transducer molecules themselves are the receptors for serine and aspartate taxis (5; M. L. 902
Hedblom and J. Adler, Fed. Proc. 39:2103, 1980). Chemotaxis to L-proline in E. coli is unusual in that it must be induced by growing the bacteria in media containing proline and in that it is not eliminated by defects in any of the known transducer proteins (13). We have prepared a set of strains of E. coli K-12 with well-defined genetic lesions in the enzymes of proline transport and utilization (24, 25). This paper describes the use of those strains to assess the roles of the proline utilization enzymes in proline chemotaxis. MATERIALS AND METHODS Materials. Microbiological media were from Difco Laboratories (Detroit, Mich.), and L-proline, L-aspartate, L-tryptophan, and thiamine hydrochloride were from Sigma Chemical Co. (St. Louis, Mo.). All other reagents were of the highest grade available. Bacterial strains. The strains used in this study, all derivatives of E. coli K-12, are listed in Table 1. A motile isolate of each strain was prepared by selecting bacteria that formed chemotactic rings in tryptone agar swarm plates (1). Chemotaxis assay. Bacterial cultures were prepared and chemotaxis was measured by the capillary assay described by Adler (1). When added to cultures as carbon or nitrogen source, or both, or as inducers, L-proline, glycyl-L-proline, and succinate were at 0.2% (wt/vol). Chemotaxis assays proceeded for 45 min at 30°C, using bacterial suspensions containing 106 to 107 bacteria per ml. The chemotaxis assay medium did not contain added Mg2e. L-Prohne and L-aspartate were used as attractant solutions at 10-2 and 1o-3 M, respectively, unless otherwise stated. When L-proline
PROLINE CHEMOTAXIS IN E. COLI K-12
VOL. 146, 1981
903
TABLE 1. E. coli K-12 strain? Strain
Genotype
Proline utilization defect
F- trp lacZ rpsL thi CSH4 A(putPA)100
None Proline transport and proline dehydrogenase JT31 Proline dehydrogenase CSH4putAl::Tn5 JT34 CSH4 putP.3.:Tn5 Proline transport a The genetic nomenclature is that of Bachmann and Low (3). CSH4 RM2
or succinate was added to both bacterial suspension and capillary during the chemotaxis assay, each was present at 10-2 M.
RESULTS Chemotaxis assay. The capillary assay yields data as numbers of bacteria accumulated per capillary tube. In view of the large variations in absolute numbers of bacteria obtained by this method, we have tested statistically the reliability of this assay as a measure of proline and aspartate taxis. Replicate measurements were made of bacterial accumulation levels with no attractant (control) or with proline or aspartate as attractant, using a large number of independently prepared cell suspensions (17 suspensions for proline taxis; 14 for aspartate). The replicate measurements were averaged before the analysis. Consideration of the ranges and means for these sets of data revealed that the logarithms of the average numbers of bacteria accumulated should be considered rather than the absolute numbers of bacteria (18). In other words, the effect of an attractant is to cause a proportional, rather than an additive, increase in the number of bacteria accumulating per capillary. This approach is analogous to considering the ratio of the number of bacteria in an attractant-containing versus a control capillary. An analysis of variance was perforned on the log accumulation levels, using a randomized complete block design (20). The log accumulation levels in the proline- or aspartate-containing capillaries were higher than those in the control capillaries to the 99% confidence level when the treatment variance was compared with the error variance. The means of the log accumulation levels and their standard deviations were: proline, 4.5 ± 0.2, versus control, 3.5 ± 0.2; and aspartate, 4.9 ± 0.3, versus control, 3.6 ± 0.3. The data in Tables 2, 3, and 4 and in Fig. 1 were obtained in an analogous way, using data from at least two cell suspensions in each case. They are also expressed as means of log accumulation levels. Regulation of proline taxis. We confirmed the inducibility of proline taxis by comparing the activities of bacteria grown on succinate as the carbon source with or without proline as an
Source
Cold Spring Harbor Laboratory R. Menzel, University of Utah (23, 25) This laboratory (23, 25) This laboratory (23, 25)
inducer. Aspartate taxis was measured as a constitutive, control activity that is mediated by a known receptor-transducer system. Bacteria grown in medium containing proline responded to both proline and aspartate as attractants, whereas those grown in medium lacking proline responded only to aspartate (Table 2). Proline transport activity is enhanced when E. coli is grown on succinate, which relieves catabolite repression, but no proline dehydrogenase activity is observed. Both transport and proline dehydrogenase activity are induced when such growth media are supplemented with proline (24). The induction of proline taxis, therefore, parallels more closely that of proline dehydrogenase than that of proline transport. Genetic requirements for proline taxis. Mesibov and Adler have shown that proline taxis is not eliminated by any of the known transducer mutations (13). To test the roles of the proline utilization enzymes in proline taxis, we have measured the activities of strains defective at putP (lacking transport activity) or at putA (lacking proline dehydrogenase) or at both loci. Either mutation eliminates proline taxis without eliminating aspartate taxis (Table 3). Inducer exclusion prevents induction of theputA gene by proline in putP mutants, but putA is induced by glycyl-L-proline in these strains (24). Either inducer is effective in strain CSH4, but neither induces chemotactic activity to proline in any of the mutant strains. Expression of both putP and putA is thus required for proline taxis. Proline dehydrogenase is located on the inner surface of the plasma membrane (16, 24). The proline porter may therefore be required directly, as a receptor, or it may allow access of proline to proline dehydrogenase if the latter is the true mediator of the chemotactic response to proline. A diffusion gradient of attractant concentration is generated when chemotaxis is measured by the capillary assay. Catabolism of proline would be expected to steepen that gradient when proline taxis by strains bearing proline dehydrogenase is measured. The responses of strains CSH4 (putPr putA+) and JT31 (putr+ putA) were tested at a series of proline concentrations from 10' to 1 M (Fig. 1). As was shown before,
904
CLANCY, MADILL, AND WOOD the maximum response to proline by the parent strain was at 10- M proline (13). Strain JT31 did not respond at any of these concentrations or at 10-5 or 10 M proline (data not shown). The lack of proline taxis in the putA mutant strains is therefore not a consequence of a decrease in the threshold or optimum concentration for that system. Role of proline oxidation. The terminal electron acceptor for proline oxidation by proline dehydrogenase is molecular oxygen (16). Proline catabolism is therefore directly linked to oxygen consumption, and it may, in a closed system, lead to the formation of an oxygen concentration gradient. The chemotactic response to oxygen is a powerful one that overrides the responses to other nutrients. We therefore propoed that the observed response to proline was in fact oxygen taxis due to proline catabolism and its coupled oxygen consumption. The response of strain CSH4 to proline and aspartate was measured with both the capillary and the bacterial suspension containing proline (Table 4). Proline taxis was abolished but aspartate taxis was retained.
J. BACTERIOL.
The diffusion gradient of proline is therefore a requirement for proline taxis; the observed taxis cannot be attrbuted to the generation of an
gradient during proline catabolism. Direct measurements of bacterial tumble frequency have implied that the chemotactic responses to oxygen, nitrate, and fumarate depend on their utilization as terminal electron acceptors by the membrane-associated respiratory chains of S. typhimurium and E. coli (22). It was therefore suggested that bacterial tumble frequency is a function of the tranamembrane proton motive force (Ap) generated during respiration. No exogenous carbon source is added to the bacterial suspensions used in the capillary assay (1), so these bacteria are likely to poses a reduced proton motive force. Proline oxidation by proline dehydrogenase generates a proton motive force in inverted membrane vesicles from E. coli (J. Stephenson and J. M. Wood, unpublished data). Perhaps, then, the oberved chemotactic response to proline reflects a change in Ap that is linked to proline oxidation. If an alternate, oxidiable carbon source provided in the chemotaxis medium should fully energize the bacteria, eliminating the impact of proline TABLE 2. Regulation ofproline taxis in E. coli oxidation on Ap and preventing proline taxis. strain CSH4 When succinate is added to both bacterial suspension and attractant capillary during the Log accumulation level per capillary (mean) in given chemotaxis assay, aspartate taxis is retained but growth mediumb proline taxis is eliminated (Table 4). This obserAttractant *vation is consistent with the view that proline Succinate- Succinate4'pro- taxis is due to an impact of proline oxidation on Sucom44e INHline Ap, but direct measurements of Ap would be required to prove that the two are coincident. None 3.7 3.6 L-Proline 3.7 4.5 DISCUSSION L-Aspartate 4.8 4.9 Taylor et al. have examined the behavioral a Taxis was measured by the capillary assay (see responses of E. coli and S. typhimurium to nitext). bBacterial cultures were prepared as described by trate, molecular oxygen, and fumarate (22). Each Adler (1), using succinate and NH4+ as carbon and of these compounds is known to be both a chenitrogen sources and proline as an inducer. motactic attractant and the electron-accepting oxygen
so,
TABLE 3. Effects ofputP and putA mwuations on proline chemotaxisa Strain
Relevant genotype
CSH4 JT31 JT34 RM2
putP putA' putAI::Tn5 putP3,.:Tn5
CSH4 JT34 RM2 b
A(putPA)100 putP+ putA+ putP3::Tn5 A(putPA)1OO
Inducerb
L-Proline L-Probne L-Proline
L-Proline Glycyl-L-proline Glycyl-L-proline Glycyl-L-proline
Log accumulation level per capillary (mean) with attractant: None Proline Aspartate
3.6 3.6 3.7 3.8 3.0 3.0 3.3
4.5 3.5 3.6 3.5 4.4 2.9 3.3
4.9 4.9 5.0 5.2
.0 4.8 5.1
Taxis was measued by the capillary asay (see text). Bacteri cultures were prepared as described by Adler (1), using succinate and NH4+ as carbon and nitrogen
sources.
PROLINE CHEMOTAXIS IN E. COLI K-12
VOL. 146, 1981
LU
4.0
-
z
0
34
0
0. PROLINE MOLARITY FIG. 1. Chemotactic response to proline of E. coli strain CSH4, wild type with respect to proline utilization (-), and JT31, a putA mutant (0). Taxis was measured by the capillary assay (see text), using cultures prepared as described by Adler (1) with succinate and NH4' as carbon and nitrogen sources and proline as an inducer.
TABLE 4. Effects ofproline and succinate on proline and aspartate chemotaxis in strain CSH4a Addition to chemotaxis medium'
Log accumulation level per capillary (mean) with attractant: None
Proline 4.5 3.5 3.9
Aspartate
5.0 3.5 None 5.4 Proline 5.1 3.7 Succinate a Taxis was measured by the capillary assay (see text), using cultures of strain CSH4 prepared as described by Adler (1) with succinate and NH4' as carbon and nitrogen sources and proline as an inducer. 'When added to the chemotaxis medium, both proline and succinate were 10-2 M.
substrate for a membrane-associated reductase. Temporal gradients of each compound elicited a transient, smooth swimming response that was inhibited by blue light (thought to reduce electron flow via flavin enzymes) and required the function of the appropriate reductase (nitrate reductase, cytochrome oxidase, or fumarate reductase). It was suggested that the observed decreases in tumble frequency, usually correlated with positive chemotactic responses, might be a direct consequence of the increased proton motive force (Ap) resulting from increased electron flow to these terminal electron acceptors.
905
Our observations regarding the enzymatic requirements for chemotaxis to proline suggest an analogous interpretation. We have used the capillary assay to establish the genetic and biochemical requirements for accumulation of E. coli cells in response to spatial gradients of proline concentration. The data show -that chemotaxis to proline requires expression of the putP and putA genes (Tables 2 and 3). Defects in putP reduce proline transport activity, and putA encodes an oxygen-linked proline dehydrogenase associated with the cytoplasmic surface of the plasma membrane (16, 23, 24). Neither proline transport nor proline dehydrogenase activity alone is correlated with a positive chemotactic response (Tables 2 and 3, Fig. 1), and the response to proline does not result from creation of an oxygen gradient during proline utilization (Table 4). Chemotaxis to proline is not eliminated by any of the known "transducer" mutations (13), but it is eliminated when succinate is provided as an alternate electron donor in both the attractant-containing capillary and the bacterial suspension used during the chemotaxis assay (Table 4). These data are all consistent with the view that the chemotactic response to proline results from a direct impact of its oxidation on bacterial energy metabolism. Proline transport activity would therefore be required in proline chemotaxis to permit uptake and oxidation of the attractant molecule. Such a mechanism would differ radically from tha established for chemotaxis to many other organic molecules. In most cases a transport protein functions as a chemotactic receptor, but neither uptake nor metabolism of the attractant molecule is required to elicit a chemotactic response. Khan and Macnab have analyzed the dependence of steady-state flagellar rotation speed and clockwise-counterclockwise bias on Ap in E. coli, S. typhimurium and Bacillus subtilis (8). Their data reveal that the fraction of time spent by a bacterium in tumbling is a direct, saturable function of Ap rather than the inverse function implied on the basis of our data and those of Taylor et al. (22). They suggest that the transient, smooth swimming responses to gradients of electron donors are due to receptor-mediated behavioral changes that are superimposed on the steady-state energetic effects. Not only binding to the appropriate enzyme but also reduction or oxidation of the attractant molecule seems to be required for chemotaxis to the electron acceptors and proline (22; Tables 2 and 3). Furthermore, at least one of these enzymes (proline dehydrogenase) is cytoplasmic in orientation. If they do function as chemotactic receptors, then, cytochrome oxidase, nitrate reductase, fumarate reductase, and proline dehydrogenase must do
906
CLANCY, MADILL, AND WOOD
so in a manner very different from that established for other systems. Workers in a number of laboratories are now attempting to clarify the relationships among bacterial tumble frequency, Ap, and the chemotactic response under chaning, as well as steady-state, conditions (6, 8, 10, 15, 17, 21). These relationships are complex, but it can be concluded that, although changes in the components of Ap can lead to behavioral changes, changes in Ap or its components are not always required for chemotaxis (15). It is not yet clear whether the chemotactic responses to electron donors or acceptors can be ascribed directly or indirectly to the impact of their metabolism on Ap. It is clear, however, that if the response to these molecules is not mediated by changes in Ap it will be possible to detect chemotactic responses to them under conditions of fixed Ap. The roles of the dehydrogenases and reductases as chemotactic "receptors" can then be reevaluated. ACKNOWLEDGMENTM We are grateful to Michael Eisenbach, Mary Hedblom, Juliua Adler, and Brian Allan, with whom this work-was discussed, to Rolf Menzel for strain RM2, and to Flo Rayner for preparation of the manupt. This research was supported by a grant from the Natural Sciences and Engneeri Research Council of Canada. LTERATURE CITED 1. Adler, J. 1973. A method for measuing chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. Gen. Microbiol. 74: 77-91. 2. Anderson, R. R., R. Menzel, and J. M. Wood. 1980. Biochemistry and regulation of a second L-proline tranport system in SalmoneUa t)phimurium. J. Bacteriol. 141:1071-1076. 3. Bachmann- B. J., and K. B. Low. 1980. Linkage map of Escherichia coli K-12, edition 6. Microbiol. Rev. 44:156. 4. Berg, H. C., and D. W. Brown. 1972. Chemotaxis in Eaerchia coli analyzed by three dimensional tracking. Nature (London) 239:600-504. 5. Clarke, S., and D. E. Kosland, Jr. 1979. Membrane receptors for aspartate and smne in bacterial chemotaxis. J. Biol. Chem. 234:9696-9702. 6. DeJong, M. IL, and C. van der Drift 1978. Control of the chemotactic behavior of Bacilus subtilis cells. Arch. Microbiol. 116:1-8. 7. Hazelbauer, G. L*, and J. S. Parkinson. 1977. Bacterial
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chemotaxis, p. 59-98. In J. Reisaig (ed.), Microbial interactions (receptors and recognition). Chapman and Hall, London. Khan, S., and R. KL Macnab. 1980. The steady state ounterclockwise/clockwise ratio of bacterial flagellar motors is regulated by protonmotive force. J. Mol. Biol. 138:563-597. Khan, S., and R. M. Macnab. 1980. Proton chemical potential, proton electrical potential and bacterial motility. J. Mol. Biol. 138:599-614. Laslo, D. J., and B. L. Taylor. 1981. Aerotaxis in SalmoneUa typhiurium: role of electron transport. J. Bacteriol. 145:900-1001. Macnab, R. M. 1978. Bacterial motility and chemotaxis. molecular biology of a behaviorial system. Crit. Rev. Biochem. 5:291-342. Macnab, R. M., and D. E. Koshland, Jr. 1972. The gradient-seing mechanism in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 69:2609-2562. Mesibov, R., and J. Adler. 1972. Chemotaxis toward amino acids in Escherichia coli. J. Bacteriol. 112:315-
326. 14. Mesibov, R., G. W. Ordal, and J. Adler. 1973. The range of attractant concentrations for bacterial chemotaxis and the threshold and size of response over this range. J. Gen. Physiol. 62:203-223. 15. Miller, J. B., and D. E. Kohland Jr. 1980. Protonmotive force and bacterial sensing. J. Bacteriol. 141:26-32. 16. Searpula, R. C., and R. L. Soffer. 1978. Membrane bound proline dehydrogenase from Escherichia coli. J. Biol. Chem. 253:5997-6001. 17. Shioi, J.-L, V. Imae, and F. Oosawa. 1978. Protonmotive force and motility of Bacilus subtilis. J. Bacteriol. 133:1083-1988. 18. Snedecor, G. W., and W. G. Cochrane. 1967. Statistical methods, 6th ed., p. 329-330. The Iowa State University Press, Ames. 19. Spudich, J. L, and D. E. Koshland, Jr. 1975. Quantitation of the sensory response in bacterial chemotaxis. Proc. Natl. Acad. Sci U.S.A. 72:710-713. 20. Steel, R. G. D., and J. H. Torrie. 1980. Principles and procedures of statistics-a biometrical approach, 2nd ed., p. 195-221. McGraw-Hill Book Co., New York. 21. Smeleman, S., and J. Adler. 1976. Change in membrane potential during bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 73:43874391. 22. Taylor, B. L, J. B. Miller, H. M. Warrick, and D. E. Koshland, Jr. 1979. Electron acceptor taxis and the blue light effect on bacterial chemotaxis. J. Bacteriol. 140:567-573. 23. Wood, J. IL 1980. Genetics of L-proline utilization in Ewcherichia coL K-12. J. Bacteriol. 145:896-901. 24. Wood, J. NL, and D. Zadworny. 1979. Characterization of an inducible porter required for L-proline catabolism by Ewherichia coi K12. Can. J. Biochem. 57:11911199. 25. Wood, J. M., and D. Zadworny. 1980. Amplification of the put genes and identification of the put gene products in Escherichia coli K12. Can. J. Biochem. 58:787796.
