Journal of Bacteriology, January 2004, p . 588-592, Vol . 186, No . 2

Biphasic Excitation by Leucine in Escherichia coli Chemotaxis

Shahid Khan^1* and David R . Trentham²

Molecular Biology Consortium, Chicago, Illinois 60612,¹ National Institute for Medical Research, London NW7 1AA, United Kingdom²

Received 29 July 2003/ Accepted 10 October 2003

 
Leucine concentration jumps (applied by photolysis of inert derivatives) triggered swim or tumble responses in Escherichia coli mutants lacking Tsr or Tar, respectively . Wild-type E . coli bacteria were attracted in spatial assays when the initial leucine concentration difference was 5 to 120 µM but were repulsed when it was over 0.5 mM . Their responses to concentration jumps confirmed earlier deductions regarding biphasic excitation .

 
Among several hydrophobic amino acids that repel Escherichia coli, L- (or D-) leucine is the most potent . A microfluidic assay developed by Mao et al . (11) showed that at low concentrations L-leucine was also an attractant . We reached a similar conclusion upon analysis, using the photorelease assay, (7) of E . coli chemotactic excitation behavior and have used this finding to characterize biphasic excitation (8) . Photolabile derivatives of leucine, O-2,6-dinitrobenzyl-L-leucine (DNB-Leu), and N-1-(2-nitrophenyl)ethoxycarbonyl-L-leucine (NPEC-Leu) were synthesized for rapid photogeneration of leucine (Fig . 1) . The methyl-accepting chemotaxis protein (MCP) Tsr mediates repulsion from leucine (15) . Tsr was the predominant receptor in the tar strain RP2361 lacking the other major MCP, Tar . The rate of change of direction (RCD) (a measure of the population angular speed) (7) of this strain increased upon leucine photorelease, as expected . A saturation response was obtained upon photorelease of 0.5 mM leucine (Fig . 2A) . No repellent response was seen in the tsr strain RP5700 . Furthermore, as anticipated on the basis of the results of a previous study (11), a swim response (i.e., decreased RCD) was obtained (Fig . 2B) . A jump in concentration from 0 to 5 µM elicited a saturation smooth-swim response; a jump in concentration from 0 to 50 nM elicited a detectible response . Thus, the attractant excitation response to leucine is comparable in strength to that seen upon serine photorelease (7) .

 
The tsr strain responded by swimming smoothly when either DNB-Leu or NPEC-Leu was used . Photorelease of protons is known to elicit smooth swim responses in tsr strains (8) . This was ruled out as a potential cause of the response seen upon leucine photorelease as follows . First, increasing the buffer concentration of morpholineethanesulfonic acid (MES) from 10 to 100 mM did not alter the response . Second, although DNB-Leu photolysis liberates protons, NPEC-Leu photolysis results in net hydroxide ion release during the 2-s observation time following photolysis (Fig . 1) . The tar tsr mutant RP3851 did not respond (Fig . 2C) . Thus, Tar was the major determinant for the swim response . The cheRcheB strain RP2859 has normal wild-type bias but greatly reduced response to aspartate (9, 14) . This strain also did not respond . Leucine response sensitivity must therefore involve a role for the MCP methylesterase CheB and/or methyltransferase CheR, as seen for aspartate .

Spatial assays were conducted (as described previously) (1, 15) to explore the consequences of dual-signal generation for chemotactic migration . The half-maximal doses (L_1/2) for repulsion from plugs containing leucine were 10 and 3.6 mM for wild-type and tar strains, respectively . The higher L_1/2 observed for the wild type (relative to that observed for tar E . coli) may be due to attenuation of the Tsr repellent response by Tar-mediated attraction . The tsr strain did not respond (Fig . 3A) . Capillary, rather than plug, assays provided a better test for attraction . The tar strain was repelled, but wild-type and tsr E . coli bacteria accumulated in the capillary when the initial concentration difference between it and the pond was 0 to 120 µM or lower (Fig . 3B) . Accumulation would decrease at higher concentrations, since the concentration gradient centered on the L_1/2 would move further away from the capillary mouth by the end of the assay (6) . However, the observed decrease was more severe than expected on this basis . It was similar to that observed for competition of the attractant, aspartate, with the repellent, valine (2) . The fact that the response declined for tsr as well as wild-type strains in the spatial assay (Fig . 3B) suggests that repulsion from leucine is not mediated solely by Tsr . The wild-type response to aspartate (Fig . 3B inset) was an order of magnitude stronger and was maintained over a larger concentration range than the leucine attractant response (consistent with attenuation of the Tar-mediated swim signal by the Tsr tumble signal) . Our spatial assay data broadly agree with previously reported leucine plug and aspartate capillary assay results (2, 15) and with the findings of Mao et al . (11), who recorded attractant responses with concentrations of leucine down to 1 µM .

 
Biphasic excitation was first observed for protons (8) (sensed by Tar as attractant and by Tsr as repellent) . Leucine seems to behave similarly (Fig . 4A) . As argued previously (8), the smooth-swim phase of biphasic excitation is due to generation of a second signal and not to adaptation of the initial repellent signal, since the tumble response in tar strains lasted for many seconds . Since the Tar L_1/2 was lower than the Tsr L_1/2, we expected that a small (0 to 5 µM leucine) jump would predominantly elicit swimming and that a prestimulus background leucine concentration between the Tar L_1/2 and Tsr L_1/2 concentrations would greatly reduce the proportion of the swim signal in comparison to the tumble signal . These expectations were met (Fig . 4B and C) . In the case of protons, it was not feasible to adjust prestimulus pH over a wide range to separate the two signals; effects of the pH jumps on motor operation and/or flagellar bundle formation were a concern . Thus, this study extends our earlier work to establish biphasic excitation as a consequence of antagonistic signal generation .

 
The action of the repellent amino acids may be due to nonspecific effects on membrane properties, since their L_1/2 values are two orders of magnitude larger then those for amino acid attractants . However, Eisenbach et al . (5) have argued against this explanation for leucine . In addition, leucine, while most potent, is less hydrophobic than some other amino acid repellents (15) . Repulsion at high leucine concentrations, together with strong attraction at low concentrations, may keep expression of the leucine/Lrp regulon, a key control element in amino acid biosynthesis (4), in an optimal range (analogous to those of pH and temperature taxis) . Leucine may be added to the list of effectors that trigger antagonistic signals from different MCPs (10, 13, 16, 17) .

Biphasic excitation reveals that integration of signals from different MCPs is not complete at the receptor level . This observation raises interesting issues regarding receptor-receptor interactions if, as believed, different MCPs are part of the same receptor cluster activating a common kinase (3, 12) . Quantitative analysis of biphasic excitation (made possible by availability of the caged leucines) should be valuable for deciphering these interactions . In addition, biphasic excitation may now serve as a rapid diagnostic for dual-signal generation . The Tsr receptor also mediates responses towards the other repellent amino acids (15) . It would be of interest to determine whether these compounds also attract, utilizing other MCP family members to do so .

 
We thank Meghan Gleason for assistance with behavioral assays and Gordon Reid for synthesis of the caged leucines .

This work was supported by grant R01-GM49319 from the National Institutes of Health .

 
* Corresponding author . Mailing address: Molecular Biology Consortium, Chicago Technology Park, 2201 W . Campbell Park Dr., Chicago, IL 60612 . Phone: (312) 942-9017 . Fax: (312) 829-4069 . E-mail: kh01@tigger.uic.edu .

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