Fructose is an attractant to E. coli
We used a wild-type strain JY26 (ΔfliC), which is a derivative of E. coli K12 strain RP437, for this experiment. The plasmid pKAF131 which constitutively expresses the sticky filament FliCst was transformed into JY26 in this study. We studied its response to a stepwise stimulus of fructose under various concentrations (0, 0.1 mM, 1 mM, 10 mM, 20 mM, 50 mM). The kinetics of the flagellar motor was monitored with a bead assay, with the motor CW bias (the probability of the flagellar motor rotating clockwise) was used as the indicator of the chemotactic output (16). The motor CW bias was stable without the addition of fructose as shown in Fig. 1A. The value of the motor CW bias was around 0.15, which is in consistent with the previous result. In contrast, the motor CW bias abruptly dropped after the stepwise stimulus of fructose at time 180 s, then it slowly recovered to its pre-stimulus level (Fig. 1B-F). After the removal of the stepwise stimulus of fructose at time 660 s, the motor CW bias increased rapidly to the peak and decrease to its pre-stimulus level (Fig. 1B-F). The results above suggested fructose is an attractant to E. coli.
Our measurements showed that fructose is not only an attractant to E. coli, but also influences the response degree and recovery time of E. coli under various concentrations. As shown in Fig. 1B-D, the motor CW bias added with the stepwise stimulus of fructose (0.1 mM, 1 mM, 10 mM) dropped to the level above 0, while the CW bias dropped to 0 after the addition of the stepwise stimulus of fructose (20 mM, 50 mM) in Fig. 1E-F. Thus, E. coli showed a stronger response to the higher concentration of fructose. The recovery time was defined as the duration time from the time adding the stimulus to the time CW bias returned back to its pre-stimulus level. The recovery time were 59±10.2 s, 113±9.1 s, 171±13.3 s, 105±11.1 s, 77±5.8 s, corresponding to the concentration of fructose 0.1 mM, 1 mM, 10 mM, 20 mM, 50 mM, respectively, in which, cells in medium of 0.1 mM and 50 mM fructose recovered faster than cells in medium of 1 mM, 10 mM, 20 mM. Interestingly, we also observed the overshoot phenomenon (14) which has been observed before in the step response of E. coli to 50 mM fructose.
CheY is essential for the chemotaxis of E. coli to fructose
To test the effect of CheY on the chemotaxis of E. coli to fructose, we measured the CW bias of the strain JY27 (ΔfliC ΔCheY) which is derived from JY26 and transformed with the plasmid pKAF131 to express FliCst. The motor CW bias was 0 and unchanged with the addition of the stimulus of 50 mM fructose as shown in Fig. 2A, which is distinct from the result of wild-type strain JY26. The motor CW bias was determined by the coupling of phosphorylated CheY (CheY-P) and motor switch complex, the more CheY-P binds to motor switch complex, the higher motor CW bias. Since CheY was deleted in JY27, there were no CheY molecules could be phosphorylated in the cells of JY27, thus the motor CW bias was 0. In addition, the cells showed no response to fructose for the reason no CheY-P could be dephosphorylated in the cells of JY27.
E. coli needs chemoreceptors to sense fructose
The strain HCB429 (Δ(tar-tap) Δtsr Δtrg) and HCB316(Δ(tar-tap) and Δtsr), both of which derived from E. coli RP437, were used in the experiment. The plasmid pKAF131 which constitutively expresses the sticky filament FliCst was transformed into HCB429, and the plasmid pFD313 which constitutively expresses sticky filament FliCst was transformed into HCB316 in this study. Considering the cells of wild type strain response significantly to the stimulus of fructose at the concentration of 50 mM, we used it as the stimulus to the cells of HCB429. The motor CW bias of HCB429 cells was unchanged with the addition of stimulus and removal of stimulus, as shown in Fig. 2C. To further verify it, we performed the experiment with the stimulus of fructose at the concentration of 1 mM, the result of which shown in Fig. 2B was in consistent with the result shown in Fig. 2C. The number of CW bias was 0 all the time remarkably.
The stimulus of fructose (1 mM, 10 mM, 50 mM) were used in the chemotactic experiment of HCB316 with the response of cells to the stimulus of fructose (1 mM, 10 mM, 50 mM) were different.
The CW bias shown in Fig. 3A was the same with the result in Fig. 2 under the stimulus of fructose 1 mM, while the CW bias shown in Fig. 3B under the stimulus of fructose 10 mM was stable above 0 before and after the addition of the stimulus, and gradually increased after the removal of the stimulus. The CW bias shown in Fig. 3C under the stimulus of fructose 50 mM was stable above 0 before the addition of the stimulus, and gradually decreased to 0 after the stimulus, then recovered to the pre-stimulus level.
All of the results above showed the cells of E. coli deleted all chemoreceptors cannot respond to the stimulus of fructose, along with the cells of E. coli deleted chemoreceptors except for Trg can response to the stimulus of fructose in high concentration. Thus, E. coli needs chemoreceptors to sense fructose.
Effect of fructose on the motility of E. coli
The strain JY26 transformed with the plasmid pKAF131 were used in the experiment. Figure 4A and Fig. 4B showed the speed: the average motor speeds of the wild-type strain (JY26) in CCW direction were 63.83±8.62 Hz, 54.18±12.38 Hz, 57.93±10.24 Hz, 55.11±10.27 Hz, 60.73±9.04 Hz, 61.98±7.84 Hz, while the average motor speeds of the wild-type strain in CW direction were 55.75±8.84, 52.68±11.16 Hz, 54.2±9.66 Hz, 52.58±10.14 Hz, 57.97±8.53 Hz, 58.01±7.39 Hz, under the concentrations of fructose from 0 mM to 0.1 mM, 1 mM, 10 mM, 20 mM, 50 mM, respectively. The CCW motor speeds of JY26 in 0.1 mM concentration of fructose decreased dramatically, which was reduced by 15.6% compared to the control in 0 mM concentration of fructose. In contrast, the CW motor speeds of JY26 showed no distinct difference. Therefore, the fructose had an influence on the motor speed with the influence on CCW rotation was greater than on CW rotation. The CW biases of the motors of the wild-type strain JY26 were shown in Fig. 4C. Obviously, the CW bias of the motor increased with the addition of fructose. We also measured the motor speeds of the strains that were defective in chemotaxis, as shown in Fig. S1. The average motor speeds of the strain JY27 in CCW direction were 48.94±10.32 Hz, 49.75±12.91 Hz, corresponding to 0 mM and 50 mM concentration of fructose. The average motor speeds of the strain HCB429 in CCW direction were 49.37±7.66 Hz, 47.46±6.27 Hz, 50.04±2.43 Hz, corresponding to 0 mM, 1 mM and 50 mM concentration of fructose. Both of these results suggested that fructose has no effect on the motor speeds of the strains that were defective in chemotaxis.
Fructose promotes bacterial aggregation on surface by lowering the bacteria motor speed
The phenomenon that the bacteria accumulated on the surface when they swim near a surface, was clearly revealed by measuring the steady-state cell distribution of the cells swimming between two parallel surfaces, with cell density increased rapidly near the surface (15, 17). The reduction of motor speed would further help the aggregation near a surface. We found the motor speeds of wild-type strain reduced with the addition of fructose. To test whether the reduction of the motor speeds influenced the aggregation of the cells near a surface, we measured the steady-state cell distribution of bacteria swimming between two parallel surfaces in a depth of 150 µm. We compared the distributions for wild-type cells (JY26) in different concentrations of fructose (0 mM, 0.1 mM, 1 mM, 10 mM, 20 mM, 50 mM), finding that the cells with the addition of fructose accumulated more significantly, as shown in Fig. 5. To verify whether the fructose affects the steady-state cell distribution of the strains that were chemotactic defective, we measured the distributions for cells (JY27 and HCB429) in different concentrations of fructose (0 mM, 0.1 mM, 1 mM, 10 mM, 20 mM, 50 mM). The cell accumulation of the bacteria that were chemotactic defective showed no difference as shown in Fig. S2, which was distinct compared to wild-type cells. Therefore the fructose promotes the aggregation of the bacteria near a surface by lowering the motor speed of bacteria.