Having a linker between the FP and MotB improves motor functions

The previous results were based on the direct fusion between the FP and MotB. In this section, we studied how a linker (of 5AA and 15 AA) between the FP and the N-terminus of MotB influences the torque-generation and the switching ability of the BFM. Four types of linkers were tested: short and flexible (GGGGS), short and rigid (EAAAK), and two longer versions of them (GGGGS x3 and EAAAK x3) [32]. The functional effects of the linker were primarily focused on Dendra2 fusion stator, because they exhibited most impaired functions in both torque generation and switching ability. Dendra2 fusion stators with four different linkers were constructed and their motilities were investigated. Functional effects of the linkers on YPet motors were also investigated since they showed the most WT-like motor functions. The list of the fusion stators with a linker that we studied are: Dendra2-EAAAK-MotB, Dendra2-GGGGS-MotB, Dendra2-EAAAKx3-MotB, Dendra2-GGGGSx3-Dendra2-EAAAKx3-MotB, YPet-EAAAKx3-Dendra2-EAAAKx3-MotB, YPet-EAAAK-Dendra2-EAAAKx3-MotB, YPet-GGGGS-MotB.

Chemotaxis motilities of the fusion stators with a linker were compared to the fusion stators without a linker (figure 3.17). In Dendra2 fusion group, Dendra2 with a long and rigid linker (EAAAKx3) showed the largest chemotaxis diameter, followed in order by Dendra2-GGGGSx3, Dendra2-EAAAK, Dendra2-GGGGS and Dendra2. The fusions with a linker showed improved chemotaxis motilities and the two longer linkers (3x) improved chemotaxis more than the shorter versions (1x). In YPet fusion group, YPet with the rigid linker (EAAAK) also showed the largest chemotaxis diameter compared to the flexible linker (GGGGS). In both fusion groups, having a linker improved chemotaxis and the rigid linkers (EAAAK) always improved better than the flexible linkers (GGGGS).

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Figure 3.17. Chemotaxis speed comparisons of the Dendra2 and YPet fusion stators with linkers. Soft agar plate images are shown on the top and the averaged chemotaxis diameters of the three replicates experiments are shown in the bottom. The chemotaxis of the YPet fusion with a long and rigid linker was not tested since this fusion construct was made later. The chemotaxis assay was performed on the 0.25~0.223 % agar plates with 0.01% arabinose induction condition for 8 hours at 37 C. Error bars are standard deviations.

The improved chemotaxis ability by the presence of a linker was also demonstrated in the single motor bead assays: the four linker Dendra2 fusions increased rotation speeds (figure 3.18).

Especially, the Dendra2 with a longer and rigid linker (EAAAKx3) showed distinctively improved motor rotation speeds, nearly by a factor of two in average. The fastest cell observed by this strain showed a rotation speed of ~55 Hz, demonstrating that they can generate high enough torque to the wild-type motor (~46 Hz in average). This torque generation is also reflected on the steps in the torque signal of this fusion motor (figure 3.19). In comparison to the Dendra2 fusion without a linker, the torque generation by single stator improved nearly by a factor of two in average as well:

from 65 ± 38 pN nm to 113 ± 35 pN nm.

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The Dendra2 motors with linkers also improved the degree of ASW. The switching events observed in Dendra2 without a linker were rather ‘pauses’ (i.e. switch to a speed close to zero) than actual reversal rotations. However, the Dendra2 with linkers could rotate in both directions; in other words, the asymmetric switch (defined above) becomes less severe. These improved asymmetric switches can be seen by the two bleu peaks in the speed histograms in figure 3.18a. Although Dendra2 showed improved asymmetric switches, the cells still could not fully restore the symmetric switches. The Dendra2 fusion with the most improved asymmetric switches was EAAAKx3 linker fusion, but its ratio of the mean CCW and CW rotation speeds were 0.18, meaning that the motors rotate at CW direction with the 18% mean rotation speed of CCW direction in average.

Figure 3.18. Histograms of rotation speeds of the Dendra2 motors and the Dendra2 motors with linkers (A) and their mean rotation speeds in a bar graph (B). The zero speed is indicated by yellow dotted lines.

The Dendra2 motors without a linker and with CCW bias (blue) do not rotate in negative speed much (<5%). The Dendra2 motors with linkers can rotate in negative speeds (which can be seen by the small peaks in negative speed at 2~5 Hz in the histograms and the orange bars in the bar graph). The fusion with EAAAKx3 linker improved both positive and negative speed the most (with speeds of 5 Hz in CW and in 28 Hz in CCW). The number of cells measured were 54, 23, 20, 30, 30 in the right to left order in (B).

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Figure 3.19. Speed per single stator of Dendra2 3x EAAAK fusion motors. (A) a step wise speed increment by a Dendra2 3x EAAAK linker motor, rotating a 1.1 µm bead. The maximum rotation speed was around 45 Hz. The speed increments for the trace in (A) are indicated in the range box in (B). (B) pair-wise (single stator) torque distance from the 10 individual Dendra2 EAAAKx3 motors.

The Dendra2 fusion motors with linkers increased their switching frequencies as well, except for the Dendra2 stator with a long and flexible linker (GGGGSx3) (figure 20). The two rigid EAAAK linkers improved the switching frequencies better than the GGGGS linkers. Consistently, Dendra2- EAAAK x3 showed the most improved switching frequency.

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Figure 3.20 Switching frequencies of the Dendra2 fusion stators. The histograms show the distributions of the switching frequencies for each Dendra2 fusion motors, and the mean switching frequencies are shown in the histogram. The EAAAK x3 showed the highest switching frequency while GGGGS x3 showed the lowest. The red bar indicates the cells did not show switching events during the time course of recording.

The YPet fusion stators with linkers did not show a significant improvement in rotation speeds (~43 Hz in average) since the YPet motors without any linker already showed a torque similar to WT. As seen above by the Dendra2 stators with linkers, however, the long and rigid linker (EAAAKx3) consistently exhibited a positive effect on the switching mechanism. YPet-EAAAKx3-MotB fusion improved, but not fully, asymmetric switch (figure 3.21). YPet fusion without a linker showed ~27 % symmetrical rotation speeds in both directions, while YPet-EAAAKx3 fusion improved to ~ 40 %. The other two YPet fusions with short linkers (GGGGS and EAAAK) did not show such improved asymmetric switches (data not shown).

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Figure 3.21 The EAAAKx3 linker improved asymmetric switching. (A) Ratio of the CW mean rotation speeds to the CCW mean rotation speeds (blue dots), and ratio of the CCW mean rotation speeds to the CW mean rotation speeds (red dots); less-bias direction over bias direction. Each dot represents individual motors. Average of the mean ratios (excluding the zero ratios) are shown on the graphs. The data is from the high induction condition cells. The same speed ratio plot for the YPet-MotB fusion is shown in fig 3.8. Having the linker EAAAKx3 improved asymmetric switches (ASW); change of the mean ratio of 0.27 to 0.40. (B) Example speed-time traces of the YPet-3xEAAAK fusion and the YPet fusion. The fusion with EAAAKx3 switched from +30 Hz to -20 Hz, while the same YPet fusion without a linker switched from +35 Hz to -15 Hz.

In addition to the improved asymmetric switching, the dwell time (resident time) in CW and CCW states of the YPet-EAAAKx3-MotB fusion motors were comparable to the WT motors.

The other three YPet fusion motors (YPet-MotB, YPet-EAAAK-MotB, YPet-GGGGS-MotB) often exhibited extended CW or CCW resident time (for CCW bias or CW bias cell, respectively), whereas the YPet-EAAAKx3-MotB fusion motors did not show this extended resident time anymore (fig 3.22a). The switching frequencies of all the YPet fusions were lower than that of the WT motors. In general, having the EAAAK linkers showed a higher switching frequency than having a GGGGS linker (fig 3.22b). Taking all together, the YPet-EAAAKx3 linker motor functions like WT motor, except their not fully recovered ASW performance and reduced switching frequency. In conclusion, having a linker can improve 1) torque generation per stator, 2) asymmetric switches (but not fully) 3) switching frequencies and 4) the CW and CCW resident time.

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Figure 3.22 (A) The mean (non-bias rotation direction) resident time of the WT and the YPet fusion motors are shown. Non-bias rotation direction indicates either CW rotation for the CCW bias motors or CCW rotations for the CW bias motors. Its resident time is the time spent in the least visited (non-bias) direction of rotation. Unlike the other YPet fusion motors, the EAAAKx3 linker fusion of the YPet motor showed the mean non-bias rotation direction resident time of 0.3 s, which is comparable to WT.

(B) The distributions of switching frequencies for each strain are shown. The numbers on the distributions indicate the mean switching frequencies. The number of cells used were: YPet (59 cells), YPet-EAAAKx3 (32 cells), YPet-GGGGS (49 cells), YPet-EAAAK (30 cells) and WT (56 cells).

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