Ligand sensing enhances bacterial flagellar motor output via stator recruitment
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Summary: This is an interesting study reporting an increase in the rotation speed of the E. coli flagellar motor upon the sensing of a non-metabolizable glucose analog (2Dg) by the cell. The authors conclude that this increase is due to an increase in the number of torque-generating stator complexes that drive the motor. Knockout of the trg gene abolished this effect, suggesting that sensing of 2Dg by the Trg chemosensor is responsible. Involvement of membrane potential, the PTS pathway, and the chemotaxis response regulator CheY is ruled out. The manuscript is well-written, and the data are convincing. But the mechanism remains unclear.
Reviewer #3 opted to reveal their name to the authors in the decision letter after review.
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Abstract
It is well known that flagellated bacteria, such as Escherichia coli , sense chemicals in their environment by a chemoreceptor and relay the signals via a well-characterized signaling pathway to the flagellar motor. It is widely accepted that the signals change the rotation bias of the motor without influencing the motor speed. Here, we present results to the contrary and show that the bacteria is also capable of modulating motor speed on merely sensing a ligand. Step changes in concentration of non-metabolizable ligand cause temporary recruitment of stator units leading to a momentary increase in motor speeds. For metabolizable ligand, the combined effect of sensing and metabolism leads to higher motor speeds for longer durations. Experiments performed with mutant strains delineate the role of metabolism and sensing in the modulation of motor speed and show how speed changes along with changes in bias can significantly enhance response to changes in its environment.
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Reviewer #3:
This manuscript presents its two main results in Figure 3:
In response to a non-hydrolysable glucose analogue, E. coli cells show...
(1) Increase in fluorescence intensity of motors with labelled stator proteins, (2) Increase in speed of motor rotation and swimming
Sufficient controls are described to rule out possible indirect explanations of this effect, via buffer refreshment, metabolism of glucose, proton motive force (Fig 3D) and rotation direction (Fig 4F), and by contrast the effect is demonstrated to depend upon the chemotaxis receptor for glucose (Fig 4B) and the phosphotransferase system (Fig 4D), which is supports the chemotaxis system. These results are interpreted as evidence for a direct effect of the chemotaxis system upon the number of independent stator units, and thereby upon motor and swimming speeds.
This…
Reviewer #3:
This manuscript presents its two main results in Figure 3:
In response to a non-hydrolysable glucose analogue, E. coli cells show...
(1) Increase in fluorescence intensity of motors with labelled stator proteins, (2) Increase in speed of motor rotation and swimming
Sufficient controls are described to rule out possible indirect explanations of this effect, via buffer refreshment, metabolism of glucose, proton motive force (Fig 3D) and rotation direction (Fig 4F), and by contrast the effect is demonstrated to depend upon the chemotaxis receptor for glucose (Fig 4B) and the phosphotransferase system (Fig 4D), which is supports the chemotaxis system. These results are interpreted as evidence for a direct effect of the chemotaxis system upon the number of independent stator units, and thereby upon motor and swimming speeds.
This is a novel finding, and with better statistics (more repeats of fluorescence experiments) and better presentation of the findings (see below), the paper would be an important contribution to the field of bacterial chemotaxis. However, especially without presenting nor postulating a mechanism for the proposed direct effect, the paper might be more suitable for a more specialist journal.
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Reviewer #2:
The authors hint towards the involvement of c-di-GMP signaling via the YcgR protein. This hypothesis can be tested by knocking down the ycgr gene and repeating the assay, but this has not been done or reported. Addition of these data to the manuscript would make the paper significantly stronger.
Do other chemoreceptors (Tar, Tsr, Tap) also act in the same way with their respective ligands? It would be useful to know if this effect is specific to Trg or if it is also found in the other chemoreceptors.
In figure 3C, what is the reason that the GFP intensity and the speed do not have the same range? In other words, why is the slope not equal to 1? Since there is 1:1 correspondence between the number of MotB and the number of GFP, shouldn't the slope be 1?
The authors do not cite or discuss the recent literature on load-dependent …
Reviewer #2:
The authors hint towards the involvement of c-di-GMP signaling via the YcgR protein. This hypothesis can be tested by knocking down the ycgr gene and repeating the assay, but this has not been done or reported. Addition of these data to the manuscript would make the paper significantly stronger.
Do other chemoreceptors (Tar, Tsr, Tap) also act in the same way with their respective ligands? It would be useful to know if this effect is specific to Trg or if it is also found in the other chemoreceptors.
In figure 3C, what is the reason that the GFP intensity and the speed do not have the same range? In other words, why is the slope not equal to 1? Since there is 1:1 correspondence between the number of MotB and the number of GFP, shouldn't the slope be 1?
The authors do not cite or discuss the recent literature on load-dependent stator remodeling (e.g. PMIDs: 29183968, 31142644). It would be helpful to have a more in-depth discussion on how the observed stator unit recruitment relates to stator remodeling in response to load.
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Reviewer #1:
Bacterial chemotaxis is a well-studied process at many levels, from the chemical networks that control the rotation of the flagella to the fluid dynamics of the motility itself. In the present paper the authors address the widely held view that ligand sensing is responsible only for changing the rotational bias of the motor driving flagellar motion, and not its speed. Using a well-established method of quantifying motor activity by monitoring the rotation of the cell body when the flagella are stuck to a surface, a fluorescent labelling technique to determine the membrane potential, a mutant with fluorescently labelled stator units, and direct measurements of swimming speed, the authors show that the sensing of a non-metabolizable analogue of glucose leads to a momentary increase in motor speed and stator unit numbers. At …
Reviewer #1:
Bacterial chemotaxis is a well-studied process at many levels, from the chemical networks that control the rotation of the flagella to the fluid dynamics of the motility itself. In the present paper the authors address the widely held view that ligand sensing is responsible only for changing the rotational bias of the motor driving flagellar motion, and not its speed. Using a well-established method of quantifying motor activity by monitoring the rotation of the cell body when the flagella are stuck to a surface, a fluorescent labelling technique to determine the membrane potential, a mutant with fluorescently labelled stator units, and direct measurements of swimming speed, the authors show that the sensing of a non-metabolizable analogue of glucose leads to a momentary increase in motor speed and stator unit numbers. At the same time, control experiments make it clear that this is purely as a consequence of ligand sensing. This behaviour is indeed contrary to the accepted view, and although the fundamental mechanism is as yet unclear, this is an important result.
On the whole I am very supportive of this work, which has been done with great care and clear logic. My only suggestion for improvement would be to make quantitative the changes in chemotactic behaviour that would be expected as a consequence of the motor speed changes revealed in this research. That is, can the authors put some numbers into a standard analysis of run-and-tumble dynamics to quantify any improvement in chemotactic efficiency or speed under such changes?
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Summary: This is an interesting study reporting an increase in the rotation speed of the E. coli flagellar motor upon the sensing of a non-metabolizable glucose analog (2Dg) by the cell. The authors conclude that this increase is due to an increase in the number of torque-generating stator complexes that drive the motor. Knockout of the trg gene abolished this effect, suggesting that sensing of 2Dg by the Trg chemosensor is responsible. Involvement of membrane potential, the PTS pathway, and the chemotaxis response regulator CheY is ruled out. The manuscript is well-written, and the data are convincing. But the mechanism remains unclear.
Reviewer #3 opted to reveal their name to the authors in the decision letter after review.
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