Potassium-mediated bacterial chemotactic response

Reviewer #1 (Public Review):

Summary:

This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with an amplitude larger than aspartate, and cells can quickly adapt (but possibly imperfectly). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

Strengths:

The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH.

Weaknesses:

The authors show that changes in pH impact fluorescent protein brightness and modify the FRET signal; this measurement explains the apparent imprecise adaptation they measured. However, this effect reduces confidence in the quantitative accuracy of the FRET measurements. For example, part of the potassium response curve (Fig. 4B) can be attributed to chemotactic response and part comes from the pH modifying the FRET signal. Measuring the full potassium response curve of the no-receptor mutants as a control would help quantify the true magnitude of the chemotactic response and the adaptation precision to potassium.

The measured response may also be impacted by adaptation. For other strong attractant stimuli, the response typically shows a low plateau before it recovers (adapts). However, in the case of Potassium, the FRET signal does not have an obvious plateau following the stimuli. Do the authors have an explanation for that? One possibility is that the cells may have already partially adapted when the response reaches its minimum, which could indicate a different response and/or adaptation dynamics from that of a regular chemo-attractant? In any case, directly measuring the response to potassium in mutants without adaptation enzymes (CheR, CheB) and with the receptors in different methylation levels would shed more light on the problem.

There seems to be an inconsistency between the FRET and bead assay measurements, the CW bias shows over-adaptation, while the FRET measurement does not. The small hill coefficient of the potassium response curve and the biphasic response of the Tar-only strain, while both very interesting, require further explanation since these are quite different than responses to more conventional chemoattractants.

Reviewer #2 (Public Review):

Summary:

Zhang et al investigated the biophysical mechanism of potassium-mediated chemotactic behavior in E coli. Previously, it was reported by Humphries et al that the potassium waves from oscillating B subtilis biofilm attract P aeruginosa through chemotactic behavior of motile P aeruginosa cells. It was proposed that K+ waves alter PMF of P aeruginosa. However, the mechanism was this behaviour was not elusive. In this study, Zhang et al demonstrated that motile E coli cells accumulate in regions of high potassium levels. They found that this behavior is likely resulting from the chemotaxis signalling pathway, mediated by an elevation of intracellular pH. Overall, a solid body of evidence is provided to support the claims. However, the impacts of pH on the fluorescence proteins need to be better evaluated. In its current form, the evidence is insufficient to say that the fluoresce intensity ratio results from FRET. It may well be an artefact of pH change. Nevertheless, this is an important piece of work. The text is well written, with a good balance of background information to help the reader follow the questions investigated in this research work.

In my view, the effect of pH on the FRET between CheY-eYFP and CheZ-eCFP is not fully examined. The authors demonstrated in Fig. S3 that CFP intensity itself changes by KCl, likely due to pH. They showed that CFP itself is affected by pH. This result raises a question of whether the FRET data in Fig3-5 could result from the intensity changes of FPs, but not FRET. The measured dynamics may have nothing to do with the interaction between CheY and CheZ. It should be noted that CFP and YFP have different sensitivities to pH. So, the measurement is likely confounded by the change in intracellular pH. Without further experiments to evaluate the effect of pH on CFP and YFP, the data using this FRET pair is inconclusive.

The data in Figure 1 is convincing. It would be helpful to include example videos. There is also ambiguity in the method section for this experiment. It states 100mM KCl was flown to the source channel. However, it is not clear if 100 mM KCl was prepared in water or in the potassium-depleted motility buffer. If KCl was prepared with water, there would be a gradient of other chemicals in the buffer, which confound the data.

The authors show that the FRET data with both KCl and K2SO4, and concluded that the chemotactic response mainly resulted from potassium ions. However, this was only measured by FRET. It would be more convincing if the motility assay in Fig1 is also performed with K2SO4.

Methods:

- Please clarify the promotes used for the constitutive expression of FliCsticky and LacI.
- Fluorescence filters and imaging conditions (exposure time, light intensity) are missing.
- Please clarify if the temperature was controlled in motility assays.
- L513. It is not clear how theta was selected. Was theta set to be between 0 and pi? If not, P(theta) can be negative?
- Typo in L442 (and) and L519 (Koff)