Thermosynechococcus switches the direction of phototaxis by a c-di-GMP-dependent process with high spatial resolution
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Evaluation Summary:
This manuscript by Nakane et al investigates phototaxis of the rod shaped bacteria Thermosynechococcus vulcanus. This is important because most our knowledge on phototaxis is only emerging on round-shaped cyanobacteria. In the study, the authors demonstrate that T. vulcanus can chemotax positively or negatively to light depending on the light source. They identify a photoreceptor complex that drives negative phototaxis and propose that it impacts motility by increasing cdiGMP levels, which in turn would regulate the motility complex, formed by bi-polar Type-IV pili. Provided that the link between light induced cdi-GMP and spatial TFP activity is established, the work would provide a new mechanistic framework to explain TFP-driven phototaxis. This is an important topic because TFPs are emerging as spatially-regulated motility machineries in a large number of bacterial systems and linking their activity to receptors and secondary messengers is a current unresolved question.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.
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Author Response:
Reviewer #1 (Public Review):
The observation that the cells are able to steadily move along the light axis but perpendicular to their long axis is very interesting considering the T4P appear to be bipolarly localized. There is some discussion on the micro-optic effect in single cells but it does not include the observation that the negative phototaxis to green light occurs no matter where the direction of blue light comes from or the micro-optic effect in a microcolony.
We have added the following sentences in the Discussions part (p16 L363-372) in the Related Manuscript File: “The focused green light would excite yet unknown photosensory molecules to induce spatially localized signalling, whereas the position of the focused blue light is not crucial for directional switching. As we showed, the direction of blue …
Author Response:
Reviewer #1 (Public Review):
The observation that the cells are able to steadily move along the light axis but perpendicular to their long axis is very interesting considering the T4P appear to be bipolarly localized. There is some discussion on the micro-optic effect in single cells but it does not include the observation that the negative phototaxis to green light occurs no matter where the direction of blue light comes from or the micro-optic effect in a microcolony.
We have added the following sentences in the Discussions part (p16 L363-372) in the Related Manuscript File: “The focused green light would excite yet unknown photosensory molecules to induce spatially localized signalling, whereas the position of the focused blue light is not crucial for directional switching. As we showed, the direction of blue light illumination did not influence directionality of movement, because cells do not move in random orientation (Figure 2 – figure supplement 6). Thus, blue light does not control the directional light-sensing capability, instead it provides the signal for the switch between positive and negative phototaxis. This is very similar to the situation in Synechocystis where the blue light receptor PixD controls the switch between negative and positive phototaxis independently of the position of the blue-light source (Sugimoto et al., 2017).”
Reviewer #2 (Public Review):
I- The author's attribute the defect of negative phototaxis observed in the SesA mutant to the level of C-di-GMP in the cell, mainly because a SesA mutant shows a two fold decrease in C-di-GMP concentration upon blue light treatment. However, this measurement has been realised in a batch culture and normalised to dry cell mass. At the opposite, the negative phototaxis observed at single cell level occurs in a range of less than a minute (Figure 2). It would be therefore important for the author's to strength the implication of C-di-GMP in the phototaxis regulation. For example, the author's could ectopically modulate the level of C-di-GMP in the cell, via the expression of ectopic a diguanylate cyclase or phosphodiesterase enzymes, and observe its effect on phototaxi
We highly appreciate your evaluation and comments. As we pointed out in our response to reviewer 1, utilizing heterologous expression systems in T. vulcanus is challenging, maybe due to the cultivation of cells at of 45°C. However, we were lucky in isolating a spontaneous mutant (named WT_N) that shows constitutive negative phototaxis under lateral light illumination. By comparative genomics, we identified the frameshift mutation that confers an increase of the intracellular concentration of c-di-GMP and which was accompanied by negative phototaxis under the condition where the WT cells showed positive phototaxis (Figure 4). We have added a paragraph in the Results part for these experiments on p9-10 (L201-219). See also our comments to the other reviewers and the editor concerning these new experiments, which support the role of c-di-GMP in directional switching. In addition, the figure formerly assigned as Figure 3 – figure supplement 1 was moved to the main manuscript as Figure 3C, because we think that the data of the intracellular concentration of c-di-GMP are very important to support our conclusions.
II- The author's used fluorescent beads to visualize T4P dynamics. As it was previously described, the author's show that it is specific of the T4P activity and it also can reveal T4P retraction. Then, the author's used this method to convincingly show that cells that move perpendicular of the light source have only active pili at one half of the both cell poles (Fig6). It is an interesting observation but again it gets short of details.
-The manuscript would definitively benefit from more general analysis of T4P dynamics during phototaxis. For example, during the switch from positive to negative phototaxis. What are the behaviours (T4P pole activation) of cells parallel to the light source?
-Beside, as suggested by the author's in the discussion, having the intracellular localisation of the Atpase PilB would definitively be a plus.
-Moreover, in the discussion section the author proposed the existence of "a specific signalling system with high special resolution" to explain the asymmetric polar T4P activation. Why could it not be a molecular mechanism similar to the one observed in round cell such as Synechocystis, where the light receptor PixD regulates T4P function at some part of the cell according to the direction of the light.
In order to get more direct insights into T4P dynamics, we have performed additional experiments, which are summarized in Figure 8 and Movies S17-20. Importantly, we succeeded in visualizing T4P filaments by PilA1 labelling using live cells. The T4P filaments were bipolarly localized and showed dynamics of assembly and retraction at both cell poles. When the cells moved perpendicular to their long axis, the T4P filaments at both poles showed biased distribution towards the same direction of cellular movement. These results support our idea that T4P are asymmetrically activated within a single cell pole. This asymmetric activation can rely on the localization of PilB ATPase. We would like to address how a molecular machinery such as PilB governs directional switching events. However, GFP-tagging has not been established in thermophilic cyanobacteria so far. We have added a chapter in the Results part for these experiments p13-14 (L296-322) in the Related Manuscript File. Please, also pay attention to our answers to similar comments of the other reviewers.
Our results suggest that the T. vulcanus cell can actuate the spatially resolved signaling even within a cell pole to activate the pilus activity at only one side of a cell pole to enable biased cellular movements. This finding means that the cell harnesses "a specific signalling system with high special resolution" compared to other rod-shaped bacteria showing pole-to-pole regulation of cell polarity. We do not exclude that a system which works similar to the PixD/PixE complex in Synechocystis contributes to the asymmetric localization of the pili in Thermosynechococcus motility. Thermosynechococcus encodes a PixD protein but no PixE homolog. For Synechocystis, it was shown very recently that PATAN domain response regulators (including PixE) bind PilB1 and PilC and can switch the direction of movement (Han et al. Mol. Microbiol. 2021). Thermosynechococcus encodes homologs of such PATAN-domain response regulators, but at the moment, we do not know whether they have a similar function in both cyanobacteria.
III- The links between the C-di-GMP concentration and T4P dynamics during the switch from positive to negative phototaxis is absent. The author's proposed in the discussion a potential binding of C-di-GMP to PilB as previously shown for some T4P. Could it be tested here by the author's since they seem to be able to handle C-di-GMP?
The experimental verification of the binding of c-di-GMP to PilB is ongoing work, but it seems that direct binding of c-di-GMP to PilB is either very weak or does not happen in our setup. Thus, detailed molecular events of c-di-GMP signaling are out of the scope of the current study. However, we do show in the revised version of the manuscript that pilus extension and retraction dynamics are not different between positive and negative phototaxis (Figure 7 − figure supplement 2), suggesting that c-di-GMP most probably does not affect the activity of the PilB protein. Therefore, we have modified the sentence about the binding of c-di-GMP to PilB in the Discussion part as follows. See p17 L391-394: “Since we did not observe a change in pilus dynamics under green and green/blue light illumination (Figure 7 − figure supplement 2), the T4P regulation in T. vulcanus may not be explained simply by a specific activation of PilB (Floyd et al., 2020, Hendrick et al., 2017).”
In addition, we have performed experiments to show additional data that the c-di-GMP levels switch the direction of T4P-dependent phototaxis (new Figure 4). We also performed additional experiments to visualize T4P dynamics by PilA labeling (new Figure 8), which suggest asymmetric activation of pili and most probably of the motor ATPases as well.
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Evaluation Summary:
This manuscript by Nakane et al investigates phototaxis of the rod shaped bacteria Thermosynechococcus vulcanus. This is important because most our knowledge on phototaxis is only emerging on round-shaped cyanobacteria. In the study, the authors demonstrate that T. vulcanus can chemotax positively or negatively to light depending on the light source. They identify a photoreceptor complex that drives negative phototaxis and propose that it impacts motility by increasing cdiGMP levels, which in turn would regulate the motility complex, formed by bi-polar Type-IV pili. Provided that the link between light induced cdi-GMP and spatial TFP activity is established, the work would provide a new mechanistic framework to explain TFP-driven phototaxis. This is an important topic because TFPs are emerging as spatially-regulated …
Evaluation Summary:
This manuscript by Nakane et al investigates phototaxis of the rod shaped bacteria Thermosynechococcus vulcanus. This is important because most our knowledge on phototaxis is only emerging on round-shaped cyanobacteria. In the study, the authors demonstrate that T. vulcanus can chemotax positively or negatively to light depending on the light source. They identify a photoreceptor complex that drives negative phototaxis and propose that it impacts motility by increasing cdiGMP levels, which in turn would regulate the motility complex, formed by bi-polar Type-IV pili. Provided that the link between light induced cdi-GMP and spatial TFP activity is established, the work would provide a new mechanistic framework to explain TFP-driven phototaxis. This is an important topic because TFPs are emerging as spatially-regulated motility machineries in a large number of bacterial systems and linking their activity to receptors and secondary messengers is a current unresolved question.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors use a custom optical set up to visualize individual cells and microcolonies of T. vulcanus phototaxing towards teal/green light and away from blue-green light. This directed motility is dependent on T4P. They went on to identify the photoreceptors, SesA, SesB, and SesC which are required for negative phototaxis and these photoreceptors contain c-di-GMP synthesis and degradation domains. They showed that the concentration of the second messenger c-di-GMP correlates with the direction of phototaxis, with higher levels of c-di-GMP resulting in negative phototaxis. They also observed that the rod-shaped cells moving along the incident light axis can move consistently perpendicular to their long axis during phototaxis and suggest that this observation shows a polarity regulation coined "within-a-pole" …
Reviewer #1 (Public Review):
The authors use a custom optical set up to visualize individual cells and microcolonies of T. vulcanus phototaxing towards teal/green light and away from blue-green light. This directed motility is dependent on T4P. They went on to identify the photoreceptors, SesA, SesB, and SesC which are required for negative phototaxis and these photoreceptors contain c-di-GMP synthesis and degradation domains. They showed that the concentration of the second messenger c-di-GMP correlates with the direction of phototaxis, with higher levels of c-di-GMP resulting in negative phototaxis. They also observed that the rod-shaped cells moving along the incident light axis can move consistently perpendicular to their long axis during phototaxis and suggest that this observation shows a polarity regulation coined "within-a-pole" polarity regulation of the polarly localized T4P.
The conclusions of this paper are mostly supported by the data. A major strength of this study is the careful microscopy and analysis. The experiments are clearly described in the text and the results are communicated well in the figures. The kymographs and movies are striking with the results of cells phototaxing towards green light and away from green light when blue light is present. The assays with the changing wavelengths and cell velocity responses are very clear. It is convincing that T4P are required for phototaxis of T. vulcanus.
The genetic link to phototaxis showing that SesA, SesB and SesC is convincing, but some details could strengthen the link. For example, the microcolony aggregation is an interesting but confounding factor. The conclusions would be strengthened if the authors could decouple the microcolony formation from phototaxis.
The observation that the cells are able to steadily move along the light axis but perpendicular to their long axis is very interesting considering the T4P appear to be bipolarly localized. There is some discussion on the micro-optic effect in single cells but it does not include the observation that the negative phototaxis to green light occurs no matter where the direction of blue light comes from or the micro-optic effect in a microcolony.
This work nicely illustrates how observing single cells can inform macroscopic phenotypes in the lab or in natural habitats.
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Reviewer #2 (Public Review):
In this manuscript Nakane et al investigate the phototaxis of the rod shaped bacteria Thermosynechococcus vulcanus. This is important because most our knowledge on phototaxis bacteria originates from study on round shaped cell. More precisely, the author's firstly convincingly demonstrate a positive and a negative phototaxis in T. vulcanus, regulated by a green to blue light ratio. Secondly, the author's show the implication of SesABC proteins in the negative phototaxis regulation, via a potential modulation of the C-di-GMP level in the cell. Finally, the author's characterised the T4P-dependant motility as the output for phototactic behaviours in T. vulcanus. The author's further investigated the T4P dynamics during negative phototaxis using fluorescent beads.
Overall the work present in the manuscript is …Reviewer #2 (Public Review):
In this manuscript Nakane et al investigate the phototaxis of the rod shaped bacteria Thermosynechococcus vulcanus. This is important because most our knowledge on phototaxis bacteria originates from study on round shaped cell. More precisely, the author's firstly convincingly demonstrate a positive and a negative phototaxis in T. vulcanus, regulated by a green to blue light ratio. Secondly, the author's show the implication of SesABC proteins in the negative phototaxis regulation, via a potential modulation of the C-di-GMP level in the cell. Finally, the author's characterised the T4P-dependant motility as the output for phototactic behaviours in T. vulcanus. The author's further investigated the T4P dynamics during negative phototaxis using fluorescent beads.
Overall the work present in the manuscript is well carry out and conclusions drawn from experiments are largely correct. Nevertheless, my general impression is that the proposed manuscript is at this stage premature. The present work needs further experiments to strength the actual conclusions but also to get more molecular details. Ultimately, a final working model would be beneficial to summarise the proposed molecular links from SesABC to T4P regulation.I- The author's attribute the defect of negative phototaxis observed in the SesA mutant to the level of C-di-GMP in the cell, mainly because a SesA mutant shows a two fold decrease in C-di-GMP concentration upon blue light treatment. However, this measurement has been realised in a batch culture and normalised to dry cell mass. At the opposite, the negative phototaxis observed at single cell level occurs in a range of less than a minute (Figure 2). It would be therefore important for the author's to strength the implication of C-di-GMP in the phototaxis regulation. For example, the author's could ectopically modulate the level of C-di-GMP in the cell, via the expression of ectopic a diguanylate cyclase or phosphodiesterase enzymes, and observe its effect on phototaxis.
II- The author's used fluorescent beads to visualize T4P dynamics. As it was previously described, the author's show that it is specific of the T4P activity and it also can reveal T4P retraction. Then, the author's used this method to convincingly show that cells that move perpendicular of the light source have only active pili at one half of the both cell poles (Fig6). It is an interesting observation but again it gets short of details.
-The manuscript would definitively benefit from more general analysis of T4P dynamics during phototaxis. For example, during the switch from positive to negative phototaxis. What are the behaviours (T4P pole activation) of cells parallel to the light source?
-Beside, as suggested by the author's in the discussion, having the intracellular localisation of the Atpase PilB would definitively be a plus.
-Moreover, in the discussion section the author proposed the existence of "a specific signalling system with high special resolution" to explain the asymmetric polar T4P activation. Why could it not be a molecular mechanism similar to the one observed in round cell such as Synechocystis, where the light receptor PixD regulates T4P function at some part of the cell according to the direction of the light.III- The links between the C-di-GMP concentration and T4P dynamics during the switch from positive to negative phototaxis is absent. The author's proposed in the discussion a potential binding of C-di-GMP to PilB as previously shown for some T4P. Could it be tested here by the author's since they seem to be able to handle C-di-GMP?
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