The GGDEF protein Dgc2 suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana

ABSTRACT Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 (LBDG_02920) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation. IMPORTANCE Self-propelled bacteria often exhibit complex collective behaviors, such as formation of dense-moving clusters, which are exemplified by wandering comet-like and rotating disk-like colonies; however, the molecular details of how these structures are formed are scant. We found that a strain of the filamentous cyanobacterium Leptolyngbya deficient in the GGDEF protein gene dgc2 elicits motility and complex and dynamic colony pattern formation, including comet-like and disk-like clusters. Although c-di-GMP has been reported to activate biofilm formation in some bacterial species, disruption of dgc2 unexpectedly enhanced it, suggesting a novel role for this GGDEF protein for inhibiting both colony pattern formation and biofilm formation.

In this work, the authors reported that a mutation in the dgc2 gene renders the ability of motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana.The rigorous genetic and microscopic characterizations support the idea that the c-di-GMP-producing DGC2 protein inhibits motility and biofilm formation in their wild-type strain.The manuscript is wellwritten, the explanations and logic are clear, and many interesting and quantitative data well support the conclusions.The authors could consider the following minor points to be addressed to improve the manuscript.
Line 53: Ref ( 20) could be wrong.Line 128: "show exhibit" remove either verb.Line 170: The authors could briefly mention whether the cellular movement is phototactic or not, as many cyanobacteria exhibit phototactic motility.Line 243: Though the mechanism of preference for CCW in Leptolyngbya movement is reasonably out of the scope of the current manuscript, the authors could explicitly mention that they did the experiments in the northern hemisphere.Line 285: "we initially confirmed that dgc2 expression is exclusively observed in the Δdgc2 strain under solid media."What did the author mean?Why is it transcribed when the gene is gone?Line 295: Given the result of Figure 7C, the authors could consider weakening the title of this chapter, as the GGDEF domain is not the primarily responsible region for complementing the phenotypes.Line 306: "Compared with the" check the font size Line 314: "GGDE" -> "GGDEF" Line 342: "This was surprising..." The authors did not observe a decrease in c-di-GMP levels in the ∆dgc2 strain (Figure 8).Therefore, the authors could be more cautious with such a statement.For example, the disruption of the dgc2 gene could activate type IV pili in a c-di-GMP-independent manner, and then the active surface motility with type IV pili led to a stable establishment of a biofilm-like structure on a glass surface, as adhesive pili are known to be crucial for biofilm formation of Pseudomonas or Clostridium.Indeed Figure 7C (Δdgc2;dgc2[Δcat]ex) apparently implies that the GGDEF domain of Dgc2 is not crucial for complementing the disruption of dgc2.Line 368: The authors used the dg5 strain as "wild-type" in this work, but it is already one of the spontaneous "mutant" acquiring the ability to grow in the dark in the presence of carbon sources.The authors could comment how the current findings could be relevant in an ecological setting.Does the same mutation render the same motility phenotype in the original "wild-type" background?Line 400: "which contrasted..." The same as line 342.Line 402: Add a reference.Line 404: "T4P, which is required for motility, may also promote adhesion of cells to solid-phase surfaces."This is already known in other bacteria.Please add some references.
Figure 1D: Why did the authors detect the transcript of dgc2 from ∆the dgc2 strain, as almost all the ORF region is deleted (Figure 1B)? Figure 2B: Verocity.Explain more details about the calculation.Total distances of the movement?Or displacement of the cells at the final frame from the first frame?What is the frame rate of the acquired movies, as it is crucial regarding the significant digits?Moreover, from Movie S1, some cells of dgc-strain seem immotile.Is that true also for ∆dgc2-?Please add a short comment on that.The manuscript by Toida et al., describes the isolation of a spontaneous gain of function mutation in Leptolyngbya boryana, a filamentous cyanobacterium.The commonly used lab strain of Leptolyngbya boryana is not motile, where the authors isolated a mutant (dgc2-) that shows motility of the filaments and unique colony morphologies.The authors used whole genome sequencing to identify potential mutations that led to the observed phenotype.They could identify a transposon insertion into a putative diguanylate cyclase gene (dgc2).The responsibility of the dgc2 gene for the observed phenotypes was confirmed using an independently generated ∆dgc2 mutant and various complementation assays which included overexpression of the dgc2 gene as wells as catalytically inactive variants.The second messenger c-di-GMP, produced by diguanylate cyclases, is known to be involved in the lifestyle decisions (motility, biofilm formation, biofilm dispersion and virulence) in many bacteria.Low cellular levels of c-di-GMP are known to facilitate motility, whereas a high c-di-GMP inhibits motility and promotes biofilm formation.Interestingly, the authors describe that the dgc2 mutant of Leptolyngbya boryana shows enhanced surface attachment and has decreased content of c-di-GMP compared to the wild type lab strain.Therefore, they conclude a novel role of c-di-GMP in biofilm formation in Leptolyngbya boryana.The authors intensively analyze the motility of ∆dgc2 filaments and their unique colony patterns in a quantitative and qualitative manner using time-lapse imaging, kymograph analyses and tracking of trajectories combined with robust statistical analyses.They further show that the mutant strain secretes a significant higher amount of EPS than the wild type lab strain.Toida et al. further compared the transcript abundance of genes that are assigned with type IV pili, assembly of these cell appendages and secretion of polysaccharides in filamentous cyanobacteria.The abundances of the pilA mRNA, major subunit of type IV pili, and the hspE mRNA showed a light dependent increase in the ∆dgc2 mutant.

Major points:
The authors conclude based on their experimental results that the Dgc2 protein plays a role in suppressing both gliding motility and biofilm formation.However, the presented results are partially lacking mechanistic details of the c-di-GMP dependent regulation and biofilm formation in Leptolyngbya boryana.How does the only slightly lowered c-di-GMP content in the mutant strain affect the biofilm formation and enhance surface attachment while simultaneously enhancing motility on agar plates?Overexpression of an EAL-domain containing c-di-GMP phosphodiesterase (like YhjH from E. coli) in Leptolyngbya could help to demonstrate the novel role of c-di-GMP in this cyanobacterium.The authors provide different scenarios to explain the observed phenotype of the ∆dgc2 mutant.The use of fluorescently labelled protein involved in gliding motility and polysaccharide secretion may clarify whether the Dgc2 protein interacts directly with the motility apparatus or acts in more globally.The authors provide evidence for an enhanced secretion of polysaccharides based on their transcriptome analyses and EPS staining; but the effect of the ~5-fold increase in the pilA mRNA abundance is not considered in detail.Are there known c-di-GMP-related domains coupled to transcription factors or other components of signaling pathways?Also, different culture condition (constant light versus diurnal light-dark cycles) for the time-lapse imaging and the transcriptome analyses/ c-di-GMP quantification were used, making the interpretation of the presented results more difficult.Diurnal changes can have an influence on the overall RNA amount of the cell.The results of the quantification of c-di-GMP in WT and ∆dgc2 strain seem be close to lower limit of quantification and only two replicates (biological/technical?) were used.The authors might try to optimize the method to achieve more reliable results.The massive overexpression of the catalytically inactive GGDEF domain of Dgc2 could sequester the cellular c-di-GMP as the isite of the GGDEF domain is still present.The use of the native promoter only might give more clear results.Also, watching the movie S3 and the figure S6, I had the impression that there is a difference in the occurrence/frequency of comets and disks in the dgc2-strain compared to the ∆dgc2 strain.Did the authors check whether the dgc2 gene is still transcribed in the dgc2 mutant?
The authors may provide more information on the physiological role of the observed colony patterns formation and their dynamic.In addition, when watching movie S2, I noticed some kind of temporal periodicity of the filament movement (phases of movement and phases of non-movement).Are the cells synchronized in regard to cell division?Furthermore, a graphic summary of the findings and discussion would enhance the manuscript.
In principle, all references should be thoroughly checked again, since citations are present in the text, but not in actual reference library.In addition, some references refer to incorrect publication or publications that do not cover the specified topic.Here are just a few examples.Line: 44: there are no reference #7, #9 and #10 in the reference library, but cited in the text Line 53: reference #20 does not deal with type IV pili-based motility of Synechocystis at all, but with photosynthesis related genes Line 285: "dgc2 expression is exclusively observed in the ∆dgc2 strain grown on solid media".I assume the authors are actually referring to the WT strain here?Line 401: reference #64 specifically deals with the occurrence of c-di-GMP modulating output domains in cyanobacteria; Pseudomonas aeruginosa is not mentioned at all, only Pseudomonas stutzeri is mentioned once Minor points: All references include the web links to the corresponding websites.Is this data intended in this way?Line 16: formation of bacteria is manifested in... Line 24: encodes a protein containing the GGDEF motif, which is conserved in the catalytic domain... Line 26: for this DGC Line34: lacking the DGC gene dgc2 elicits... Line 45: the basis for the formation of complex... Line 109: without changing the amino acid sequence of the protein Line 114: Thereby designating LBDG_02920 as dgc2... Shouldn't the assignment of gene names be based on the nomenclature for bacterial gene names?(dgcA to dgcI instead of dgc1 to dgc9) Line 118: No significant difference in cell morphology were observed at higher magnification.Line 153: genetically different from the... Line 273: encodes a subunit of a glycosyl transferase... Line 277: except picocyanobacterial.The HmpF proteins forms a fibrous structure within... Line 320: Since the UEA-I was also weakly observed... Line 33: indicating that motility Line 380: The dramatic increase Line 382: Considering the presence of multiple copies of dgc genes in Leptolyngbya, the effect of a single deletion of the dgc2 gene... Line 384: While the mechanism is currently unknown, several scenarios...

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Figure 7A :
Figure 7A: What is the small orange rectangle?Movie S2: ∆dgc2 strain moved better after 1 d incubation after spotting (as this time point is used by the authors for e.g., Fig 2B).The cell movement looked worse at 2 d, but again got better at 3 d (or more).Could the authors possibly comment on possible reasons for that?
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred