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1. A DnaK(Hsp70) Chaperone System Connects Type IV Pilus Activity to Polysaccharide Secretion in Cyanobacteria

   https://doi.org/10.1128/mbio.00514-22  

   McDonald, Heather J.; Kweon, HoJun; Kurnfuli, Shadi; Risser, Douglas D. (April 2022, mBio)

   Sogaard-Andersen, Lotte (Ed.)

   ABSTRACT Surface motility powered by type IV pili (T4P) is widespread among bacteria, including the photosynthetic cyanobacteria. This form of movement typically requires the deposition of a motility-associated polysaccharide, and several studies indicate that there is complex coregulation of T4P motor activity and polysaccharide production, although a mechanistic understanding of this coregulation is not fully defined. Here, using a combination of genetic, comparative genomic, transcriptomic, protein-protein interaction, and cytological approaches in the model filamentous cyanobacterium N. punctiforme , we provided evidence that a DnaK-type chaperone system coupled the activity of the T4P motors to the production of the motility-associated hormogonium polysaccharide (HPS). The results from these studies indicated that DnaK1 and DnaJ3 along with GrpE comprised a chaperone system that interacted specifically with active T4P motors and was required to produce HPS. Genomic conservation in cyanobacteria and the conservation of the protein-protein interaction network in the model unicellular cyanobacterium Synechocystis sp. strain PCC 6803 imply that this system is conserved among nearly all motile cyanobacteria and provides a mechanism to coordinate polysaccharide secretion and T4P activity in these organisms. IMPORTANCE Many bacteria, including photosynthetic cyanobacteria, exhibit type IV pili (T4P) driven surface motility. In cyanobacteria, this form of motility facilitates dispersal, phototaxis, the formation of supracellular structures, and the establishment of nitrogen-fixing symbioses with eukaryotes. T4P-powered motility typically requires the deposition of motility-associated polysaccharides, and previous studies indicate that T4P activity and polysaccharide production are intimately linked. However, the mechanism by which these processes are coupled is not well defined. Here, we identified and characterized a DnaK(Hsp70)-type chaperone system that coordinates these two processes in cyanobacteria. 

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2. Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus

   https://doi.org/10.1073/pnas.1812871115  

   Yang, Yiling; Lam, Vinson; Adomako, Marie; Simkovsky, Ryan; Jakob, Annik; Rockwell, Nathan C.; Cohen, Susan E.; Taton, Arnaud; Wang, Jingtong; Lagarias, J. Clark; et al (December 2018, Proceedings of the National Academy of Sciences)

   Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacteriumSynechocystissp. strain PCC 6803, but the rod-shapedSynechococcus elongatusPCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate ofS. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJ_Se(Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ fromSynechocystis. Plate-based phototaxis assays indicate that UTEX 3055 uses PixJ_Seto sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJ_Secontrols both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis inSynechocystis. 

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3. Phototaxis in Cyanobacteria: From Mutants to Models of Collective Behavior

   https://doi.org/10.1128/mBio.02398-21  

   Menon, Shakti N.; Varuni, P.; Bunbury, Freddy; Bhaya, Devaki; Menon, Gautam I. (December 2021, mBio)

   Harwood, Caroline S. (Ed.)

   ABSTRACT Cyanobacteria rely on photosynthesis, and thus have evolved complex responses to light. These include phototaxis, the ability of cells to sense light direction and move towards or away from it. Analysis of mutants has demonstrated that phototaxis requires the coordination of multiple photoreceptors and signal transduction networks. The output of these networks is relayed to type IV pili (T4P) that attach to and exert forces on surfaces or other neighboring cells to drive “twitching” or “gliding” motility. This, along with the extrusion of polysaccharides or “slime” by cells, facilitates the emergence of group behavior. We evaluate recent models that describe the emergence of collective colony-scale behavior from the responses of individual, interacting cells. We highlight the advantages of “active matter” approaches in the study of bacterial communities, discussing key differences between emergent behavior in cyanobacterial phototaxis and similar behavior in chemotaxis or quorum sensing. 

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4. Cyanoexosortase B is essential for motility, biofilm formation, and scytonemin production in a filamentous cyanobacterium

   https://doi.org/10.1128/msphere.01006-24  

   Parrett, Gabriel A; Haft, Daniel H; Ruiz, Maida; Garcia-Pichel, Ferran; Ebmeier, Christopher C; Risser, Douglas D (June 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Exosortases are involved in trafficking proteins containing PEP-CTERM domains to the exterior of gram-negative bacterial cells. The role of these proteins in cyanobacteria, where such homologs are common, has not been defined. The filamentous cyanobacteriumNostoc punctiformecontains a single putative exosortase, designated cyanoexosortase B (CrtB), implicated by previous work both in motility and in the production of the UV-absorbing pigment, scytonemin. To determine the role ofcrtBinN. punctiforme, acrtB-deletion strain (ΔcrtB) was generated. ΔcrtBpresented the loss of motility, biofilm formation, and scytonemin production. In the case of motility, the ΔcrtBmutant exhibited a specific defect in the ability of hormogonia (specialized motile filaments) to adhere to hormogonium polysaccharide (HPS), and several PEP-CTERM proteins expressed in motile hormogonia were differentially abundant in the exoproteome of the wild-type compared with the ΔcrtBstrain. These results are consistent with the hypothetical role of CrtB in the processing and export of PEP-CTERM proteins that play a critical role in stabilizing the interaction between the filament surface and HPS to facilitate motility and biofilm formation. In the case of scytonemin—the late biosynthetic steps of which occur in the periplasm and whose operon contains several putative PEP-CTERM proteins—ΔcrtBfailed to produce it. Given the abundance of putative PEP-CTERM proteins encoded in theN. punctiformegenome and the fact that this study only associates a fraction of them with biological functions, it seems likely that CrtB may play an important role in other biological processes in cyanobacteria.IMPORTANCEIn gram-negative bacteria, exosortases facilitate the trafficking of proteins to the exterior of the cell where they have been implicated in stabilizing the association of extracellular polymeric substances (EPS) with the cell surface to facilitate biofilm formation and flocculation, but the role of exosortases in cyanobacteria has not been explored. Here, we characterize the role of cyanoexosortase B (CrtB) in the filamentous cyanobacteriumNostoc punctiforme, demonstrating thatcrtBis essential for motility, biofilm formation, and the production of the sunscreen pigment scytonemin. These findings have important implications for understanding motility and biofilm formation in filamentous cyanobacteria as well as efforts toward the heterologous production of scytonemin in non-native hosts. 

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5. Identification of a hormogonium polysaccharide‐specific gene set conserved in filamentous cyanobacteria

   https://doi.org/10.1111/mmi.14566  

   Zuniga, Esthefani G.; Boateng, Kelvin K. A.; Bui, Nhi U.; Kurnfuli, Shadi; Muthana, Sawsan M.; Risser, Douglas D. (July 2020, Molecular Microbiology)

   Abstract Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst‐forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P‐driven mechanism. Here, several novelhpsloci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild‐type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co‐occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy‐type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P‐mediated mechanism. 

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