Gen Enomoto

The model cyanobacterium Synechocystis sp. PCC 6803 encodes, in addition to the major pilin of the Type IV pilus filament, an extensive, partially uncharacterized repertoire of minor pilins. For those cyanobacterial minor pilins that have been characterized, their roles span a surprisingly diverse range of functions. To elucidate the roles of uncharacterized minor pilins in a systematic way, we applied structural phylogenomics across 90 genomes, classifying cyanobacterial pilins into six conserved families that form two distinct putative core priming complexes. We demonstrate that these complexes initiate the assembly of two morphologically distinct Type IV pilus filaments: short, hyper-dynamic pili for natural competence, and long, adhesive pili for motility and phototaxis. Structural analysis revealed a conserved β-solenoid domain in PilX subunits, which we propose fulfills the capping function of PilY1 homologs. Proteomic data indicate that the minor pilin PilX2 facilitates the assembly of motility pili, which is required to maintain DnaJ3 co-chaperone levels and trigger cAMP-dependent surface sensing. These findings challenge the concept of a single multipurpose pilus, establishing that cyanobacteria utilize a shared machinery to assemble two specialized filaments, each primed by distinct core minor pilin complexes to meet the conflicting biophysical requirements of DNA uptake and surface motility.

Ligilactobacillus ruminis is a flagellated lactic acid bacterium found in the intestines of various mammals, including humans. Although this species harbors a complete flagellar gene cluster, flagella formation has not been observed in human-derived strains, and the underlying regulatory mechanisms remain unknown. Here, we isolated a motility-acquired mutant of L. ruminis ATCC 25644 that exhibited full flagellation and a measurable chemotactic response under acidic conditions (pH 3.0). Whole-genome sequencing revealed a ~35 kb deletion encompassing multiple regulatory genes. Functional complementation identified a single response regulator, designated FpsR (flagellation-piliation switchover regulator), as a central switch that suppresses flagella formation while promoting pilus expression. The motility-acquired mutant displayed reduced pilus production, diminished adhesion to murine intestinal mucus and fibronectin, and increased susceptibility to acid (pH 3.0) and bile (0.25-0.5%), resulting in a complete loss of intestinal colonization in a murine model. Furthermore, while flagellin from the motile strain activated TLR5 and induced proinflammatory responses comparable to those of pathogenic bacteria, no such inflammation was observed in vivo, likely due to the strain's colonization failure. These findings reveal FpsR as a previously unrecognized genetic mechanism that coordinates motility and mucosal colonization in a human commensal bacterium and provide insight into how flagella are regulated and silenced in the gut environment to support host-microbe symbiosis.

Type IV pili are versatile prokaryotic cell appendages that are extremely widespread in the bacterial and archaeal domains of life. The structure, dynamics, and functions of type IV pili have been most intensively studied in several species of heterotrophic bacteria, but these appendages also appear universal in cyanobacteria. Cyanobacterial type IV pili have much in common with those found in other bacteria, but they also show some unique features that may be crucial for facilitating the photoautotrophic lifestyles of cyanobacteria. Here, we discuss what is known of the structure and dynamic organization of cyanobacterial type IV pili. We discuss the multiple roles of cyanobacterial type IV pili in motility and phototaxis, sensory signal transduction, DNA uptake, and the formation of cell aggregates and biofilms. We conclude with some ideas on the likely importance of cyanobacterial type IV pilus functions in the natural environment and for biotechnological applications.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation. Graphical 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.

Photoreceptors are proteins that sense incident light and then trigger downstream signaling events. Phytochromes are linear tetrapyrrole-binding photoreceptors present in plants, algae, fungi, and various bacteria. Most phytochromes respond to red and far-red light signals. Among the phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show much more diverse optical properties covering the entire visible region. Both phytochromes and cyanobacteriochromes share the GAF domain scaffold to cradle the chromophore as the light-sensing region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here we utilize ancestral sequence reconstruction and report that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore enables the photoreceptor to perceive green light. The ancestral cyanobacteriochromes show modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense green-rich irradiance rather than red light, which is preferentially utilized for photosynthesis. In contrast to plants and green algae, many cyanobacteria can utilize green light for photosynthesis with their special light-harvesting complexes, phycobilisomes. The evolution of green/red sensing cyanobacteriochromes may therefore have allowed ancient cyanobacteria to acclimate to different light environments by rearranging the absorption capacity of the cyanobacterial antenna complex by chromatic acclimation. Significance Statement: Light serves as a crucial environmental stimulus affecting the physiology of organisms across all kingdoms of life. Photoreceptors serve as important players of light responses, absorbing light and actuating biological processes. Among a plethora of photoreceptors, cyanobacteriochromes arguably have the wealthiest palette of color sensing, largely contributing to the success of cyanobacteria in various illuminated habitats. Our ancestral sequence reconstruction and the analysis of the resurrected ancestral proteins suggest that the very first cyanobacteriochrome most probably responded to the incident green-to-red light ratio, in contrast to modern red/far-red absorbing plant phytochromes. The deprotonation of the light-absorbing pigment for green light-sensing was a crucial molecular event for the invention of the new class of photoreceptors with their huge color tuning capacity.

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. Furthermore, we reveal 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.

Summary: Photoautotrophic cyanobacteria have developed sophisticated light response systems to capture and utilize the energy and information of incident light [1]. Cyanobacteria-specific photoreceptors cyanobacteriochromes (CBCRs) are distantly related to more widespread phytochromes. CBCRs show the most diverse spectral properties among the naturally occurring photoreceptors, typified by a unique and prevailing blue/green light-absorbing variant [2–6]. However, where the CBCR-mediated ‘colorful’ signaling systems function in nature has been elusive. We previously reported that the three CBCRs SesA/B/C synthesize/degrade a bacterial second messenger cyclic diguanylate (c-di-GMP) in response to blue/green light [6–8]. The cooperative action of SesA/B/C enables blue light-ON and green light-OFF regulation of the c-di-GMP-dependent cell aggregation of the thermophilic cyanobacteriumThermosynechococcus vulcanus[8, 9]. Here, we report that SesA/B/C can serve as a physiological sensor of cell density. Because cyanobacterial cells show lower transmittance of blue light than green light, higher cell density gives more green light-enriched irradiance to cells. The cell density-dependent suppression of cell aggregation under blue/green-mixed light and white light conditions support this idea. Such a sensing mechanism may provide information about the cell position in cyanobacterial mats in hot springs, the natural habitat ofThermosynechococcus. This cell position-dependent SesA/B/C-mediated regulation of cellular sessility (aggregation) might be ecophysiologically essential for the reorganization and growth of phototrophic mats. We also report that the green light-induced dispersion of cell aggregates requires red light-driven photosynthesis. Blue/green CBCRs might work as shade detectors in a different niche than red/far-red phytochromes, which may be why CBCRs have evolved in cyanobacteria.