Background: Cryoconite granules are mineral–microbial aggregates found on glacier surfaces worldwide and are hotspots of biogeochemical reactions in glacier ecosystems. However, despite their importance within glacier ecosystems, the geographical diversity of taxonomic assemblages and metabolic potential of cryoconite communities around the globe remain unclear. In particular, the genomic content of cryoconite communities on Asia’s high mountain glaciers, which represent a substantial portion of Earth’s ice masses, has rarely been reported. Therefore, in this study, to elucidate the taxonomic and ecological diversities of cryoconite bacterial consortia on a global scale, we conducted shotgun metagenomic sequencing of cryoconite acquired from a range of geographical areas comprising Polar (Arctic and Antarctic) and Asian alpine regions.Results: Our metagenomic data indicate that compositions of both bacterial taxa and functional genes are particularly distinctive for Asian cryoconite. Read abundance of the genes responsible for denitrification was significantly more abundant in Asian cryoconite than the Polar cryoconite, implying that denitrification is more enhanced in Asian glaciers. The taxonomic composition ofCyanobacteria, the key primary producers in cryoconite communities, also differs between the Polar and Asian samples. Analyses on the metagenome-assembled genomes and fluorescence emission spectra reveal that Asian cryoconite is dominated by multiple cyanobacterial lineages possessing phycoerythrin, a green light-harvesting component for photosynthesis. In contrast, Polar cryoconite is dominated by a single cyanobacterial speciesPhormidesmis priestleyithat does not possess phycoerythrin. These findings suggest that the assemblage of cryoconite bacterial communities respond to regional- or glacier-specific physicochemical conditions, such as the availability of nutrients (e.g., nitrate and dissolved organic carbon) and light (i.e., incident shortwave radiation).Conclusions: Our genome-resolved metagenomics provides the first characterization of the taxonomic and metabolic diversities of cryoconite from contrasting geographical areas, highlighted by the distinct light-harvesting approaches ofCyanobacteriaand nitrogen utilization between Polar and Asian cryoconite, and implies the existence of environmental controls on the assemblage of cryoconite communities. These findings deepen our understanding of the biodiversity and biogeochemical cycles of glacier ecosystems, which are susceptible to ongoing climate change and glacier decline, on a global scale.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae , whereas no orthologous relationships were found between C. caldarium Lhcr1–3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a “bucket” consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pK_a, whereas they are directly hydrogen bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the “leaky bucket” structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs. Significance Green/red CBCRs are one of the most important CBCR subfamilies owing to their physiological roles in cyanobacteria phylum and optogenetic applications. They are known to utilize a change in the bilin protonation state to cause the marked change in green/red absorption, but the structural bases of the protochromic photocycle are not well understood. Here, we have determined the crystal structure of the chromatic acclimation sensor RcaE of this subfamily in the Pr photoproduct state, identifying a unique conformation of the bilin and its interacting residues. In addition, we provide direct evidence of the protonation state of the bilin via NMR analysis. These findings bring insight to our understanding of the molecular mechanisms underlying the spectral diversity of CBCRs.

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin–thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state (^15ZPb) and orange-absorbing 15E state (^15EPo). pH titration analysis revealed that ^15ZPb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, ^15EPo harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state (^15ZPg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in ^15ZPb and bilin protonation in ^15EPo, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated ^15ZPg and protonated ^15EPr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed ^15ZPg formation as an intermediate in the ^15EPr–to–^15ZPg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP–to–15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue–absorbing CBCR subfamilies.