Probing bilin-protein interaction in the protochromic photocycle of cyanobacteriochrome RcaE by site-directed mutagenesis.

Cyanobacteriochromes (CBCRs) are members of the phytochrome superfamily of photosensor proteins that bind a bilin chromophore. CBCRs exhibit substantial diversity in their absorption wavelengths through a variety of bilin-protein interactions. RcaE is the first discovered cyanobacteriochrome as a regulator of chromatic acclimation, where cyanobacteria optimize the absorption wavelength of their photosynthetic antenna. RcaE undergoes a reversible photoconversion between a green-absorbing (Pg) and a red-absorbing (Pr) states, where the bilin chromophore adopts a deprotonated C15-Z,anti and a protonated C15-E,syn structures, respectively. This photocycle is designated as "protochromic photocycle" as the change of the bilin protonation state is responsible for the large absorption shift. With the guidance of recently determined Pg and Pr structures of RcaE, in this study, we investigated bilin-chromophore interaction by site-directed mutagenesis on three key residues referred to as a protochromic triad and also other conserved residues interacting with the bilin. Among the protochromic triad residues, Glu217 and Lys261 are critical for the formation of the Pr state, while Leu249 is critical for the formation of both Pg and Pr states. Substitution in other conserved residues, including Val218, Phe219, and Pro220 in the wind-up helix and Phe252, Phe214, and Leu209 in a part of the bilin-binding pocket, had less substantial effects on the spectral sensitivity in RcaE. These data provide insights into our understanding of the bilin-chromophore interaction in the protochromic photocycle and also its evolution in the CBCRs.

Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains.

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid-based reporter assays in budding yeast using a RNR3 promoter-driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid-based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid-based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. KEY POINTS: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast.

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE.

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 pKa, whereas they are directly hydrogen bonded in the ß-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.

A hybrid type of chromatic acclimation regulated by the dual green/red photosensory systems in cyanobacteria.

Cyanobacteria are phototrophic bacteria that perform oxygenic photosynthesis. They use a supermolecular light-harvesting antenna complex, the phycobilisome (PBS), to capture and transfer light energy to photosynthetic reaction centers. Certain cyanobacteria alter the absorption maxima and/or overall structure of their PBSs in response to the ambient light wavelength-a process called chromatic acclimation (CA). One of the most well-known CA types is the response to green and red light, which is controlled by either the RcaEFC or CcaSR photosensory system. Here, we characterized a hybrid type of CA in the cyanobacterium Pleurocapsa sp. Pasteur Culture Collection (PCC) 7319 that uses both RcaEFC and CcaSR systems. In vivo spectroscopy suggested that strain PCC 7319 alters the relative composition of green-absorbing phycoerythrin and red-absorbing phycocyanin in the PBS. RNA sequencing and promoter motif analyses suggested that the RcaEFC system induces a gene operon for phycocyanin under red light, whereas the CcaSR system induces a rod-membrane linker gene under green light. Induction of the phycoerythrin genes under green light may be regulated through a yet unidentified photosensory system called the Cgi system. Spectroscopy analyses of the isolated PBSs suggested that hemidiscoidal and rod-shaped PBSs enriched with phycoerythrin were produced under green light, whereas only hemidiscoidal PBSs enriched with phycocyanin were produced under red light. PCC 7319 uses the RcaEFC and CcaSR systems to regulate absorption of green or red light (CA3) and the amount of rod-shaped PBSs (CA1), respectively. Cyanobacteria can thus flexibly combine diverse CA types to acclimate to different light environments.

Pressurized Liquid Extraction of a Phycocyanobilin Chromophore and Its Reconstitution with a Cyanobacteriochrome Photosensor for Efficient Isotopic Labeling.

Linear tetrapyrrole compounds (bilins) are chromophores of the phytochrome and cyanobacteriochrome classes of photosensors and light-harvesting phycobiliproteins. Various spectroscopic techniques, such as resonance Raman, Fourier transform-infrared and nuclear magnetic resonance, have been used to elucidate the structures underlying their remarkable spectral diversity, in which the signals are experimentally assigned to specific structures using isotopically labeled bilin. However, current methods for isotopic labeling of bilins require specialized expertise, time-consuming procedures and/or expensive reagents. To address these shortcomings, we established a method for pressurized liquid extraction of phycocyanobilin (PCB) from the phycobiliprotein powder Lina Blue and also the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PCB was efficiently cleaved in ethanol with three extractions (5 min each) under nitrogen at 125�C and 100 bars. A prewash at 75�C was effective for removing cellular pigments of Synechocystis without PCB cleavage. Liquid chromatography and mass spectrometry suggested that PCB was cleaved in the C3-E (majority) and C3-Z (partial) configurations. 15N- and 13C/15N-labeled PCBs were prepared from Synechocystis cells grown with NaH13CO3 and/or Na15NO3, the concentrations of which were optimized based on cell growth and pigmentation. Extracted PCB was reconstituted with a recombinant apoprotein of the cyanobacteriochrome-class photosensor RcaE. Yield of the photoactive holoprotein was improved by optimization of the expression conditions and cell disruption in the presence of Tween 20. Our method can be applied for the isotopic labeling of other PCB-binding proteins and for the commercial production of non-labeled PCB for food, cosmetic and medical applications.

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.

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 ( 15Z Pb) and orange-absorbing 15E state ( 15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state ( 15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to- 15Z Pg 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.

Yeast-based genotoxicity tests for assessing DNA alterations and DNA stress responses: a 40-year overview.

By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.

Development and evaluation of yeast-based GFP and luciferase reporter assays for chemical-induced genotoxicity and oxidative damage.

We aimed to develop the bioassays for genotixicity and/or oxidative damage using the recombinant yeast. A genotoxicity assay was developed using recombinant Saccharomyces cerevisiae strain BY4741 with a green fluorescent protein (GFP) reporter plasmid, driven by the DNA damage-responsive RNR3 promoter. Enhanced fluorescence induction was observed in DNA repair-deficient strains treated with methyl methanesulfonate, but not with hydrogen peroxide. A GFP reporter yeast strain driven by the oxidative stress-responsive TRX2 promoter was newly developed to assess oxidative damage, but fluorescence was poorly induced by oxidants. In place of GFP, yeast strains with luciferase gene reporter plasmids (luc2 and luc2CP, encoding stable and unstable luciferase, respectively) were prepared. Transient induction of luciferase activity was clearly detected only in a TRX2 promoter-driven luc2CP reporter strain within 90 min of oxidant exposure. However, luciferase was strongly induced by hydroxyurea in the RNR3 promoter-driven luc2 and GFP reporter strains over 8 h after the exposure, suggesting that the RNR3 promoter is continuously upregulated by DNA damage, whereas the TRX2 promoter is transiently activated by oxidative agents. Luciferase activity levels were also increased in a TRX2-promoter-driven luc2CP reporter strain treated with tert-butyl hydroperoxide and menadione and weakly induced with diamide and diethyl maleate. Weakly enhanced luciferase activity induction was detected in the sod1Δ, sod2Δ, and rad27Δ strains treated with hydrogen peroxide compared with that in the wild-type strain. In conclusion, tests using GFP and stable luciferase reporters are useful for genotoxicity, and oxidative damage can be clearly detected by assay with an unstable luciferase reporter.

Green/red light-sensing mechanism in the chromatic acclimation photosensor.

Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.

Sensing chemical-induced genotoxicity and oxidative stress via yeast-based reporter assays using NanoLuc luciferase.

Mutagens and oxidative agents damage biomolecules, such as DNA; therefore, detecting genotoxic and oxidative chemicals is crucial for maintaining human health. To address this, we have developed several types of yeast-based reporter assays designed to detect DNA damage and oxidative stress. This study aimed to develop a novel yeast-based assay using a codon-optimized stable or unstable NanoLuc luciferase (yNluc and yNluCP) gene linked to a DNA damage- or oxidative stress-responsive promoter, enabling convenient sensing genotoxicity or oxidative stress, respectively. End-point luciferase assays using yeasts with a chromosomally integrated RNR3 promoter (PRNR3)-driven yNluc gene exhibited high levels of chemiluminescence via NanoLuc luciferase and higher fold induction by hydroxyurea than a multi-copy plasmid-based assay. Additionally, the integrated reporter system detected genotoxicity caused by four different types of chemicals. Oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and menadione) were successfully detected through transient expressions of luciferase activity in real-time luciferase assay using yeasts with a chromosomally integrated TRX2 promoter (PTRX2)-linked yNlucCP gene. However, the luciferase activity was gradually induced in yeasts with a multi-copy reporter plasmid, and their expression profiles were notably distinct from those observed in chromosomally integrated yeasts. The responses of yNlucCP gene against three oxidative chemicals, but not diamide and zinc oxide suspension, were observed using chromosomally integrated reporter yeasts. Given that yeast cells with chromosomally integrated PRNR3-linked yNluc and PTRX2-linked yNlucCP genes express strong chemiluminescence signals and are easily maintained and handled without restrictive nutrient medium, these yeast strains with NanoLuc reporters may prove useful for screening potential genotoxic and oxidative chemicals.

Distinct prokaryotic and eukaryotic communities and networks in two agricultural fields of central Japan with different histories of maize-cabbage rotation.

Crop rotation is an important agricultural practice for homeostatic crop cultivation. Here, we applied high-throughput sequencing of ribosomal RNA gene amplicons to investigate soil biota in two fields of central Japan with different histories of maize-cabbage rotation. We identified 3086 eukaryotic and 17,069 prokaryotic sequence variants (SVs) from soil samples from two fields rotating two crops at three different growth stages. The eukaryotic and prokaryotic communities in the four sample groups of two crops and two fields were clearly distinguished using ß-diversity analysis. Redundancy analysis showed the relationships of the communities in the fields to pH and nutrient, humus, and/or water content. The complexity of eukaryotic and prokaryotic networks was apparently higher in the cabbage-cultivated soils than those in the maize-cultivated soils. The node SVs (nSVs) of the networks were mainly derived from two eukaryotic phyla: Ascomycota and Cercozoa, and four prokaryotic phyla: Pseudomonadota, Acidobacteriota, Actinomycetota, and Gemmatimonadota. The networks were complexed by cropping from maize to cabbage, suggesting the formation of a flexible network under crop rotation. Ten out of the 16 eukaryotic nSVs were specifically found in the cabbage-cultivated soils were derived from protists, indicating the potential contribution of protists to the formation of complex eukaryotic networks.

Raman Spectroscopy of an Atypical C15-E,syn Bilin Chromophore in Cyanobacteriochrome RcaE.

Cyanobacteriochromes (CBCRs) belong to the phytochrome superfamily of photoreceptors, the members of which utilize a linear tetrapyrrole (bilin) as a chromophore. RcaE is a representative member of a green/red-type CBCR subfamily that photoconverts between a green-absorbing dark state and red-absorbing photoproduct (Pr). Our recent crystallographic study showed that the phycocyanobilin (PCB) chromophore of RcaE adopts a unique C15-E,syn configuration in the Pr state, unlike the typical C15-E,anti configuration for the phytochromes and other CBCRs. Here, we measured Raman spectra of the Pr state of RcaE with 1064 nm excitation and explored the structure of PCB and its interacting residues under physiologically relevant aqueous conditions. We also performed measurements of RcaE in D2O as well as the sample reconstituted with the PCB labeled with 15N or with both 13C and 15N. The observed Raman spectra were analyzed by quantum mechanics/molecular mechanics (QM/MM) calculations together with molecular dynamics simulations. The Raman spectra and their isotope effects were well-reproduced by the simulated spectra of fully protonated PCB with the C15-E,syn configuration and allowed us to assign most of the observed bands. The present vibrational analysis of the all syn bilin chromophore using the QM/MM method will advance future studies on CBCRs and the related proteins by vibrational spectroscopy.

Sensitive detection of chemical-induced genotoxicity by the Cypridina secretory luciferase reporter assay, using DNA repair-deficient strains of Saccharomyces cerevisiae.

Yeast-based reporter assays are useful for detecting various genotoxic chemicals. We established a genotoxicity assay using recombinant strains of Saccharomyces cerevisiae, each containing a reporter plasmid with the secretory luciferase gene from Cypridina noctiluca, driven by a DNA damage-responsive promoter of the yeast RNR3 gene. This system detected the genotoxicity of methyl methanesulphonate (MMS) as sensitively as conventional yeast-based reporter assays, using the ß-galactosidase gene in a concentration-dependent manner; it also detects four other genotoxic chemicals, allowing us to monitor DNA damage easily by skipping the cell extraction process for the assay. We examined Cypridina luciferase levels induced by MMS and three antitumour agents using a set of BY4741-derived deletion mutants, each defective in a DNA repair pathway or DNA damage checkpoint. Luciferase activities were particularly enhanced in mutant strains with mms2 Δ and mag1 Δ by exposure to MMS, rad59 Δ and mlh1 Δ to camptothecin and mms2 Δ and mlh1 Δ to mitomycin C, respectively, compared with their parent strains. Enhanced reporter activities were also found in some DNA repair mutants with cisplatin. These observations suggest that this Cypridina secretory luciferase reporter assay using yeast DNA repair mutants offers convenient and sensitive detection of the potential genotoxicity of numerous compounds, including antitumour drugs and studying the mechanisms of DNA damage response in yeast.

Distinct community structures of soil nematodes from three ecologically different sites revealed by high-throughput amplicon sequencing of four 18S ribosomal RNA gene regions.

Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1-4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer-persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing.

Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing.

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1-4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade.

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.

Taxonomic profiling of individual nematodes isolated from copse soils using deep amplicon sequencing of four distinct regions of the 18S ribosomal RNA gene.

Nematodes are representative soil metazoans with diverged species that play crucial roles in nutrient recycling in the pedosphere. Qualitative and quantitative information on nematode communities is useful for assessing soil quality, and DNA barcode-mediated taxonomic analysis is a powerful tool to investigate taxonomic compositions and changes in nematode communities. Here, we investigated four regions (regions 1-4) of the 18S small subunit ribosomal RNA (SSU) gene as PCR targets of deep amplicon sequencing for the taxonomic profiling of individual soil nematodes. We determined the sequence variants (SVs) of 4 SSU regions for 96 nematodes (total 384 amplicons) isolated from copse soils and assigned their taxonomy using the QIIME2 software with dada2 or deblur algorithm and the SILVA database. Dada2 detected approximately 2-fold more nematode-derived SVs than deblur, and a larger number of SVs were obtained in regions 1 and 4 than those in other regions. These results and sufficient reference sequence coverage in region 4 indicated that DNA barcoding using a primer set for region 4 followed by dada2-based analysis would be most suitable for soil nematode taxonomic analysis. Eighteen SSU-derived operational taxonomic units (rOTUs) were obtained from 68 isolates, and their orders were determined based on the phylogenetic trees built by four regional sequences of rOTUs and 116 nematode reference species as well as the BLASTN search. The majority of the isolates were derived from three major orders Dorylaimida (6 rOTUs, 51.5% in 68 isolates), Rhabditida (4 rOTUs, 29.4%), and Triplonchida (7 rOTUs, 17.6%). The predicted feeding types of the isolates were fungivores (38.2% in total nematodes), plant feeders (32.4%), and 14.7% for both bacterivores and omnivores/predators. Additionally, we attempted to improve the branch structure of phylogenetic trees by using long nucleotide sequences artificially prepared by connecting regional sequences, but the effect was limited.

Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans.

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs.

A specific combination of dual index adaptors decreases the sensitivity of amplicon sequencing with the Illumina platform.

Amplicon sequencing is a powerful approach in microbiome studies as it detects live organisms with high sensitivity. This approach determines the composition of sequence variants of marker genes using high-throughput DNA sequencers. The use of dual index adaptors is the fundamental technique for pooling DNA libraries for Illumina sequencers and is believed not to affect the results. However, here, we observed a decrease of sequence quality in samples containing a specific combination of indexes, namely N704 and S507 in Nextera kits, in multiple runs on the Illumina MiSeq sequencer operated in different facilities. This decrease was also observed when sequencing randomly fragmented DNA of Escherichia coli and was not observed when either individual adaptor was used. Each end of the DNA library with this index combination contains a complementary sequence motif, which potentially inhibits proper cluster generation and/or subsequent sequencing. Community analysis of the 16S and 18S rRNA amplicons using QIIME2 revealed significant decreases in α-diversity in the samples containing the N704/S507 index combination, resulting from loss of low-abundance sequence variants during denoising. Our data underscore the importance of quality validation of sequence reads in developing dual index techniques and suggest cautious interpretation of microbiome data containing low-quality sequence reads.