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A ubiquitous and pink-pigmented facultatively methylotrophic bacterium, designated LRY1–08 (=JCM 33120), was isolated from a lichen in Thailand. Strain LRY1–08 and Methylobacterium durans NBRC 112876T shared 99.92 % similarity based on the 16S rRNA gene sequence. The draft genome of LRY1–08 was 5.26 Mbp with 4,952 protein-coding sequences and an average G + C content of 70.0 mol%. Comparing strain LRY1–08 to M. durans NBRC 112876T, the ANIb, ANIm, AAI, and digital DNA-DNA hybridization values were 96.29 %, 97.10 %, 96.7 %, and 82.29 %, respectively. Based on the phenotypic characteristics and genome analysis, it was identified as M. durans. Its genomic sequence data revealed the PHB and CoQ10 biosynthesis genes. Therefore, the results offer suggestions for further investigation into possible applications of this bacterium in biotechnology. The draft genome was deposited at DDBJ/EMBL/GenBank (DNA Databank of Japan/European Molecular Biology Laboratory/Genbank) (JAYEEX000000000).

Abstract It is generally assumed that all bacteria must have at least one rRNA operon (rrn operon) on the chromosome, but some strains of the genera Aureimonas and Oecophyllibacter carry their sole rrn operon on a plasmid. However, other related strains and species have chromosomal rrn loci, suggesting that the exclusive presence of rrn operons on a plasmid is rare and unlikely to be stably maintained over long evolutionary periods. Here, we report the results of a systematic search for additional bacteria without chromosomal rrn operons. We find that at least four bacterial clades in the phyla Bacteroidota, Spirochaetota, and Pseudomonadota (Proteobacteria) lost chromosomal rrn operons independently. Remarkably, Persicobacteraceae have apparently maintained this peculiar genome organization for hundreds of millions of years. In our study, all the rrn-carrying plasmids in bacteria lacking chromosomal rrn loci possess replication initiator genes of the Rep_3 family. Furthermore, the lack of chromosomal rrn operons is associated with differences in copy numbers of rrn operons, plasmids, and chromosomal tRNA genes. Thus, our findings indicate that the absence of rrn loci in bacterial chromosomes can be stably maintained over long evolutionary periods.

Archaeal cells are typically enveloped by glycosylated S-layer proteins. Archaeal protein glycosylation provides valuable insights not only into their adaptation to their niches but also into their evolutionary trajectory. Notably, thermophilic Thermoproteota modify proteins with N-glycans that include two GlcNAc units at the reducing end, resembling the ''core structure'' preserved across eukaryotes. Recently, Asgard archaea, now classified as members of the phylum Promethearchaeota, have offered unprecedented opportunities for understanding the role of archaea in eukaryogenesis. Despite the presence of genes indicative of protein N-glycosylation in this archaeal group, these have not been experimentally investigated. Here we performed a glycoproteome analysis of the firstly isolated Asgard archaeon Promethearchaeum syntrophicum. Over 700 different proteins were identified through high-resolution LC-MS/MS analysis, however, there was no evidence of either the presence or glycosylation of putative S-layer proteins. Instead, N-glycosylation in this archaeon was primarily observed in an extracellular solute-binding protein, possibly related to chemoreception or transmembrane transport of oligopeptides. The glycan modification occurred on an asparagine residue located within the conserved N-X-S/T sequon, consistent with the pattern found in other archaea, bacteria, and eukaryotes. Unexpectedly, three structurally different N-glycans lacking the conventional core structure were identified in this archaeon, presenting unique compositions that included atypical sugars. Notably, one of these sugars was likely HexNAc modified with a threonine residue, similar to modifications previously observed in mesophilic methanogens within the Methanobacteriati. Our findings advance our understanding of Asgard archaea physiology and evolutionary dynamics.

Microbially influenced corrosion (MIC) may contribute significantly to corrosion-related failures in injection wells and iron pipes of iodine production facilities. In this study, the iron (Fe0) corroding activity of strain Q-1 isolated from iodide-rich brine in Japan and two Iodidimonas strains phylogenetically related to strain Q-1 were investigated under various culture conditions. Under aerobic conditions, the Fe0 foil in the culture of strain Q-1 was oxidized in the presence of nitrate and yeast extract, while those of two Iodidimonas strains were not. The amount of oxidized iron in this culture was six times higher than in the aseptic control. Oxidation of Fe0 in aerobic cultures of nitrate-reducing bacterium Q-1 was dependent on the formation of nitrite from nitrate. This Fe0 corrosion by nitrate-reducing bacterium Q-1 started after initial nitrite accumulation by day 4. Nitrate reduction in strain Q-1 is a unique feature that distinguishes it from two known species of Iodidimonas. Nitrite accumulation was supported by the encoding of genes for nitrate reductase and the missing of genes for nitrite reduction to ammonia or nitrogen gas in its genome sequence. Phylogenetic position of strain Q-1 based on the 16S rRNA gene sequence was with less than 96.1% sequence similarity to two known Iodidimonas species, and digital DNA–DNA hybridization (dDDH) values of 17.2-19.3%, and average nucleotide identity (ANI) values of 73.4-73.7% distinguished strain Q-1 from two known species. In addition of nitrate reduction, the ability to hydrolyze aesculin and gelatin hydrolysis and cellular fatty acid profiles also distinguished strain Q-1 from two known species. Consequently, a new species, named Iodidimonas nitroreducens sp. nov., is proposed for the nitrate-reducing bacterium strain Q-1T.

Abstract A novel type of agarose gel microcapsule (AGM), consisting of an alginate picolitre sol core and an agarose gel shell, was developed to obtain high-quality, single-cell, amplified genomic DNA of bacteria. The AGM is easy to prepare in a stable emulsion with oil of water-equivalent density, which prevents AGM aggregation, with only standard laboratory equipment. Single cells from a pure culture of Escherichia coli, a mock community comprising 15 strains of human gut bacteria, and a termite gut bacterial community were encapsulated within AGMs, and their genomic DNA samples were prepared with massively parallel amplifications in a tube. The genome sequencing did not need second-round amplification and showed an average genome completeness that was much higher than that obtained using a conventional amplification method on the microlitre scale, regardless of the genomic guanine–cytosine content. Our novel method using AGM will allow many researchers to perform single-cell genomics easily and effectively, and can accelerate genomic analysis of yet-uncultured microorganisms.

ABSTRACT Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

ABSTRACT Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

Summary: Raman microspectroscopy is a powerful tool for obtaining biomolecular information from single microbial cells in a nondestructive manner. Here, we detail steps to discriminate prokaryotic species using single-cell Raman spectra acquisitions followed by data preprocessing and random forest model tuning. In addition, we describe the steps required to evaluate the model. This protocol requires minimal preprocessing of Raman spectral data, making it accessible to non-spectroscopists, yet allows intuitive visualization of feature importance.For complete details on the use and execution of this protocol, please refer to Kanno et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Diverse yet-uncultivated bacteria and archaea, i.e., microbial dark matter, are present in terrestrial hot spring environments. Numerous metagenome-assembled genomes (MAGs) of these uncultivated prokaryotes by short-read metagenomics have been reported so far, suggesting their metabolic potential. However, more reliable MAGs, i.e., circularized complete MAGs (cMAGs), have been rarely reported from hot spring environments. Here, we report 61 high-quality (HQ)-MAGs, including 14 cMAGs, of diverse uncultivated bacteria and archaea retrieved from hot spring sediment (52°C, pH 7.2) by highly accurate long-read sequencing using PacBio Sequel II. The HQ MAGs were affiliated with one archaeal and 13 bacterial phyla. Notably, nine of the 14 cMAGs were the first reported cMAGs for the family- to class-level clades that these cMAGs belonged to. The genome information suggests that the bacteria represented by MAGs play a significant role in the biogeochemical cycling of carbon, nitrogen, iron, and sulfur at this site. In particular, the genome analysis of six HQ MAGs including two cMAGs of Armatimonadota, of which members are frequently abundant in hot spring environments, predicts that they are aerobic, moderate thermophilic chemoorganoheterotrophs, and potentially oxidize and/or reduce iron. This prediction is consistent with the environmental conditions where they were detected. Our results expand the knowledge regarding the ecological potential of uncultivated bacteria in moderately-high-temperature environments.

ABSTRACT Many insects harbor bacterial endosymbionts that supply essential nutrients and enable their hosts to thrive on a nutritionally unbalanced diet. Comparisons of the genomes of endosymbionts and their insect hosts have revealed multiple cases of mutually-dependent metabolic pathways that require enzymes encoded in 2 genomes. Complementation of metabolic reactions at the pathway level has been described for hosts feeding on unbalanced diets, such as plant sap. However, the level of collaboration between symbionts and hosts that feed on more variable diets is largely unknown. In this study, we investigated amino acid and vitamin/cofactor biosynthetic pathways in Blattodea, which comprises cockroaches and termites, and their obligate endosymbiont Blattabacterium cuenoti (hereafter Blattabacterium). In contrast to other obligate symbiotic systems, we found no clear evidence of “collaborative pathways” for amino acid biosynthesis in the genomes of these taxa, with the exception of collaborative arginine biosynthesis in 2 taxa, Cryptocercus punctulatus and Mastotermes darwiniensis. Nevertheless, we found that several gaps specific to Blattabacterium in the folate biosynthetic pathway are likely to be complemented by their host. Comparisons with other insects revealed that, with the exception of the arginine biosynthetic pathway, collaborative pathways for essential amino acids are only observed in phloem-sap feeders. These results suggest that the host diet is an important driving factor of metabolic pathway evolution in obligate symbiotic systems. IMPORTANCE The long-term coevolution between insects and their obligate endosymbionts is accompanied by increasing levels of genome integration, sometimes to the point that metabolic pathways require enzymes encoded in two genomes, which we refer to as “collaborative pathways”. To date, collaborative pathways have only been reported from sap-feeding insects. Here, we examined metabolic interactions between cockroaches, a group of detritivorous insects, and their obligate endosymbiont, Blattabacterium, and only found evidence of collaborative pathways for arginine biosynthesis. The rarity of collaborative pathways in cockroaches and Blattabacterium contrasts with their prevalence in insect hosts feeding on phloem-sap. Our results suggest that host diet is a factor affecting metabolic integration in obligate symbiotic systems.

Abstract Background Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract

Summary: Accessing enormous uncultivated microorganisms (microbial dark matter) in various Earth environments requires accurate, nondestructive classification, and molecular understanding of the microorganisms in in situ and at the single-cell level. Here we demonstrate a combined approach of random forest (RF) machine learning and single-cell Raman microspectroscopy for accurate classification of phylogenetically diverse prokaryotes (three bacterial and three archaeal species from different phyla). Our RF classifier achieved a 98.8 ± 1.9% classification accuracy among the six species in pure populations and 98.4% for three species in an artificially mixed population. Feature importance scores against each wavenumber reveal that the presence of carotenoids and structure of membrane lipids play key roles in distinguishing the prokaryotic species. We also find unique Raman markers for an ammonia-oxidizing archaeon. Our approach with moderate data pretreatment and intuitive visualization of feature importance is easy to use for non-spectroscopists, and thus offers microbiologists a new single-cell tool for shedding light on microbial dark matter.

ABSTRACT Fe(II)-oxidizing microorganisms and Fe(III)-reducing microorganisms, which drive the biogeochemical Fe cycle on the Earth’s surface, are phylogenetically and ecologically diverse. However, no single organism capable of aerobic Fe(II) oxidation and anaerobic Fe(III) reduction at circumneutral pH have been reported so far. Here, we report a novel neutrophilic Fe(II)-oxidizing Rhodoferax bacterium, strain MIZ03, isolated from an iron-rich wetland in Japan. Our cultivation experiments demonstrate that MIZ03 represents a much more versatile metabolism for energy acquisition than previously recognized in the genus Rhodoferax. MIZ03 can grow chemolithoautotrophically at circumneutral pH by oxidation of Fe(II), H2, or thiosulfate as the sole electron donor under (micro)aerobic conditions (i.e., using O2 as the sole electron acceptor). In addition, it can reduce Fe(III) or nitrate under anaerobic conditions. Thus, this is the first report demonstrating the presence of a single bacterium capable of both Fe(II) oxidation and Fe(III) reduction at circumneutral pH. The observed physiology was consistent with its 4.9-Mbp complete genome encoding key genes for iron oxidation/reduction (foxEY and mtrABC), for nitrate reduction (narGHI), for thiosulfate oxidation (soxABCDXYZ), and for carbon fixation via the Calvin cycle. Our metagenomic survey suggests that there are more Rhodoferax members capable of Fe(II) oxidation and Fe(III) reduction. Such bifunctional Rhodoferax may have an ecological advantage in suboxic/anoxic environments at circumneutral pH by recycling of Fe as the electron donor and acceptor. IMPORTANCE The biogeochemical cycle of iron (Fe) via reactions of oxidation, reduction, precipitation, and dissolution is involved in the cycle of other ecologically relevant elements, such as C, N, P, S, As, Co, Ni, and Pb. The Fe cycle on the Earth’s surface is driven by a variety of Fe(II)-oxidizing microorganisms and Fe(III)-reducing microorganisms. Here, we discovered a novel bacterium, Rhodoferax sp. strain MIZ03, capable of both Fe(II) oxidation and Fe(III) reduction at circumneutral pH, and we report its physiological characteristics and complete genome sequence. The unexpected capability of this bacterium provides novel insights into the Fe cycle in the environment. Moreover, this bacterium will help to better understand the molecular mechanisms of microbial Fe redox cycling as a model organism.

Abstract Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate respiration‐ and Fe0‐corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate‐reducing strains was enhanced by nitrate, which was not observed in the two strains unable to reduce nitrate. When the nitrate‐reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20‐fold compared to that in aseptic controls. This enhancement was not observed in cultures of the strains unable to reduce nitrate. The oxidation of Fe0 in the anaerobic cultures of nitrate‐reducing strains occurred concomitantly with the formation of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0‐ and carbon steel by the nitrate‐reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.

Butyrate produced by gut microbiota has multiple beneficial effects on host health, and oligosaccharides derived from host diets and glycans originating from host mucus are major sources of its production. A significant reduction of butyrate-producing bacteria has been reported in patients with inflammatory bowel diseases and colorectal cancers. Although gut butyrate levels are important for host health, oligosaccharide metabolic properties in butyrate producers are poorly characterized. We studied the metabolic properties of fructooligosaccharides (FOSs) and other prebiotic oligosaccharides (i.e. raffinose and xylooligosaccharides; XOSs) in gut butyrate producers. 1-Kestose (kestose) and nystose, FOSs with degrees of polymerization of 3 and 4, respectively, were also included. Fourteen species of butyrate producers were divided into four groups based on their oligosaccharide metabolic properties, which are group A (two species) metabolizing all oligosaccharides tested, group F (four species) metabolizing FOSs but not raffinose and XOSs, group XR (four species) metabolizing XOSs and/or raffinose but not FOSs, and group N (four species) metabolizing none of the oligosaccharides tested. Species assigned to groups A and XR are rich glycoside hydrolase (GH) holders, whereas those in groups F and N are the opposite. In total, 17 enzymes assigned to GH32 were observed in nine of the 14 butyrate producers tested, and species that metabolized FOSs had at least one active GH32 enzyme. The GH32 enzymes were divided into four clusters by phylogenetic analysis. Heterologous gene expression analysis revealed that the GH32 enzymes in each cluster had similar FOS degradation properties within clusters, which may be linked to the conservation/substitution of amino acids to bind with substrates in GH32 enzymes. This study provides important knowledge to understand the impact of FOS supplementation on the activation of gut butyrate producers. Abbreviations: SCFA, short chain fatty acid; FOS, fructooligosaccharide; XOS, xylooligosaccharide; CAZy, Carbohydrate Active Enzymes; CBM, carbohydrate-binding module; PUL, polysaccharide utilization locus; S6PH sucrose-6-phosphate hydrolase.

Abstract A large variety of microbes are present in the human gut, some of which are considered to interact with each other. Most of these interactions involve bacterial metabolites. Phascolarctobacterium faecium hardly uses carbohydrates for growth and instead uses succinate as a substrate. This study investigated the growth behavior of the co‐culture of the succinate‐specific utilizer P. faecium and the succinogenic gut commensal Bacteroides thetaiotaomicron. Succinate production by B. thetaiotaomicron supported the growth of P. faecium and concomitant propionate production via the succinate pathway. The succinate produced was completely converted to propionate. This result was comparable with the monoculture of P. faecium in the medium supplemented with 1% (w/v) succinate. We analyzed the transcriptional response (RNA‐Seq) between the mono‐ and co‐culture of P. faecium and B. thetaiotaomicron. Comparison of the expression levels of genes of P. faecium between the mono‐ and co‐cultured conditions highlighted that the genes putatively involved in the transportation of succinate were notably expressed under the co‐cultured conditions. Differential expression analysis showed that the presence of P. faecium induced changes in the B. thetaiotaomicron transcriptional pattern, for example, expression changes in the genes for vitamin B12 transporters and reduced expression of glutamate‐dependent acid resistance system‐related genes. Also, transcriptome analysis of P. faecium suggested that glutamate and succinate might be used as sources of succinyl‐CoA, an intermediate in the succinate pathway. This study revealed some survival strategies of asaccharolytic bacteria, such as Phascolarctobacterium spp., in the human gut.

Plasmids are extrachromosomal DNA that can be horizontally transferred between different bacterial cells by conjugation. Horizontal gene transfer of plasmids can promote rapid evolution and adaptation of bacteria by imparting various traits involved in antibiotic resistance, virulence, and metabolism to their hosts. The host range of plasmids is an important feature for understanding how they spread in environmental microbial communities. Earlier bioinformatics studies have demonstrated that plasmids are likely to have similar oligonucleotide (k-mer) compositions to their host chromosomes and that evolutionary host ranges of plasmids could be predicted from this similarity. However, there are no complementary studies to assess the consistency between the predicted evolutionary host range and experimentally determined replication/transfer host range of a plasmid. In the present study, the replication/transfer host range of a model plasmid, pSN1216-29, exogenously isolated from cow manure as a newly discovered self-transmissible plasmid, was experimentally determined within microbial communities extracted from soil and cow manure. In silico prediction of evolutionary host range was performed with the pSN1216-29 using its oligonucleotide compositions independently. The results showed that oligonucleotide compositions of the plasmid pSN1216-29 had more similarities to those of hosts (transconjugants genera) than those of non-hosts (other genera). These findings can contribute to the understanding of how plasmids behave in microbial communities, and aid in the designing of appropriate plasmid vectors for different bacteria.

Two evolutionarily distinct systems for cytochrome c maturation in mitochondria—Systems I and III—have been found among diverse aerobic eukaryotes. System I requires a set of proteins including mitochondrion-encoded CcmA, CcmB, CcmC, and CcmF (or a subset of the four proteins). On the other hand, System III is operated exclusively by nucleus-encoded proteins. The two systems are mutually exclusive among eukaryotes except a single organism possessing both. Recent advances in understanding both diversity and phylogeny of eukaryotes united cryptophytes, goniomonads, Hemiarma marina, kathablepharids and Palpitomonas bilix into one of the major taxonomic assemblages in eukaryotes (Cryptista). Among the lineages/species in Cryptista, the mitochondrial genomes (mtDNAs) have been completed for multiple cryptophytes, a goniomonad and P. bilix, and ccm genes were found solely in the P. bilix mtDNA. Nevertheless, we have been unsure whether H. marina and/or kathablepharids use System I for cytochrome c maturation, as no mtDNA was available for either of the two cryptist members. In this study, we determined the complete sequences of the mtDNAs of H. marina and a kathablepharid Leucocryptos marina, and found ccmA, ccmC, and ccmF in the former genome. Curiously, the ccm genes found in the H. marina mtDNA showed no phylogenetic affinity to the P. bilix homologs, unfavoring vertical inheritance of the ccm genes in the mtDNA evolution in Cryptista. Finally, we propose revised scenarios for the evolution of cytochrome c maturation systems in Cryptista by incorporating the mtDNA data obtained in this study.

The diversity of sporocarp-inhabiting fungi (SCIF) was examined using six samples of xylarialean fungi from two different forests in Ibaraki Prefecture, Japan: a moist forest in the Sakuragawa area and an urban dry forest in the Tsukuba area. These fungi were enumerated using direct observation and dilution plate methods. We obtained 44 isolates, and careful morphological and molecular phylogenetic studies of these isolates revealed that approximately 30% of the operating taxonomic units were undescribed or cryptic species related to known fungi. Although typical mycoparasitic fungi, such as helotialean fungi and Trichoderma spp., were not isolated, the genera Acremonium, Acrodontium, and Simplicillium were detected. Comparisons of SCIF communities between the two forests suggested that the number of isolated species in the Sakuragawa area was lower than that in the Tsukuba area. Soil-borne fungi, such as Aspergillus, Beauveria, Penicillium, and Talaromyces, or polypores/corticioid mushrooms, are frequently detected in the Tsukuba area. Factors affecting SCIF communities in the two forests are discussed. Some noteworthy fungi are briefly described with notes on taxonomy, ecology, and molecular phylogeny.

Rocky outcrops covered with thick Fe and Mn oxide coatings, which are known as ferromanganese (Fe-Mn) crusts, are commonly found on slopes of aged seamounts in bathyal and abyssal zones. Although the presence of diverse microorganisms on these Fe-Mn crusts has been reported, little is known about their metabolism. Here, we report the metabolic potential of the microbial community in an abyssal crust collected in the Takuyo-Daigo Seamount, in the north-western Pacific. We performed shotgun metagenomic sequencing of the Fe-Mn crust, and detected putative genes involved in dissolution and precipitation of Fe and Mn, nitrification, sulfur oxidation, carbon fixation, and decomposition of organics in the metagenome. In addition, four metagenome-assembled genomes (MAGs) of abundant members in the microbial community were recovered from the metagenome. The MAGs were affiliated with Thaumarchaeota, Alphaproteobacteria, and Gammaproteobacteria, and were distantly related to previously reported genomes/MAGs of cultured and uncultured species. Putative genes involved in the above reactions were also found in the crust MAGs. Our results suggest that crust microbial communities play a role in biogeochemical cycling of C, N, S, Fe, and Mn, and imply that they contribute to the growth of Fe-Mn crusts.

The purpose of this study was to investigate the ability of ascomycetous yeasts to assimilate/ferment d-fructose. This ability of the vast majority of yeasts has long been neglected since the standardization of the methodology around 1950, wherein fructose was excluded from the standard set of physiological properties for characterizing yeast species, despite the ubiquitous presence of fructose in the natural environment. In this study, we examined 388 strains of yeast, mainly belonging to the Saccharomycetes (Saccharomycotina, Ascomycota), to determine whether they can assimilate/ferment d-fructose. Conventional methods, using liquid medium containing yeast nitrogen base +0.5% (w/v) of d-fructose solution for assimilation and yeast extract-peptone +2% (w/v) fructose solution with an inverted Durham tube for fermentation, were used. All strains examined (n = 388, 100%) assimilated d-fructose, whereas 302 (77.8%) of them fermented d-fructose. In addition, almost all strains capable of fermenting d-glucose could also ferment d-fructose. These results strongly suggest that the ability to assimilate/ferment d-fructose is a universal phenotype among yeasts in the Saccharomycetes. Furthermore, the fructophilic behavior of Ambrosiozyma platypodis JCM 1843 and Cyberlindnera americana JCM 3592 was characterized by sugar consumption profiles during fermentation.

Bamboo hemicellulose hydrolysate (BHH) may possess antihypercholesterolemic activity; however, this activity requires further comprehensive study to assess the prebiotic mechanisms of BHH in vivo. Here, we used high-throughput 16S rRNA gene sequencing to preliminarily investigate the correlations between BHH and the fecal microbiomes of three groups of mice fed either a normal diet, a high-fat diet, or a high-fat diet supplemented with 5% BHH for 5 weeks. Alpha diversity (within community) was nonsignificant for all groups; however, beta diversity analysis among communities showed that 5% BHH suppressed the significant changes induced by the high-fat diet. The Firmicutes/Bacteroidetes ratio, the family S24-7 within the order Bacteroidales, the family Lachnospiraceae and several cellulolytic taxa were slightly ameliorated in the BHH group. These results indicated that BHH supplementation influenced the gut bacterial community and suppressed the high-fat diet-induced alterations. Additionally, BHH significantly lowered the serum cholesterol levels and fecal pH. Improving short-chain fatty acid production for all of the bacterial communities in the mouse guts may induce this effect. Thus, the prebiotic potential of BHH should be evaluated considering the gut microbial communities and their interactions.

Bacterial stress responses are closely associated with the survival and colonization of anaerobes in the human gut. Mesosutterella multiformis JCM 32464T is a novel member of the family Sutterellaceae, an asaccharolytic bacterium. We previously demonstrated energy generation via heme biosynthesis, which is coupled with nitrate reductase. Here, physiological and morphological changes in M. multiformis induced by exposure to nitrate were investigated. The ability of M. multiformis to reduce nitrate was determined using a colorimetric assay. A unique morphology was observed during nitrate reduction under anaerobic conditions. The association between nitrate concentration and cell size or cellular fatty acid composition was evaluated. Nitrate-induced responses of M. multiformis were compared to those of related species. An increase in cellular filamentation and the ratio of saturated: unsaturated fatty acids was mediated specifically by nitrate. This indicates a decrease in cell fluidity and low leakage. Furthermore, a similar response was not observed in other related species cultured in the presence of nitrate. Hence, the nitrate-induced stress response in new anaerobes such as M. multiformis was demonstrated. The response could also be involved in the conservation of menaquinones and the maximization of nitrate reduction.

Novel self-transmissible plasmids were exogenously captured from environmental samples by triparental matings with pBBR1MCS-2 as a mobilizable plasmid and Pseudomonas resinovorans as a recipient. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The whole nucleotide sequences of six plasmids were determined, including one IncP-1 plasmid (pSN1104-59), four PromA-like plasmids (pSN1104-11, pSN1104-34, pSN0729-62, and pSN0729-70), and one novel plasmid (pSN1216-29), whose incompatibility group has not been previously identified. No previously known antibiotic resistance genes were found in these plasmids. In-depth phylogenetic analyses showed that the PromA-like plasmids belong to subgroups of PromA (designated as PromAγ and PromAδ) different from previously proposed subgroups PromAα and PromAβ. Twenty-four genes were identified as backbone genes by comparisons with other PromA plasmids. The nucleotide sequences of pSN1216-29 share high identity with those found in clinical isolates. A minireplicon of pSN1216-29 was successfully constructed from repA encoding a replication initiation protein and oriV. All the captured plasmids were found to have a broad host range and could be transferred to and replicated in different classes of Proteobacteria. Notably, repA and oriV of pSN1216-29 showed high similarity with one of two replication systems of pSRC119-A/C, known as a plasmid with multidrug resistance genes found in Salmonella enterica serovar Senftenberg. Our findings suggest that these “cryptic” but broad-host-range plasmids may be important for spreading several genes as “vehicles” in a wider range of bacteria in natural environments.

[This corrects the article DOI: 10.1371/journal.pone.0186248.].

The yeast strains IPM32-16, ISM28-8sT, and IPM46-17, isolated from plant and soil samples from Iriomote Island, Japan, were explored in terms of lipid production during growth in a mixture of glucose and xylose. Phylogenetically, the strains were most closely related to Cystobasidium slooffiae, based on the sequences of the ITS regions and the D1/D2 domain of the LSU rRNA gene. The strains were oleaginous, accumulating lipids to levels > 20% dry cell weight. Moreover, kinetic analysis of the sugar-to-lipid conversion of a 1:1 glucose/xylose mixture showed that the strains consumed the two sugars simultaneously. IPM46-17 attained the highest lipid content (33%), mostly C16 and C18 fatty acids. Thus, the yeasts efficiently converted lignocellulosic sugars to lipids, aiding in biofuel production (which benefits the environment, promotes rural jobs, and strengthens fuel security). The strains constituted a novel species of Cystobasidium, for which we propose the name Cystobasidium iriomotense (type strain ISM28-8sT = JCM 24594T = CBS 15015T).

Calliandra calothyrsus preserved in silage is an alternative method for improving the crude protein content of feeds for sustainable ruminant production. The aim of this research was to evaluate the quality of silage which contained different levels of C. calothyrsus by examining the fermentation characteristics and microbial diversity. Silage was made in a completely randomized design consisting of five treatments with three replications i.e.: R0, Pennisetum purpureum 100%; R1, P. purpureum 75%+C. calothyrsus 25%;, R2, P. purpureum 50%+C. calothyrsus 50%; R3, P. purpureum 25%+C. calothyrsus 75%; and R4, C. calothyrsus 100%. All silages were prepared using plastic jar silos (600 g) and incubated at room temperature for 30 days. Silages were analyzed for fermentation characteristics and microbial diversity. Increased levels of C. calothyrsus in silage had a significant effect (p

Self-transmissible plasmids are classified into two types based on their sex pili: short and rigid pili, and long and flexible pili. The transferability of two plasmids with different types of sex pili, pBP136 and pCAR1, was compared in stirring liquid conditions with different cell density. The most probable number method to count transconjugants could detect differences in the transfer frequency with higher resolution in comparison with the conventional CFU counting method. Both plasmids showed higher transfer frequency in high stirring rates than static liquid conditions when the donor and recipient density was 106-107 CFU mL-1. The probability of donor-initiated plasmid transfer was investigated by a single-cell-level analysis using a cell sorter. The probability was >36-fold higher for pBP136 than for pCAR1; thus, the simulated transfer frequency of pBP136 was much higher than that of pCAR1 in stirring liquid conditions. Nevertheless, the transfer frequency of pCAR1 was as high as that of pBP136 when the donor and recipient cell density was 106 CFU mL-1. This fact indicates that the lower probability of the donor pCAR1 to initiate transfer could be overcome by its high tolerance to the shearing force between donor and recipient cells under higher stirring liquid conditions. Our findings can explain the different survival strategies of these two types of plasmids based on their preferences of transfer conditions.

Background: Peri-implantitis (PI) is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified. Objective: The purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences. Design: Three subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique. Results: A total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46%) were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions. Conclusions: The biofilm in PI showed a more complex microbiota when compared to periodontitis and periodontally healthy teeth, and it was mainly composed of Gram-negative anaerobic bacteria. Common periodontopathic bacteria showed low prevalence, and several bacteria were identified as candidate pathogens in PI.

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146T (97.8% similarity) and V. agarivorans CECT 5085T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085T, V. hangzhouensis JCM 15146T V. maritimus LMG 25439T, and V. variabilis LMG 25438T). In silico DDH data also supported the genomic relationship. The strains C7T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1T, V. brasiliensis LMG 20546T, V. orientalis ATCC 33934T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058T and V. astriarenae C7T are members of the newest clade of Vibrionaceae named Agarivorans.

The order Trichosporonales (Tremellomycotina, Basidiomycota) includes various species that have clinical, agricultural and biotechnological value. Thus, understanding why and how evolutionary diversification occurred within this order is extremely important. This study clarified the phylogenetic relationships among Tricosporonales species. To select genes suitable for phylogenetic analysis, we determined the draft genomes of 17 Trichosporonales species and extracted 30 protein-coding DNA sequences (CDSs) from genomic data. The CDS regions of Trichosporon asahii and T. faecale were identified by referring to mRNA sequence data since the intron positions of the respective genes differed from those of Cryptococcus neoformans (outgroup) and are not conserved within this order. A multiple alignment of the respective gene was first constructed using the CDSs of T. asahii, T. faecale and C. neoformans, and those of other species were added and aligned based on codons. The phylogenetic trees were constructed based on each gene and a concatenated alignment. Resolution of the maximum-likelihood trees estimated from the concatenated dataset based on both nucleotide (72,531) and amino acid (24,173) sequences were greater than in previous reports. In addition, we found that several genes, such as phosphatidylinositol 3-kinase TOR1 and glutamate synthase (NADH), had good resolution in this group (even when used alone). Our study proposes a set of genes suitable for constructing a phylogenetic tree with high resolution to examine evolutionary diversification in Trichosporonales. These can also be used for epidemiological and biogeographical studies, and may also serve as the basis for a comprehensive reclassification of pleomorphic fungi.

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.

he Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial 'Candidatus Liberibacter asiaticus' and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium 'Candidatus Profftella armatura' in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium 'Candidatus Carsonella ruddii' in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT) after L. crescens diverged from other Liberibacter lineages. K A/K S analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector's symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.

BackgroundInferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa-in particular determining the root-is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences.Principal findingsActin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported.Conclusions/significanceWe conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids.

An understanding of the role of yeasts in the environment has been uncertain because estimates of population size and diversity have often been based on species identifications that were determined from a limited number of phenotypic characteristics. DNA-based species identification has now become widely used, allowing an accurate assessment of species in different habitats. However, there are still problems in classification because some genera are polyphyletic. Consequently, the identification of yeasts and measurement of their diversity at the genus level remains difficult, as does assignment of genera to higher taxonomic ranks.A total of 1021 yeast strains was isolated from soil samples and plant materials collected from Japan's subtropical Iriomote Island and the cool temperate Rishiri Island. Based on sequence analyses of the D1/D2 domain of the LSU rRNA gene, these 1021 strains were tentatively classified into 183 species, with apparent new species accounting for approximately half of the total species isolated (60 and 46, Iriomote and Rishiri, respectively). The yeast species composition was statistically different between the two sites with only 15 species in common. Rarefaction curves of respective sources/areas gave distinctive patterns when the threshold of sequence identity became broader, indicating that the yeast diversity was distinct at the different taxonomic levels compared.Our isolation study of yeasts in Japan has enabled us to expand the inventory of species diversity because a large number of new species was observed in the sampling areas. Further, we propose use of a particular diversity threshold as an "indicator" to recognize species, genera and higher taxonomic ranks.

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.

Genomic information is essential for taxonomic, phylogenetic, and functional studies to comprehensively decipher the characteristics of microorganisms, to explore microbiomes through metagenomics, and to answer fundamental questions of nature and human life. However, large gaps remain in the available genomic sequencing information published for bacterial and archaeal species, and the gaps are even larger for fungal type strains. The Global Catalogue of Microorganisms (GCM) leads an internationally coordinated effort to sequence type strains and close gaps in the genomic maps of microorganisms. Hence, the GCM aims to promote research by deep-mining genomic data.

An endophytic actinomycete, strain 3MP-14 isolated from the root of Mimosa pudica belonged to the genus Streptomyces and was closely related to Streptomyces mimosae JCM 33328T (100%) based on 16S rRNA gene sequences. The strain had genome sizes of 7.2 Mb with genomic G+C contents of 73.4 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) of strain 3MP-14 were 100% when compared with S. mimosae JCM 33328T. Based on the polyphasic approach, dDDH, and ANI, strain 3MP-14 was identified as Streptomyces mimosae. Twenty-four putative secondary metabolite biosynthetic gene clusters (BGCs) were identified from the draft genome analysis of strain 3MP-14. In the course of chemical investigation for the products of the strain, a physicochemical (PC) screening was conducted, and a new compound, designated as pudicin, and two known antimicrobials, ikarugamycin and fumaquinone, were identified. The structure elucidation of pudicin revealed a unique structurally new compound with N-acetylcysteine moiety using one-dimensional and two-dimensional nuclear magnetic resonance (1D and 2D NMR spectroscopy) and Electrospray Ionization Mass Spectrometry (ESI-MS). Pudicin showed slight antioxidative activity based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method with the IC50 value of 394.89 ± 3.88 µg/ml. [ABSTRACT FROM AUTHOR]