Metagenomic approaches are now commonly used in microbial ecology to study microbial communities in more detail, including many strains that cannot be cultivated in the laboratory. Bioinformatic analyses make it possible to mine huge metagenomic datasets and discover general patterns that govern microbial ecosystems. However, the findings of typical metagenomic and bioinformatic analyses still do not completely describe the ecology and evolution of microbes in their environments. Most analyses still depend on straightforward sequence similarity searches against reference databases. We herein review the current state of metagenomics and bioinformatics in microbial ecology and discuss future directions for the field. New techniques will allow us to go beyond routine analyses and broaden our knowledge of microbial ecosystems. We need to enrich reference databases, promote platforms that enable meta- or comprehensive analyses of diverse metagenomic datasets, devise methods that utilize long-read sequence information, and develop more powerful bioinformatic methods to analyze data from diverse perspectives.

A facultative anaerobic bacterium, designated as strain SCS5, was isolated from the anodic biofilm of a mediator-less microbial fuel cell using acetate as the electron donor and α-FeOOH as the electron acceptor. The isolate was Gram-negative, motile, and shaped as short rods (0.9–1.3 μm in length and 0.4–0.5 μm in width). A phylogenetic analysis of the 16S rRNA, gyrB, and rpoD genes suggested that strain SCS5 belonged to the Aeromonas genus in the Aeromonadaceae family and exhibited the highest 16S rRNA gene sequence similarity (99.45%) with Aeromonas jandaei ATCC 49568. However, phenotypic, cellular fatty acid profile, and DNA G+C content analyses revealed that there were some distinctions between strain SCS5 and the type strain A. jandaei ATCC 49568. The optimum growth temperature, pH, and NaCl (%) for strain SCS5 were 35°C, 7.0, and 0.5% respectively. The DNA G+C content of strain SCS5 was 59.18%. The isolate SCS5 was capable of reducing insoluble iron oxide (α-FeOOH) and transferring electrons to extracellular material (the carbon electrode). The electrochemical activity of strain SCS5 was corroborated by cyclic voltammetry and a Raman spectroscopic analysis. The cyclic voltammogram of strain SCS5 revealed two pairs of oxidation-reduction peaks under anaerobic and aerobic conditions. In contrast, no redox pair was observed for A. jandaei ATCC 49568. Thus, isolated strain SCS5 is a novel exoelectrogenic bacterium phylogenetically related to A. jandaei, but shows distinct electrochemical activity from its close relative A. jandaei ATCC 49568.

The transcription patterns of [FeFe]-hydrogenase genes (hydA), which encode the enzymes responsible for H₂ production, were investigated during rice straw decomposition in paddy soil using molecular biological techniques. Paddy soil amended with and without rice straw was incubated under anoxic conditions. RNA was extracted from the soil, and three clone libraries of hydA were constructed using RNAs obtained from samples in the initial phase of rice straw decomposition (day 1 with rice straw), methanogenic phase of rice straw decomposition (day 14 with rice straw), and under a non-amended condition (day 14 without rice straw). hydA genes related to Proteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, and Thermotogae were mainly transcribed in paddy soil samples; however, their proportions markedly differed among the libraries. Deltaproteobacteria-related hydA genes were predominantly transcribed on day 1 with rice straw, while various types of hydA genes related to several phyla were transcribed on day 14 with rice straw. Although the diversity of transcribed hydA was significantly higher in the library on day 14 with rice straw than the other two libraries, the composition of hydA transcripts in the library was similar to that in the library on day 14 without rice straw. These results indicate that the composition of active H₂ producers and/or H₂ metabolic patterns dynamically change during rice straw decomposition in paddy soil.

Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF.

The genus Acidithiobacillus includes iron-oxidizing lithoautotrophs that thrive in acidic mine environments. Acidithiobacillus ferrooxidans is a representative species and has been extensively studied for its application to the bioleaching of precious metals. In our attempts to cultivate the type strain of A. ferrooxidans (ATCC 23270^T), repeated transfers to fresh inorganic media resulted in the emergence of cultures with improved growth traits. Strains were isolated from the resultant culture by forming colonies on inorganic silica-gel plates. A representative isolate (strain NU-1) was unable to form colonies on agarose plates and was more sensitive to organics, such as glucose, than the type strain of A. ferrooxidans. Strain NU-1 exhibited superior growth traits in inorganic iron media to those of other iron-oxidizing acidithiobacilli, suggesting its potential for industrial applications. A draft genome of NU-1 uncovered unique features in catabolic enzymes, indicating that this strain is not a mutant of the A. ferrooxidans type strain. Our results indicate that the use of inorganic silica-gel plates facilitates the isolation of as-yet-unexamined iron-oxidizing acidithiobacilli from environmental samples and enrichment cultures.

Previous transcriptome analyses have suggested that a gene cluster including a transcriptional regulator (blr7984) of the tetracycline repressor family was markedly down-regulated in symbiosis. Since blr7984 is annotated to be the transcriptional repressor, we hypothesized that it is involved in the repression of genes in the genomic cluster including blr7984 in symbiotic bacteroids. In order to examine the function and involvement of the blr7984 gene in differentiation into bacteroids, we compared the free-living growth/symbiotic phenotype and gene expression between a blr7984-knockout mutant and the wild-type strain of Bradyrhizobium diazoefficiens USDA110. The mutant transiently increased the cell growth rate under free-living conditions and nodule numbers over those with the wild-type strain USDA110. The expression of three genes adjacent to the disrupted blr7984 gene was strongly up-regulated in the mutant in free-living and symbiotic cells. The mutant also induced the expression of genes for glutathione S-transferase, cytochrome c oxidases, ABC transporters, PTS sugar transport systems, and flagella synthesis under free-living conditions. bll7983 encoding glutathione S-transferase was up-regulated the most by the blr7984 disruption. Since redox regulation by glutathione is known to be involved in cell division in prokaryotes and eukaryotes, the strong expression of glutathione S-transferase encoded by the bll7983 gene may have caused redox changes in mutant cells, which resulted in higher rates of cell division.

The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state.

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.

Predation by protists is top-down pressure that regulates prokaryotic abundance, community function, structure, and diversity in natural and artificial ecosystems. Although the effects of predation by protists have been studied in aerobic ecosystems, they are poorly understood in anoxic environments. We herein studied the influence of predation by Metopus and Caenomorpha ciliates—ciliates frequently found in anoxic ecosystems—on prokaryotic community function, structure, and diversity. Metopus and Caenomorpha ciliates were cocultivated with prokaryotic assemblages (i.e., anaerobic granular sludge) in an up-flow anaerobic sludge blanket (UASB) reactor for 171 d. Predation by these ciliates increased the methanogenic activities of granular sludge, which constituted 155% of those found in a UASB reactor without the ciliates (i.e., control reactor). Sequencing of 16S rRNA gene amplicons using Illumina MiSeq revealed that the prokaryotic community in the UASB reactor with the ciliates was more diverse than that in the control reactor; 2,885–3,190 and 2,387–2,426 operational taxonomic units (>97% sequence similarities), respectively. The effects of predation by protists in anaerobic engineered systems have mostly been overlooked, and our results show that the influence of predation by protists needs to be examined and considered in the future for a better understanding of prokaryotic community structure and function.

The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading “Syntrophomonas wolfei subsp. methylbutyratica” strain JCM 14075^T (=4J5^T), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1^T, and non-BCFA-metabolizing S. wolfei subsp. wolfei strain Göttingen^T. We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems (i.e., electron-bifurcating hydrogenase, formate dehydrogenase, and membrane-bound hydrogenase) and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H₂ generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.

Pseudogulbenkiania is a relatively recently characterized genus within the order Neisseriales, class Betaproteobacteria. This genus contains several strains that are capable of anaerobic, nitrate-dependent Fe(II) oxidation (NDFO), a geochemically important reaction for nitrogen and iron cycles. In the present study, we examined denitrification functional gene diversities within this genus, and clarified whether other Pseudogulbenkiania sp. strains perform denitrification and NDFO. Seventy strains were analyzed, including two type strains, a well-characterized NDFO strain, and 67 denitrifying strains isolated from various rice paddy fields and rice-soybean rotation fields in Japan. We also attempted to identify the genes responsible for NDFO by mutagenesis. Our comprehensive analysis showed that all Pseudogulbenkiania strains tested performed denitrification and NDFO; however, we were unable to obtain NDFO-deficient denitrifying mutants in our mutagenesis experiment. This result suggests that Fe(II) oxidation in these strains is not enzymatic, but is caused by reactive N-species that are formed during nitrate reduction. Based on the results of the comparative genome analysis among Pseudogulbenkiania sp. strains, we identified low sequence similarity within the nos gene as well as different gene arrangements within the nos gene cluster, suggesting that nos genes were horizontally transferred. Since Pseudogulbenkiania sp. strains have been isolated from various locations around the world, their denitrification and NDFO abilities may contribute significantly to nitrogen and iron biogeochemical cycles.

The diversity of aerobic anoxygenic phototrophic (AAP) bacteria in freshwater environments, particularly in rivers, has not been examined in as much detail as in ocean environments. In the present study, we investigated the phylogenetic and physiological diversities of AAP bacteria in biofilms that developed on submerged stones in a freshwater river using culture methods. The biofilms collected were homogenized and inoculated on solid media and incubated aerobically in the dark. Sixty-eight red-, pink-, yellow-, orange-, or brown-colored colonies were isolated, and, of these, 28 isolates contained the photosynthetic pigment, bacteriochlorophyll (BChl) a. Phylogenetic analyses based on 16S rRNA gene sequences showed that the isolates were classified into 14 groups in 8 operational taxonomic units (OTUs) and distributed in the orders Rhodospirillales, Rhodobacterales, and Sphingomonadales of Alphaproteobacteria and in Betaproteobacteria. Physiological analyses confirmed that none of the representative isolates from any of the groups grew under anaerobic phototrophic conditions. Seven isolates in 4 OTUs showed a 16S rRNA gene sequence identity of 98.0% or less with any established species, suggesting the presence of previously undescribed species of AAP bacteria. Six isolates in 2 other OTUs had the closest relatives, which have not been reported to be AAP bacteria. Physiological comparisons among the isolates revealed differences in preferences for nutrient concentrations, BChl contents, and light-harvesting proteins. These results suggest that diverse and previously unknown AAP bacteria inhabit river biofilms.

Soil-borne diseases caused by pathogenic microorganisms are one of the main factors responsible for the decline in crop yields in farmlands. Pathogenic Fusarium oxysporum causes serious damage to various crops, and, thus, a feasible diagnostic method for soil-borne diseases is required. We herein examined a simple method to evaluate the suppressiveness of soil microorganisms against a pathogen by co-cultivating indigenous soil microorganisms and a pathogenic fungus (F. oxysporum f. sp. spinaciae). We inoculated F. oxysporum onto the center of agar medium plates mixed with a dilution series of a suspension of organic fertilizers or soil. After an approximately one-week cultivation, the growth degree of F. oxysporum was estimated based on the size of the colonies that formed on the plates. The growth degree of F. oxysporum significantly differed among the organic fertilizers tested, indicating the usefulness of the method for evaluating suppressiveness by organic fertilizers. Differences in the growth degrees of F. oxysporum were associated with the incidence of disease in spinach on soil treated with organic fertilizers and inoculated with a pathogenic F. oxysporum strain. These results suggested that this method provides some useful information on the suppressiveness of organic fertilizers and soil against Fusarium wilt.

Cockroaches are parasitized by thelastomatid nematodes, which live in an obligate manner in their hindgut and interact with the resident microbial community. In the present study, a composition analysis was performed on the gut microbiome of Periplaneta fuliginosa and P. americana to investigate natural and artificial infection by thelastomatid nematodes. Nine libraries of the 16S rRNA gene V3–V4 region were prepared for pyrosequencing. We examined the complete gut microbiome (fore-, mid-, and hindgut) of lab-reared P. fuliginosa naturally infected with the parasitic nematode Leidynema appendiculatum and those that were nematode-free, and complemented our study by characterizing the hindgut microbial communities of lab-reared P. americana naturally infected with Hammerschmidtiella diesingi and Thelastoma bulhoesi, artificially infected with L. appendiculatum, and those that were nematode-free. Our results revealed that the fore- and midgut of naturally infected and nematode-free P. fuliginosa have close microbial communities, which is in contrast with hindgut communities; the hindgut communities of both cockroaches exhibit higher microbial diversities in the presence of their natural parasites and marked differences were observed in the abundance of the most representative taxa, namely Firmicutes, Proteobacteria, and Bacteroidetes. Our results have provided basic information and encourage further studies on multitrophic interactions in the cockroach gut as well as the thelastomatid nematodes that play a role in this environment.

Plant-associated microbes have specific beneficial functions and are considered key drivers for plant health. The bacterial community structure of healthy Anthurium andraeanum L. plants was studied by 16S rRNA gene pyrosequencing associated with different plant parts and the rhizosphere. A limited number of bacterial taxa, i.e., Sinorhizobium, Fimbriimonadales, and Gammaproteobacteria HTCC2089 were enriched in the A. andraeanum rhizosphere. Endophytes were more diverse in the roots than in the shoots, whereas all shoot endophytes were found in the roots. Streptomyces, Flavobacterium succinicans, and Asteroleplasma were only found in the roots, Variovorax paradoxus only in the stem, and Fimbriimonas 97%-OTUs only in the spathe, i.e., considered specialists, while Brevibacillus, Lachnospiraceae, Pseudomonas, and Pseudomonas pseudoalcaligenes were generalist and colonized all plant parts. The anaerobic diazotrophic bacteria Lachnospiraceae, Clostridium sp., and Clostridium bifermentans colonized the shoot system. Phylotypes belonging to Pseudomonas were detected in the rhizosphere and in the substrate (an equiproportional mixture of soil, cow manure, and peat), and dominated the endosphere. Pseudomonas included nine 97%-OTUs with different patterns of distribution and phylogenetic affiliations with different species. P. pseudoalcaligenes and P. putida dominated the shoots, but were also found in the roots and rhizosphere. P. fluorescens was present in all plant parts, while P. resinovorans, P. denitrificans, P. aeruginosa, and P. stutzeri were only detected in the substrate and rhizosphere. The composition of plant-associated bacterial communities is generally considered to be suitable as an indicator of plant health.

Accretionary prisms are mainly composed of ancient marine sediment scraped from the subducting oceanic plate at a convergent plate boundary. Large amounts of anaerobic groundwater and natural gas, mainly methane (CH₄) and nitrogen gas (N₂), are present in the deep aquifers associated with an accretionary prism; however, the origins of these gases are poorly understood. We herein revealed regional variations in CH₄ and N₂ production processes in deep aquifers in the accretionary prism in Southwest Japan, known as the Shimanto Belt. Stable carbon isotopic and microbiological analyses suggested that CH₄ is produced through the non-biological thermal decomposition of organic matter in the deep aquifers in the coastal area near the convergent plate boundary, whereas a syntrophic consortium of hydrogen (H₂)-producing fermentative bacteria and H₂-utilizing methanogens contributes to the significant production of CH₄ observed in deep aquifers in midland and mountainous areas associated with the accretionary prism. Our results also demonstrated that N₂ production through the anaerobic oxidation of organic matter by denitrifying bacteria is particularly prevalent in deep aquifers in mountainous areas in which groundwater is affected by rainfall.

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi.

Elevated concentrations of atmospheric CO₂ ([CO₂]) enhance the production and emission of methane in paddy fields. In the present study, the effects of elevated [CO₂], elevated temperature (ET), and no nitrogen fertilization (LN) on methanogenic archaeal and methane-oxidizing bacterial community structures in a free-air CO₂ enrichment (FACE) experimental paddy field were investigated by PCR-DGGE and real-time quantitative PCR. Soil samples were collected from the upper and lower soil layers at the rice panicle initiation (PI) and mid-ripening (MR) stages. The composition of the methanogenic archaeal community in the upper and lower soil layers was not markedly affected by the elevated [CO₂], ET, or LN condition. The abundance of the methanogenic archaeal community in the upper and lower soil layers was also not affected by elevated [CO₂] or ET, but was significantly increased at the rice PI stage and significantly decreased by LN in the lower soil layer. In contrast, the composition of the methane-oxidizing bacterial community was affected by rice-growing stages in the upper soil layer. The abundance of methane-oxidizing bacteria was significantly decreased by elevated [CO₂] and LN in both soil layers at the rice MR stage and by ET in the upper soil layer. The ratio of mcrA/pmoA genes correlated with methane emission from ambient and FACE paddy plots at the PI stage. These results indicate that the decrease observed in the abundance of methane-oxidizing bacteria was related to increased methane emission from the paddy field under the elevated [CO₂], ET, and LN conditions.

A novel species of Cladophialophora is herein described from the natural environment of secondary forest soil in Japan, which was able to be colonized by the host plant root. Morphological observations indicated that the isolate is distinct from previously identified species, and, thus, is described as the new species, C. inabaensis sp. nov.

The effects of a precipitous decrease in the inlet organic loading rate on sludge reductions and the microbial community in a membrane bioreactor were investigated. The sludge biomass was markedly reduced to 47.4% of the initial concentration (approximately 15,000 mg L⁻¹) within 7 d after the organic loading rate was decreased by half (450 to 225 mg chemical oxygen demand L⁻¹ d⁻¹). An analysis of the microbial community structure using high-throughput sequencing revealed an increase in the abundance of facultative predatory bacteria-related operational taxonomic units as well as microorganisms tolerant to environmental stress belonging to the classes Deinococci and Betaproteobacteria.