Takashi Narihiro

Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor

Ecogenomic investigation of a methanogenic bioreactor degrading terephthalate (TA) allowed elucidation of complex synergistic networks of uncultivated microorganisms, including those from candidate phyla with no cultivated representatives. Our previous metagenomic investigation proposed that Pelotomaculum and methanogens may interact with uncultivated organisms to degrade TA; however, many members of the community remained unaddressed because of past technological limitations. In further pursuit, this study employed state-of-the-art omics tools to generate draft genomes and transcriptomes for uncultivated organisms spanning 15 phyla and reports the first genomic insight into candidate phyla Atribacteria, Hydrogenedentes and Marinimicrobia in methanogenic environments. Metabolic reconstruction revealed that these organisms perform fermentative, syntrophic and acetogenic catabolism facilitated by energy conservation revolving around H2 metabolism. Several of these organisms could degrade TA catabolism by-products (acetate, butyrate and H2) and syntrophically support Pelotomaculum. Other taxa could scavenge anabolic products (protein and lipids) presumably derived from detrital biomass produced by the TA-degrading community. The protein scavengers expressed complementary metabolic pathways indicating syntrophic and fermentative step-wise protein degradation through amino acids, branched-chain fatty acids and propionate. Thus, the uncultivated organisms may interact to form an intricate syntrophy-supported food web with Pelotomaculum and methanogens to metabolize catabolic by-products and detritus, whereby facilitating holistic TA mineralization to CO2 and CH4.

Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea.

For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers.

Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen

The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota methanogenesis. To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol coenzyme M methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for carbon fixation, nitrogen fixation and biosynthesis of many amino acids. Acetate, malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially compete with CO2-reducing methanogens and even sulfate reducers. These findings reveal a remarkably unique methanogen ‘Candidatus Methanofastidiosum methylthiophilus’ as the first insight into the sixth class of methanogens ‘Candidatus Methanofastidiosa’.

Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments

Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8–98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. Thus, the novel strategy allowed to enrich and isolate novel iron(III) reducers that were able to thrive by reducing crystalline ferric iron oxides.

Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance

ABSTRACT Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

Bacterial community characterization and dynamics of indigo fermentation

Indigo fermentation has been traditionally performed for dyeing textiles in Japan. Limited information is available on the microbiota involved and the succession of the bacterial community structure during indigo reduction. We investigated the bacterial community structure associated with indigo fermentation using denaturing gradient gel electrophoresis and clone library analyses of a PCR-amplified 16S rRNA gene in the early phase of fermentation carried out in our laboratory. A marked substitution of Halomonas spp. by Amphibacillus spp. was observed corresponding to the marked change in the state of indigo reduction. Although the reported indigo-reducing bacteria, Alkalibacterium spp., were not predominant in the early phase of fermentation, they were predominant in fermentation liquor aged for 10 months obtained from Date City, Japan, as determined by culture-dependent and culture-independent analyses. Novel indigo-reducing strains, Amphibacillus spp. strain C40 and Oceanobacillus spp. strain A21, were isolated from fermentation liquor aged for 10 months and from liquor aged for 4 days, respectively. It is considered that, in addition to the strains belonging to the genus Alkalibacterium, strains belonging to genera Amphibacillus and Oceanobacillus play important roles in sustaining the reduced state of indigo during fermentation.

Quantitative detection of culturable methanogenic archaea abundance in anaerobic treatment systems using the sequence-specific rRNA cleavage method.

A method based on sequence-specific cleavage of rRNA with ribonuclease H was used to detect almost all known cultivable methanogens in anaerobic biological treatment systems. To do so, a total of 40 scissor probes in different phylogeny specificities were designed or modified from previous studies, optimized for their specificities under digestion conditions with 32 methanogenic reference strains, and then applied to detect methanogens in sludge samples taken from 6 different anaerobic treatment processes. Among these processes, known aceticlastic and hydrogenotrophic groups of methanogens from the families Methanosarcinaceae, Methanosaetaceae, Methanobacteriaceae, Methanothermaceae and Methanocaldococcaceae could be successfully detected and identified down to the genus level. Within the aceticlastic methanogens, the abundances of mesophilic Methanosaeta accounted for 5.7–48.5% of the total archaeal populations in mesophilic anaerobic processes, and those of Methanosarcina represented 41.7% of the total archaeal populations in thermophilic processes. For hydrogenotrophic methanogens, members of the Methanomicrobiales, Methanobrevibacter and Methanobacterium were detected in mesophilic processes (1.2–17.2%), whereas those of Methanothermobacter, Methanothermaceae and Methanocaldococcaceae were detected in thermophilic process (2.0–4.8%). Overall results suggested that those hierarchical scissor probes developed could be effective for rapid and possibly on-site monitoring of targeted methanogens in different microbial environments.

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol-degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate-degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl-coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H2 /formate-generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H2 and formate generation. The genome analysis further identified a putative ion-translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.

Cultivating yet-to-be cultivated microbes: the challenge continues.

When looking into the current situation of microbial ecology, you would realize that the (meta) omics-driven studies associated with next generation sequencing technologies make the headlines in related journals. Currently, isolation and characterization of as-yet-uncultured, but functionally important microorganisms is, at least to a certain extent, being replaced by omics-driven approach without cultivation to decipher their functions. To date, more than 100 microbes have been identified as “Candidatus”, that is a provisional status for “well-characterized but as-yet uncultured organisms” (26). Together with this trend, numerous scientists still voice the importance of the cultivation and isolation of microorganisms. Indeed, the pace of proposals on novel species, genus and even higher levels has been incredibly accelerated: i.e., over the past decades, more than 6,000 prokaryotic species have been isolated and characterized on the basis of biochemical, morphological, physiological, and genetic traits (5). However, most of the described organisms are readily cultivable ones, in turn, as-yet-uncultured organisms still remain uncultivable. To fill the gap between the canonical isolation methods and the state-of-the-art technologies that circumvent isolation, developing new approaches to cultivate those as-yet-uncultured organisms in hand is one of the most intriguing challenges in microbial ecology (12, 24). Significant progress can be highlighted by the description on microorganisms within the class Anaerolineae of the phylum Chloroflexi (formerly known as Chloroflexi subphylum I). Until 2003, the subphylum I within the phylum Chloroflexi had not have any cultured representatives whereas it had been well-known as cosmopolitan based on culture-independent molecular analyses. Since the first cultured microorganism was obtained and the novel class Anaerolineae was coined, it has gained increased universality. The first cultivated organism named Anaerolinea thermophila has been followed by a number of newly isolated organisms within the new genera Bellilinea, Leptolinea, Levilinea, Longilinea, Thermanaerothrix, and Ornatilinea, all of which were within the class Anaerolineae, were difficult to isolate but were eventually isolated in pure culture (7, 23, 32). Those were isolated from anaerobic wastewater treatment process, rice paddy soil, and deep terrestrial hot aquifer, and characterized as anaerobic heterotrophic bacteria (32). Moreover, the 16S rRNA gene clones associated with the class Anaerolineae were frequently observed in anaerobic wastewater treatment processes, and they may play a role in the degradation of organic compounds such as carbohydrates and amino acids, probably to a great extent, associated with methanogens via interspecies hydrogen transfer (1, 20, 32). In the current issue of Microbes and Environments, Nunoura et al. (21) report that Anaerolineae-type organisms were predominated in an in situ colonization system placed on the shallow submarine hydrothermal vent. They successfully isolate a bacterial strain SW7 and propose the new genus Thermomarinilinea with type species T. lacunofontalis. Among a number of novel isolates described over the last decade, these organisms are exceptionally well-coordinated on nomenclature basis. Together with genome sequencing of these organisms, we will now know the entity of those organisms that allows us to know exactly who they would be and what they would do, once close relatives are isolated, or omics data that hints at the presence of relatives are obtained. Methanogenic archaea (methanogens) have a key role in anaerobic ecosystems, where electron accepters other than carbon dioxide (e.g., oxygen, sulfate, and ferric iron) are limited. Previously characterized methanogens have been classified into the orders Methanosarcinales, Methanocellales, Methanomicrobiales, Methanobacteriales, Methanococcales, and Methanopyrales of the phylum Euryarchaeota (Fig. 1). Generally, we use laborious culturing techniques with specific apparatuses (e.g., roll tube and agar shake tube) for isolation of the methanogens as well as other obligate anaerobic microbes to eliminate oxygen in the culture medium (8, 21, 30, 31). Such obstacles have led to the difficulty in isolation of obligate anaerobic microorganisms. Nakamura et al. (18) developed a simple technique for cultivation of such fastidious anaerobic organisms by using six-well plate and anaerobic gas pack system. They demonstrated the usefulness of this technique to cultivate the methanogens, syntrophic substrate-oxidizing bacteria (syntrophs), and sulfate- or thiosulfate-reducing bacteria. Subsequently, a thermophilic and hydrogenotrophic methanogen, Methanothermobacter tenebrarum strain RMAS, was successfully isolated from natural gas field by using this technique (17). More recently, a methanogenic archaeon, Methanomassiliicoccus luminyensis strain B10, was isolated from human feces (3). This strain is the first cultured methanogenic representative of the class Thermoplasmata. Thereafter, Iino et al. (9) report the methanogenic enrichment culture derived from the sludge of an anaerobic digestion process, that contains a novel methanogenic archaeon Kjm51a as a sole archaeal population. Phylogenetic analysis based on the 16S rRNA gene sequences indicates that archaeon Kjm51a is a relative of the Methanomassiliicoccus luminyensis but the identity between them is relatively low. According to the phylogenetic and physiological traits of archaeon Kjm51a, they propose “Candidatus Methanogranum caenicola” as the provisional taxonomic assignment. Together with Methanomassiliicoccus luminyensis, they also propose novel taxa, the family Methanomassiliicoccaceae and the order Methanomassiliicoccales, for a methanogenic linage of the class Thermoplasmata (Fig. 1). Fig. 1 Phylogeny of methanogens. The neighbor-joining tree was constructed on the basis of 16S rRNA gene sequences of previously known methanogens (19) and Methanomassiliicoccales-related strains (3, 9, 22) using the ARB software (15). The 16S rRNA gene sequences ... Besides the anaerobic microorganisms, remarkable efforts have been made to cultivate aerobic microorganisms. Fujitani et al. (6) develop a bioreactor-based selective culturing technique for the enrichment of Nitrospira-type nitrite-oxidizing bacteria (NOB). The affinity to nitrite strikingly affects the growth of dominant NOB, thus nitrite concentration is maintained at a low level to facilitate the specific growth of Nitrospira-type NOB and to inhibit the growth of Nitrobacter-type NOB. Because the bioreactor-based culturing strategy has the advantage of setting up the stable culture conditions suitable for targeted uncultured microbes, this approach was applied to enrich the yet-to-be cultured organisms: for example, anaerobic ammonium oxidation (anammox) bacteria in coastal sediment (13) and phylogenetically diverse anaerobic microorganisms in subseafloor sediment (10). Tanaka et al. (29) successfully isolated a novel aerobic bacterial strain YO-36 from the rhizoplane of an aquatic plant in freshwater environment by using low-nutrient agar medium. Bacterium YO-36 was assigned to the candidate phylum OP10, and proposed as Armatimonas rosea of the novel phylum Armatimonadetes (28). In addition, functionally important microorganisms have recently been isolated or enriched: ammonia-oxidizing archaeon (16), aromatic-hydrocarbon-degrading bacteria (11), cellulolytic bacteria (4), chitinolytic bacteria (25), denitrifying bacteria (27), methane-oxidizing bacteria (2), sulfate-reducing bacteria (8), uranium-tolerant bacteria (14), and uric acid-degrading bacteria (30). Clearly, cultivation and (meta) omics approaches should be complimentary. Omics information will give us a clue to the way of isolation of yet-to-be cultivated organisms, and conversely, characterization and genome information of isolates will provide convincing information that would make omics data far more robust. The challenge still continues.

The Impact of Aridification and Vegetation Type on Changes in the Community Structure of Methane-Cycling Microorganisms in Japanese Wetland Soils

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.