Complete genome sequence of Opitutales bacterium strain ASA1, isolated from soil.

We present the complete genome of Opitutales bacterium ASA1, isolated from soil. The genome is 5,821,695 bp with 4,638 protein-coding sequences. The genome data suggest that this strain belongs to the class Opitutae of the phylum Verrucomicrobiota, and its genome has six unique biosynthetic gene clusters associated with secondary metabolites.

Microbial Biomanufacturing Using Chemically Synthesized Non-Natural Sugars as the Substrate.

The bioproduction of valuable materials using biomass sugars is attracting attention as an environmentally friendly technology. However, its ability to fulfil the enormous demand to produce fuels and chemical products is limited. With a view towards the future development of a novel bioproduction process that addresses these concerns, this study investigated the feasibility of bioproduction of valuable substances using Corynebacterium glutamicum (C. glutamicum) with a chemically synthesized non-natural sugar solution. Cells were grown using the synthesized sugar solution as the sole carbon source and they produced lactate under oxygen-limited conditions. It was also found that some of the sugars produced by the series of chemical reactions inhibited cell growth since prior removal of these sugars increased the cell growth rate. The results obtained in this study indicate that chemically synthesized sugars have the potential to resolve the concerns regarding future biomass sugar supply in microbial biomanufacturing.

Transcriptional Regulation of Methanol Dehydrogenases in the Methanotrophic Bacterium Methylococcus capsulatus Bath by Soluble and Insoluble Lanthanides.

The effects of soluble and insoluble lanthanides on gene expression in Methylococcus capsulatus Bath were investigated. Genes for lanthanide-containing methanol dehydrogenases (XoxF-MDHs) and their calcium-containing counterparts (MxaFI-MDHs) were up- and down-regulated, respectively, by supplementation with soluble lanthanide chlorides, indicating that M. capsulatus has the "lanthanide switch" observed in other methanotrophs. Insoluble lanthanide oxides also induced the lanthanide switch and were dissolved by the spent medium of M. capsulatus, suggesting the presence of lanthanide-chelating compounds. A transcriptome ana-lysis indicated that a gene cluster for the synthesis of an enterobactin-like metal chelator contributed to the dissolution of insoluble lanthanides.

Construction of an autocatalytic reaction cycle in neutral medium for synthesis of life-sustaining sugars.

Autocatalytic mechanisms in carbon metabolism, such as the Calvin cycle, are responsible for the biological assimilation of CO2 to form organic compounds with complex structures, including sugars. Compounds that form C-C bonds with CO2 are regenerated in these autocatalytic reaction cycles, and the products are concurrently released. The formose reaction in basic aqueous solution has attracted attention as a nonbiological reaction involving an autocatalytic reaction cycle that non-enzymatically synthesizes sugars from the C1 compound formaldehyde. However, formaldehyde and sugars, which are the substrate and products of the formose reaction, respectively, are consumed in Cannizzaro reactions, particularly under basic aqueous conditions, which makes the formose reaction a fragile sugar-production system. Here, we constructed an autocatalytic reaction cycle for sugar synthesis under neutral conditions. We focused on the weak Brønsted basicity of oxometalate anions such as tungstates and molybdates as catalysts, thereby enabling the aldol reaction, retro-aldol reaction, and aldose-ketose transformation, which collectively constitute the autocatalytic reaction cycle. These bases acted on sugar molecules of substrates together with sodium ions of a Lewis acid to promote deprotonation under neutral conditions, which is the initiation step of the reactions forming an autocatalytic cycle, whereas the Cannizzaro reaction was inhibited. The autocatalytic reaction cycle established using this abiotic approach is a robust sugar production system. Furthermore, we found that the synthesized sugars work as energy storage substances that sustain microbial growth despite their absence in nature.

Self-cloning of the Catalase Gene in Environmental Isolates Improves Their Colony-forming Abilities on Agar Media.

Hydrogen peroxide (H2O2) inhibits microbial growth at a specific concentration. However, we previously isolated two environmental bacterial strains that exhibited sensitivity to a lower H2O2 concentration in agar plates. Putative catalase genes, which degrade H2O2, were detected in their genomes. We herein elucidated the characteristics of these putative genes and their products using a self-cloning technique. The products of the cloned genes were identified as functional catalases. The up-regulation of their expression increased the colony-forming ability of host cells under H2O2 pressure. The present results demonstrated high sensitivity to H2O2 even in microbes possessing functional catalase genes.

Genome Sequences of Comamonadaceae Bacteria OS-1 and OS-4, Two Highly H2O2-Sensitive Strains Isolated from Pond Water.

The Comamonadaceae bacterial strains OS-1 and OS-4 were isolated from pond water and were found to be highly sensitive to hydrogen peroxide in the agar plates. Here, we report the nearly complete and complete genome sequences, respectively, of these two strains.

Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains.

We previously showed that autoclaving in preparing agar media is one of the sources of hydrogen peroxide (H2O2) in the medium. This medium-embedded H2O2 was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of H2O2 detrimental to colony formation on the agar plate remain largely undetermined. Herein, we elucidated the specific effect of H2O2 on microbial colony formation on solid agar medium by external supplementation of varying amounts of H2O2. While common laboratory strains (often called domesticated microbes) formed colonies in the presence of high H2O2 concentrations (48.8 µM or higher), microbes from a freshwater sample demonstrated greatly decreased colony counts in the presence of 8.3 µM H2O2. This implies that environmental microbes are susceptible to much lower concentrations of H2O2 than laboratory strains. Among the emergent colonies on agar plates supplemented with different H2O2 concentrations, the relative abundance of betaproteobacterial colonies was found to be lower on plates containing higher amounts of H2O2. Further, the growth of the representative betaproteobacterial isolates was completely inhibited in the presence of 7.2 µM H2O2. Therefore, our study clearly demonstrates that low micromolar levels of H2O2 in agar plates critically affect growth of environmental microbes, and large portions of those are far more susceptible to the same than laboratory strains. IMPORTANCE It is well-known that most of environmental microorganisms do not form colonies on agar medium despite that agar medium is the commonly used solidified medium. We previously demonstrated the negative effects of H2O2 generation during agar medium preparation on colony formation. In the present study, we investigated the independent effect of H2O2 on microbial growth by adding different concentrations of H2O2 to agar medium. Our results demonstrate for the first time that even low micromolar levels of H2O2 in agar plates, that are far lower than previously recognized as significant, adversely affect colony number obtained from freshwater inoculum.

Draft Genome Sequence of the Alkaliphilic, Lithoautotrophic Homoacetogen Fuchsiella alkaliacetigena Z-7100T.

Fuchsiella alkaliacetigena is a spore-forming, alkaliphilic hydrogentrophic homoacetogen that was isolated from the soda lake Lake Tanatar III in Russia. The genome of the type strain Z-7100 (= DSM 24880) is 2.9 Mb, with a G+C content of 36.2%.

Draft Genome Sequence of the Extremely Haloalkaliphilic and Homoacetic Bacterium Natroniella acetigena Z-7937T.

Natroniella acetigena Z-7937T (= DSM 9952T) is a heterotrophic homoacetogenic natronophile. The draft genome sequence is 2.6 Mb in 116 contigs, with a G+C content of 34.1%.

Complete Genome Sequence of Alphaproteobacteria Bacterium Strain SO-S41, Isolated from Forest Soil.

The complete genome of hydrogen peroxide-sensitive alphaproteobacterial strain SO-S41 was sequenced. The complete genome contains a single chromosome, is 4,443,179 bp in length, contains a total of 4,632 genes, and has a G+C content of 66.2%.

Ferrihydrite Reduction by Photosynthetic Synechocystis sp. PCC 6803 and Its Correlation With Electricity Generation.

Microbial extracellular electron transfer (EET) to solid-state electron acceptors such as anodes and metal oxides, which was originally identified in dissimilatory metal-reducing bacteria, is a key process in microbial electricity generation and the biogeochemical cycling of metals. Although it is now known that photosynthetic microorganisms can also generate (photo)currents via EET, which has attracted much interest in the field of biophotovoltaics, little is known about the reduction of metal (hydr)oxides via photosynthetic microbial EET. The present work quantitatively assessed the reduction of ferrihydrite in conjunction with the EET of the photosynthetic microbe Synechocystis sp. PCC 6803. Microbial reduction of ferrihydrite was found to be initiated in response to light but proceeded at higher rates when exogenous glucose was added, even under dark conditions. These results indicate that current generation from Synechocystis cells does not always need light irradiation. The qualitative trends exhibited by the ferrihydrite reduction rates under various conditions showed significant correlation with those of the microbial currents. Notably, the maximum concentration of Fe(II) generated by the cyanobacterial cells under dark conditions in the presence of glucose was comparable to the levels observed in the photic layers of Fe-rich microbial mats.

Reductive Transformation of Fe(III) (oxyhydr)Oxides by Mesophilic Homoacetogens in the Genus Sporomusa.

Microbial reduction of iron contributes to the dissolution and transformation of iron-containing minerals in nature. Diverse groups of homoacetogenic bacteria (homoacetogens) have been reported to reduce insoluble Fe(III) oxides, such as hydrous ferric oxide (HFO), an Fe(III) mineral commonly found in soils and sediments. Several members of genus Sporomusa reportedly oxidize Fe(0), indicating the presence of an extracellular electron-uptake mechanism. However, the ability of the genus to reduce insoluble Fe(III) oxides is limited, and the underlying reduction mechanism remains to be elucidated. In this study, the HFO reduction ability of three Sporomusa spp. (Sporomusa sp. strain GT1, Sporomusa sphaeroides, and Sporomusa ovata) and a homoacetogen of a different genus (Acetobacterium woodii) were assayed under organotrophic (ethanol) and lithotrophic (H2 + CO2) conditions without a chelator or reducing reagent. All tested homoacetogens showed acetogenic growth and concomitant reduction of HFO under both organotrophic and lithotrophic conditions. Analysis of the growth stoichiometry showed that Fe(III) reduction does not support direct energy conservation, thereby indicating that Fe(III) reduction is a side reaction of acetogenesis to dissipate the excess reducing power. HFO was reduced to a soluble Fe(II) form by microbial activity. In addition, we observed that strain GT1, S. sphaeroides, and S. ovata reduced crystalline Fe(III) oxides, and HFO was reductively transformed into magnetite (Fe3O4) under phosphate-limiting conditions. Separation of HFO by a dialysis membrane still permitted Fe(II) production, although the reduction rate was decreased, suggesting that Fe(III) reduction is at least partially mediated by soluble redox compound(s) secreted from the cells. Finally, culture experiments and comparative genomic analysis suggested that electron transfer by flavins and multiheme c-type cytochrome were not directly correlated with Fe(III) reduction activity. This study reveals the capability of Sporomusa spp. in the reductive transformation of iron mineral and indicates the potential involvement of these organisms in iron and other mineral cycles in nature.

An iron corrosion-assisted H2-supplying system: a culture method for methanogens and acetogens under low H2 pressures.

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the "iron corrosion-assisted H2-supplying (iCH) system" by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifically enriched in the iCH cultures. We also observed selective enrichment of acetogen species by the iCH system (Acetobacterium spp. and Sporomusa spp.), whereas Clostridium spp. predominated in the high H2 cultures. These results demonstrate that the iCH system facilitates culture of anaerobic microorganisms under low H2 pressures, which will enable the selective culture of microorganisms adapting to low H2 environments.

Microbial Community Analysis of Anaerobic Enrichment Cultures Supplemented with Bacterial Peptidoglycan as the Sole Substrate.

Methanogenic microbial communities were enriched from rice paddy soil and anaerobic digester sludge using peptidoglycan purified from gram-negative Escherichia coli or gram-positive Micrococcus luteus as the sole substrate. Methane production data suggested the anaerobic degradation of peptidoglycan and also that peptidoglycan from E. coli had lower degradability. The community structures of enrichment cultures fed peptidoglycan from E. coli or M. luteus were similar, but distinctly different. A number of phylogenetically novel and uncultured bacteria, particularly in the phyla Bacteroidetes, WWE1, Armatimonadetes, and Verrucomicrobia, dominated the enrichment cultures, suggesting their involvement in anaerobic peptidoglycan degradation.

Rapid Enrichment and Isolation of Polyphosphate-Accumulating Organisms Through 4'6-Diamidino-2-Phenylindole (DAPI) Staining With Fluorescence-Activated Cell Sorting (FACS).

Screening for bacteria with abilities to accumulate valuable intracellular compounds from an environmental community is difficult and requires strategic methods. Combining the experimental procedure for phenotyping living cells in a microbial community with the cell recovery necessary for further cultivation will allow for an efficient initial screening process. In this study, we developed a strategy for the isolation of polyphosphate-accumulating organisms (PAOs) by combining (i) nontoxic fluorescence staining of polyphosphate granules in viable microbial cells and (ii) fluorescence-activated cell sorting (FACS) for the rapid detection and collection of target cells. To implement this screening approach, cells from wastewater sludge samples were stained with 4'6-diamidino-2-phenylindole (DAPI) to target cells with high polyphosphate (polyP) accumulation. We found a staining procedure (10 µg/ml of DAPI for 30 min) that can visualize polyP granules while maintaining viability for the majority of the cells (>60%). The polyP positive cells were recovered by FACS, purified by colony isolation and phylogenetically identified by 16S rRNA gene sequencing. Follow-up analysis confirmed that these isolates accumulate polyP, indicating that DAPI can be implemented in staining living cells and FACS can effectively and rapidly screen and isolate individual cells from a complex microbial community.

Isolation and Genomic Characterization of a Proteobacterial Methanotroph Requiring Lanthanides.

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.

Improved Isolation of Uncultured Anaerobic Bacteria using Medium Prepared with Separate Sterilization of Agar and Phosphate.

We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Conductive Iron Oxides Promote Methanogenic Acetate Degradation by Microbial Communities in a High-Temperature Petroleum Reservoir.

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Enhancement of methanogenesis by electric syntrophy with biogenic iron-sulfide minerals.

Recent studies have shown that interspecies electron transfer between chemoheterotrophic bacteria and methanogenic archaea can be mediated by electric currents flowing through conductive iron oxides, a process termed electric syntrophy. In this study, we conducted enrichment experiments with methanogenic microbial communities from rice paddy soil in the presence of ferrihydrite and/or sulfate to determine whether electric syntrophy could be enabled by biogenic iron sulfides. Although supplementation with either ferrihydrite or sulfate alone suppressed methanogenesis, supplementation with both ferrihydrite and sulfate enhanced methanogenesis. In the presence of sulfate, ferrihydrite was transformed into black precipitates consisting mainly of poorly crystalline iron sulfides. Microbial community analysis revealed that a methanogenic archaeon and iron- and sulfate-reducing bacteria (Methanosarcina, Geobacter, and Desulfotomaculum, respectively) predominated in the enrichment culture supplemented with both ferrihydrite and sulfate. Addition of an inhibitor specific for methanogenic archaea decreased the abundance of Geobacter, but not Desulfotomaculum, indicating that Geobacter acquired energy via syntrophic interaction with methanogenic archaea. Although electron acceptor compounds such as sulfate and iron oxides have been thought to suppress methanogenesis, this study revealed that coexistence of sulfate and iron oxide can promote methanogenesis by biomineralization of (semi)conductive iron sulfides that enable methanogenesis via electric syntrophy.