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.

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.

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene.

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).IMPORTANCE Methanotrophs have long been considered promising strains for biologically reducing methane from the environment and converting it into valuable products, because they can oxidize methane at ambient temperatures and pressures. Although several methodologies and tools for the genetic manipulation of methanotrophs have been developed, their mutagenic efficiency remains lower than that of tractable strains such as Escherichia coli Therefore, further improvements are still desired. The significance of our study is that we increased the efficiency of counterselection in M. capsulatus (Bath) by employing pheS*, which was newly constructed as a counterselectable marker. This will allow for the efficient production of gene-disrupted and gene-integrated mutants of M. capsulatus (Bath). We anticipate that this counterselection system will be utilized widely by the methanotroph research community, leading to improved productivity of methane-based bioproduction and new insights into methanotrophy.

Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media.

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

Specific Interaction between Redox Phospholipid Polymers and Plastoquinone in Photosynthetic Electron Transport Chain.

Redox phospholipid polymers added in culture media are known to be capable of extracting electrons from living photosynthetic cells across bacterial cell membranes with high cytocompatibility. In the present study, we identify the intracellular redox species that transfers electrons to the polymers. The open-circuit electrochemical potential of an electrolyte containing the redox polymer and extracted thylakoid membranes shift to positive (or negative) under light irradiation, when an electron transport inhibitor specific to plastoquinone is added upstream (or downstream) in the photosynthetic electron transport chain. The same trend is also observed for a medium containing living photosynthetic cells of Synechococcus elongatus PCC7942. These results clearly indicate that the phospholipid redox polymers extract photosynthetic electrons mainly from plastoquinone.

Extracellular electron transfer in acetogenic bacteria and its application for conversion of carbon dioxide into organic compounds.

Acetogenic bacteria (i.e., acetogens) produce acetate from CO2 during anaerobic chemoautotrophic growth. Because acetogens fix CO2 with high energy efficiency, they have been investigated as biocatalysts of CO2 conversion into valuable chemicals. Recent studies revealed that some acetogens are capable of extracellular electron transfer (EET), which enables electron exchange between microbial cells and extracellular solid materials. Thus, acetogens are promising candidates as biocatalysts in recently developed bioelectrochemical technologies, including microbial electrosynthesis (MES), in which useful chemicals are biologically produced from CO2 using electricity as the energy source. In microbial photoelectrosynthesis, a variant of MES technology, the conversion of CO2 into organic compounds is achieved using light as the sole energy source without an external power supply. In this mini-review, we introduce the general features of bioproduction and EET of acetogens and describe recent progress and future prospects of MES technologies based on the EET capability of acetogens.

Influence of Anode Potentials on Current Generation and Extracellular Electron Transfer Paths of Geobacter Species.

Geobacter species are capable of utilizing solid-state compounds, including anodic electrodes, as electron acceptors of respiration via extracellular electron transfer (EET) and have attracted considerable attention for their crucial role as biocatalysts of bioelectrochemical systems (BES's). Recent studies disclosed that anode potentials affect power output and anodic microbial communities, including selection of dominant Geobacter species, in various BES's. However, the details in current-generating properties and responses to anode potentials have been investigated only for a model species, namely Geobacter sulfurreducens. In this study, the effects of anode potentials on the current generation and the EET paths were investigated by cultivating six Geobacter species with different anode potentials, followed by electrochemical analyses. The electrochemical cultivation demonstrated that the G. metallireducens clade species (G. sulfurreducens and G. metallireducens) constantly generate high current densities at a wide range of anode potentials (≥-0.3 or -0.2 V vs. Ag/AgCl), while the subsurface clades species (G. daltonii, G. bemidjensis, G. chapellei, and G. pelophilus) generate a relatively large current only at limited potential regions (-0.1 to -0.3 V vs. Ag/AgCl). The linear sweep voltammetry analyses indicated that the G. metallireducens clade species utilize only one EET path irrespective of the anode potentials, while the subsurface clades species utilize multiple EET paths, which can be optimized depending on the anode potentials. These results clearly demonstrate that the response features to anode potentials are divergent among species (or clades) of Geobacter.

Electrochemical Detection of Circadian Redox Rhythm in Cyanobacterial Cells via Extracellular Electron Transfer.

Recent research on cellular circadian rhythms suggests that the coupling of transcription-translation feedback loops and intracellular redox oscillations is essential for robust circadian timekeeping. For clarification of the molecular mechanism underlying the circadian rhythm, methods that allow for the dynamic and simultaneous detection of transcription/translation and redox oscillations in living cells are needed. Herein, we report that the cyanobacterial circadian redox rhythm can be electrochemically detected based on extracellular electron transfer (EET), a process in which intracellular electrons are exchanged with an extracellular electrode. As the EET-based method is non-destructive, concurrent detection with transcription/translation rhythm using bioluminescent reporter strains becomes possible. An EET pathway that electrochemically connected the intracellular region of cyanobacterial cells with an extracellular electrode was constructed via a newly synthesized electron mediator with cell membrane permeability. In the presence of the mediator, the open circuit potential of the culture medium exhibited temperature-compensated rhythm with approximately 24 h periodicity. Importantly, such circadian rhythm of the open circuit potential was not observed in the absence of the electron mediator, indicating that the EET process conveys the dynamic information regarding the intracellular redox state to the extracellular electrode. These findings represent the first direct demonstration of the intracellular circadian redox rhythm of cyanobacterial cells.

Isolation of acetogenic bacteria that induce biocorrosion by utilizing metallic iron as the sole electron donor.

Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH4 production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion.

Microbial interspecies electron transfer via electric currents through conductive minerals.

In anaerobic biota, reducing equivalents (electrons) are transferred between different species of microbes [interspecies electron transfer (IET)], establishing the basis of cooperative behaviors and community functions. IET mechanisms described so far are based on diffusion of redox chemical species and/or direct contact in cell aggregates. Here, we show another possibility that IET also occurs via electric currents through natural conductive minerals. Our investigation revealed that electrically conductive magnetite nanoparticles facilitated IET from Geobacter sulfurreducens to Thiobacillus denitrificans, accomplishing acetate oxidation coupled to nitrate reduction. This two-species cooperative catabolism also occurred, albeit one order of magnitude slower, in the presence of Fe ions that worked as diffusive redox species. Semiconductive and insulating iron-oxide nanoparticles did not accelerate the cooperative catabolism. Our results suggest that microbes use conductive mineral particles as conduits of electrons, resulting in efficient IET and cooperative catabolism. Furthermore, such natural mineral conduits are considered to provide ecological advantages for users, because their investments in IET can be reduced. Given that conductive minerals are ubiquitously and abundantly present in nature, electric interactions between microbes and conductive minerals may contribute greatly to the coupling of biogeochemical reactions.

Regulation of the cyanobacterial circadian clock by electrochemically controlled extracellular electron transfer.

There is growing awareness that circadian clocks are closely related to the intracellular redox state across a range of species. As the redox state is determined by the exchange of the redox species, electrochemically controlled extracellular electron transfer (EC-EET), a process in which intracellular electrons are exchanged with extracellular electrodes, is a promising approach for the external regulation of circadian clocks. Herein, we discuss whether the circadian clock can be regulated by EC-EET using the cyanobacterium Synechococcus elongatus PCC7942 as a model system. In vivo monitoring of chlorophyll fluorescence revealed that the redox state of the plastoquionone pool could be controlled with EC-EET by simply changing the electrode potential. As a result, the endogenous circadian clock of S. elongatus cells was successfully entrained through periodically modulated EC-EET by emulating the natural light/dark cycle, even under constant illumination conditions. This is the first example of regulating the biological clock by electrochemistry.

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.

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.

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.

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.

Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals.

Methanogenesis is an essential part of the global carbon cycle and a key bioprocess for sustainable energy. Methanogenesis from organic matter is accomplished by syntrophic interactions among different species of microbes, in which interspecies electron transfer (IET) via diffusive carriers (e.g. hydrogen and formate) is known to be the bottleneck step. We report herein that the supplementation of soil microbes with (semi)conductive iron-oxide minerals creates unique interspecies interactions and facilitates methanogenesis. Methanogenic microbes were enriched from rice paddy field soil with either acetate or ethanol as a substrate in the absence or presence of (semi)conductive iron oxides (haematite or magnetite). We found that the supplementation with either of these iron oxides resulted in the acceleration of methanogenesis in terms of lag time and production rate, while the supplementation with an insulative iron oxide (ferrihydrite) did not. Clone-library analyses of 16S rRNA gene fragments PCR-amplified from the enrichment cultures revealed that the iron-oxide supplementation stimulated the growth of Geobacter spp. Furthermore, the addition of a specific inhibitor for methanogenesis suppressed the growth of Geobacter spp. These results suggest that Geobacter grew under syntrophic association with methanogens, and IET could occur via electric currents through (semi)conductive iron-oxide minerals (termed 'electric syntrophy'). Given the ubiquity of conductive minerals in nature, such energetic interactions may occur widely in soil and sediments and can be used to develop efficient bioenergy processes.

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%.

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%.

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.