Hot-springs are accessible yet extreme environments for studying thermophilic microbial communities and their ecological interactions. Here, we isolated and characterized two thermophilic filamentous Leptolyngbya-related cyanobacterial strains, Akita and Seranma, from geothermal hot springs in Japan. The two strains differed in morphology and pigmentation: L. sp. Akita formed linear blue-green filaments, whereas L. sp. Seranma formed loosely coiled brown filaments. Whole-genome sequencing and comparative genomic analyses revealed strain- and strain-group-specific gene enrichment patterns related to nitrogen metabolism, metal binding, mobile genetic elements, and the photosynthetic apparatus, as well as candidate genes putatively associated with thermal tolerance. L. sp. Seranma also showed light-dependent pigmentation changes and retained an rfpABC gene cluster, indicating light-responsive phenotypic and genomic variation. Intestinal 16S rRNA gene metabarcoding of sympatric Buergeria tadpoles detected Leptolyngbya-assigned reads, particularly in B. japonica from Seranma hot-spring. These reads showed a close correspondence to L. sp. Seranma, suggesting possible exposure to, or recent ingestion of, L. sp. Seranma-related cyanobacteria. Together, these findings provide genomic and metabarcoding evidence for a potential trophic association between thermophilic cyanobacteria and amphibian larvae in hot-spring ecosystems.

Interfaces that convert digitally defined signals into biological analog functions (D/A biointerfaces) remain underdeveloped compared with biosensors that digitize biological analog information (A/D biointerfaces). Here, we report direct electrochemical control of insulin secretion through membrane-potential modulation using a stable tetraethyl orthosilicate (TEOS)-incorporated PEDOT:PSS electrode that supports long-term culture of pancreatic β-cells as a model endocrine system. β-cells expressing luminescent insulin (iGL cells) were cultured on the electrode, and secretion dynamics were monitored by luminescence imaging during potential application. Application of +500 mV relative to the resting electrode potential (REST) rapidly reduced luminescence, indicating insulin release within seconds. Following this potential application, cells remained viable after additional culture for 3 days, retaining insulin synthesis and the ability to respond to subsequent induction. Fluorescence imaging with the membrane-potential dye FluoVolt™ suggested that the application of REST +200 mV induced depolarization comparable to that generally reported for activation of voltage-gated Ca2+ channels. Following a brief induction period, reapplication of the REST potential halted secretion. Alternating induction and REST pulses enabled stepwise modulation of cumulative insulin release. These results provide a basic operational principle for D/A biointerfaces that translate digitally programmed electrochemical inputs into analog, graded hormonal outputs.

Asbestos use has been banned in many countries; nevertheless, it is still present in old buildings, emphasizing the need to generate rapid and highly sensitive detection methods. In this study, we developed a novel asbestos detection method based on amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA). The method involved conjugating donor and acceptor beads with asbestos-binding proteins and mixing them with test samples. When both beads were simultaneously bound to an asbestos fiber, they were brought in close proximity to generate an amplified luminescent signal, enabling the detection of as little as 1 & micro;g of asbestos. The method successfully identified 0.1 % (w/w) asbestos contamination in talc and mortar, and in Rockwool following appropriate pretreatment. In contrast, detection performance was reduced in calcium silicate and gypsum, and the current protocol was not readily applicable to perlite and vermiculite. Further studies are needed to evaluate matrix-dependent interference and optimize pretreatment conditions. Although the method has three limitations-material-dependent applicability, semi-quantitative signal output influenced by fiber surface accessibility, and lack of morphological information-it provides a rapid, high-throughput screening approach that complements conventional quantitative and morphological asbestos analyses. This novel approach expands the scope of AlphaLISA-based detection to hazardous inorganic materials.

Harmful algal blooms negatively impact the ecosystem and fisheries in affected areas. Eutrophication is a major factor contributing to bloom occurrence, and phosphorus is particularly important in limiting the growth of bloom-forming algae. Although algae efficiently utilize orthophosphate (Pi) as a phosphorous source over other molecular forms, Pi is often limited in the marine environment. While uptake and utilization of soluble inorganic and organic phosphorous by bloom-forming algae has been extensively studied, the details of geochemical and biological phosphorous cycling remain to be elucidated. Here, we report for the first time that the bloom-forming alga Heterosigma akashiwo can phagocytose bacteria and grow under phosphate-depleted conditions. The addition of Vibrio comitans to Pi-depleted H. akashiwo enabled the alga propagate to high cell densities, whereas other bacterial strains had only a minor effect. Importantly, V. comitans accumulates polyphosphate-a linear polymer of Pi-at high levels. The extent of algal proliferation induced by the addition of Vibrio species and polyphosphate-accumulating Escherichia coli correlated strongly with their polyphosphate content, indicating that bacterial polyphosphate served as an alternative PO4 3- source for H. akashiwo. The direct uptake of polyphosphate-accumulating bacteria through algal phagocytosis may represent a novel biological phosphorous-cycling pathway in marine ecosystems. The role of polyphosphate-accumulating marine bacteria as a hidden phosphorous source required for bloom formation warrants further investigation.

Hot springs represent one of the most accessible yet extreme environments on the Earth’s surface. We isolated and characterized two novel cyanobacteria strains of genus Leptolyngbya , L . sp. Akita and L . sp. Seranma from geothermal hot-springs in Japan. These strains showed distinct morphological features; L . sp. Akita exhibited linear filamentous structures and blue-green when cultured under white light, while L . sp. Seranma exhibited coiled structures and brown color under the same conditions. Whole-genome sequencing and comprehensive genomic analyses of their genomes identified acquisition of gene clusters related to metabolic processes which may associated with the strain specific phenotypes. In addition, intestinal metagenomic analysis of tadpoles ( Buergeria buergeri and B . japonica ) sympatrically living with these strains in the hot-springs suggested that the tadpoles utilized these Leptolyngbya as temporal or regular foods in high temperature environments. These findings provide insights into the ecological significance of these cyanobacteria in extreme environments and their potential applications in biotechnology and ecological conservation of primary consumers including amphibians. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, https://ror.org/00hhkn466, 18K06365, 21K06125 New Energy and Industrial Technology Development Organization, JPNP17005 Yuzawa Geopark Academic Research Encouragement Subsidy, 196(FY2021), 308(FY2022)

A phosphite (Pt)-dependent biological containment strategy, achieved by introducing a Pt-metabolic pathway and disrupting endogenous phosphate transporters, renders Escherichia coli growth strictly dependent on Pt, a compound rarely detected in natural environments, thereby preventing unintended environmental spread. In this study, we demonstrated that expression of phosphate regulon (Pho regulon) genes was markedly upregulated in a Pt-dependent E. coli strain due to the elimination of phoU, a negative regulator of the Pho regulon, along with the high-affinity phosphate transporter pstSCAB. However, further genetic modification of this strain for detailed analysis was hindered by the presence of multiple antibiotic resistance markers. To overcome this limitation, we reconstructed a Pt-dependent E. coli strain using CRISPR-Cas12a-mediated genome editing, enabling the removal of the antibiotic resistance markers and facilitating subsequent genetic manipulation. Using this strain, we disrupted the PhoBR two-component regulatory genes and found that deletion of phoBR alleviated the constitutive overexpression of Pho regulon genes and partially restored growth of the Pt-dependent strain. These findings provide mechanistic insights and technical advances for the refinement and practical application of Pt-dependent biocontainment strategy.

The development of cell-based devices using mammalian cells is becoming increasingly feasible. To remotely control such sophisticated devices, an interface between digital computer/internet networks and cellular/organ networks is essential. This study explores the electrochemical manipulation of insulin secretion-a regulatory hormone for the control of blood sugar levels-using pancreatic β cells as a model. iGL cells, expressing insulin fused with Gaussia Luciferase (INS-GLase), were directly cultured on a custom-made cell culture device coated with a transparent poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) electrode. Luminescence imaging was employed to evaluate insulin secretion in response to applied potentials. Results showed that insulin secretion could be induced by regulating membrane potential through an applied potential. The addition of nicardipine, an L-type voltage-dependent Ca2+ channel inhibitor, suppressed insulin secretion, suggesting the involvement of Ca2+ channels in this electrochemical system. Additionally, changes in membrane potential were directly visualized with the membrane potential-sensitive dye FluoVolt™, which confirmed both the forced depolarization and the forced restoration of the membrane potential to its non-excited state upon potential application to the electrode. The reported electrochemical technique, in which cells are directly cultured on an electrode, offers significant promise for designing advanced bio-hybrid systems that integrate cellular functions with digital networks.

Synthetic biology approaches enable the creation of promising chassis for practical application in various fields, though engineering of microbial metabolism often imposes a metabolic burden, potentially driving adaptive evolution during long-term cultivation. A previously established phosphite (Pt)-dependent metabolic system has proven to be an effective strategy for the containment of genetically engineered microorganisms, although its implementation accompanied a slight growth retardation. Here, we investigated the effect of long-term serial passaging cultivation on the Pt-dependent strain of Synechococcus elongatus PCC 7942, RH714. Compared with the originally constructed RH714, the passaged population of RH714 exhibited improved growth and a higher rate of Pt consumption in culture medium. Sequence analysis revealed point mutations within the introduced htxBCDE transporter genes, which are required for selective incorporation of Pt as a phosphorus nutrition. Introduction of the mutated gene cluster into S. elongatus PCC 7942 reproduced the traits of the passaged RH714 population, suggesting that these genetic changes enhance Pt transport activity and account for the observed phenotypes. Disruption of endogenous phosphate (Pi) transporter genes in the strains expressing the mutated htxBCDE-ptxD cluster abolished growth in Pi-containing medium, suggesting that the mutations in the transporter genes did not alter substrate specificity toward Pi. These results indicated that long-term passage cultivation developed an optimized mutant capable of efficient proliferation under the Pt metabolizing conditions without compromising its biocontainment capability.

Phosphite dehydrogenase (PtxD) catalyzes NAD+-dependent oxidation of phosphite (Pt) to phosphate (Pi), offering various biotechnological applications, such as the creation of Pt-dependency for the biological containment of genetically modified organisms. Previously, we established a Pt-dependent cyanobacterial strain (RH714) by expressing PtxD and a reduced phosphorous compound-specific transporter (HtxBCDE) in Synechococcus elongatus PCC 7942 devoid of its endogenous Pi transporters. This strain demonstrated strict Pt dependency but failed to grow in unbuffered BG-11 medium supplemented with 2 % CO2 owing to medium acidification below approximately pH 6.5. The present study aimed to overcome this limitation by passaging the RH714 strain in an unbuffered growth medium, resulting in mutants capable of growing without buffering. The mutant strains carried a Gly157Ser mutation in the Rossmann fold domain of PtxD, leading to approximately five- and eight-fold higher Km values for NAD+ and Pt, respectively, compared with the wild-type enzyme. Interestingly, PtxDG157S exhibited enhanced resistance to nitrate, a major component of BG-11, suggesting that reduced substrate affinity mitigates nitrate inhibition at lower pH levels. Further kinetic analysis revealed that nitrate inhibits wild-type PtxD through an uncompetitive mechanism, targeting the enzyme-substrate complex at an allosteric site. Consequently, the PtxDG157S mutation reduces nitrate binding, facilitating sustained growth of Pt-dependent strains under conditions without pH buffering. These findings imply that PtxDG157S could significantly enhance the applicability of Pt-dependent cyanobacterial strain.

Biosilicification is the process by which organisms incorporate soluble, monomeric silicic acid, Si(OH)4, in the form of polymerized insoluble silica, SiO2. Although the mechanisms underlying eukaryotic biosilicification have been intensively investigated, prokaryotic biosilicification has only recently begun to be studied. We previously reported that biosilicification occurs in the gram-positive, spore-forming bacterium Bacillus cereus, and that silica is intracellularly deposited on the spore coat as a protective coating against acids, although the underlying mechanism is not yet fully understood. In eukaryotic biosilicifying organisms, such as diatoms and siliceous sponges, several relevant biomolecules are embedded in biogenic silica (biosilica). These biomolecules include peptides, proteins, and long-chain polyamines. In this study, we isolated organic compounds embedded in B. cereus biosilica to investigate the biomolecules involved in the prokaryotic biosilicification process and identified long-chain polyamines with a chemical structure of H2N-(CH2)4-[NH-(CH2)3]n-NH2 (n: up to 55). Our results demonstrate the common presence of long-chain polyamines in different evolutionary lineages of biosilicifying organisms, i.e., diatoms, siliceous sponges, and B. cereus, suggesting a common mechanism underlying eukaryotic and prokaryotic biosilicification.

A novel artificial insulin receptor was developed, that can self-assemble on a gold surface and alter its structure in response to insulin recognition via partial domains of the intrinsic insulin receptor.The candidates for the artificial insulin receptor were designed by fusing the αCT segment and L1CR domain of the insulin receptor with a gold-binding peptide to have self-assembling abilities on a gold surface.The proteins were termed 3GαL and αL3G, based on the order of these domains, and expressed in mammalian cells.A quartz crystal microbalance technique confirmed the ability of both proteins to self-assemble on the gold surface.Electrochemical impedance spectroscopy measurements using gold electrodes modified with these proteins revealed that 3GαL altered its structure in response to insulin recognition, even on a gold surface, confirming that it works as an artificial insulin receptor that selfassembles on a gold surface.We expect that 3GαL will contribute to the development of various biosensors that utilize gold surfaces as insulin-recognition elements.

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnapTM) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnapTM were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.

Indonesia is renowned as an agricultural powerhouse, ranking first globally in oil palm production. This prominence in agriculture leads to the consistent generation of agro-industrial waste, notably Palm Oil Mill Effluent (POME). Effectively addressing these waste concerns is important due to their adverse impacts on aquatic ecosystems and the nation’s health and economy. Anthropogenic wastewater with excessive phosphorus content can trigger eutrophication and toxic algal blooms, posing environmental risks and potentially precipitating a future clean water crisis. Thus, a comprehensive approach is necessary to restore the environment and biogeochemical cycles. Treatment efforts involving bioremediation agents aim to recycle organic and inorganic pollutants in the environment. Photosynthetic organisms like plants and microalgae serve as effective bioremediation agents, capable of absorbing excess phosphorus. They can utilize phosphate as an energy source to boost biomass. Integrating these bioremediation agents with bioengineering technology optimizes the treatment efficacy while simultaneously producing valuable biomass for products and bioenergy. This review article explores photosynthetic organisms’ multifunctional role as phosphorus bioremediation agents for wastewater treatment, minimizing environmental pollutant impacts, and providing biomass for fertilizers, polymers, bioplastics, and renewable energy. Furthermore, this study unveils opportunities for future technological advancements in this field.

In the practical scale of cyanobacterial cultivation, the golden algae Poterioochromonas malhamensis is a well-known predator that causes devastating damage to the culture, referred to as pond crash. The establishment and maintenance of monoculture conditions are ideal for large-scale cultures. However, this is a difficult challenge because microbial contamination is unavoidable in practical-scale culture facilities. In the present study, we unexpectedly observed the pond crash phenomenon during the pilot-scale cultivation of Synechococcus elongatus PCC 7942 using a 100-L photobioreactor. This was due to the contamination with P. malhamensis, which probably originated from residual fouling. Interestingly, we found that S.elongatus PCC 7942 can alter its morphological structure when subjected to continuous grazing pressure from predators, resulting in cells that were more than 100 times longer than those of the wild-type strain. These hyper-elongated S.elongatus PCC 7942 cells had mutations in the genes encoding FtsZ or Ftn2 which are involved in bacterial cell division. Importantly, the elongated phenotype remained stable during cultivation, enabling S.elongatus PCC 7942 to thrive and resist grazing. The cultivation of the elongated S.elongatus PCC 7942 mutant strain in a 100-L pilot-scale photobioreactor under non-sterile conditions resulted in increased cyanobacterial biomass without encountering pond crash. This study demonstrates an efficient strategy for cyanobacterial cell culture in practical-scale bioreactors without the need for extensive decontamination or sterilization of the growth medium and culture facility, which can contribute to economically viable cultivation and bioprocessing of microalgae.

Biological containment is a biosafety strategy that prevents the dispersal of genetically modified organisms in natural ecosystems. We previously established a biocontainment system that makes bacterial growth dependent on the avail-ability of phosphite (Pt), an ecologically rare form of phosphorus (P), by introducing Pt metabolic pathway genes and disrupting endogenous phosphate and organic phosphate transporter genes. Although this system proved highly effective, horizontal gene transfer (HGT) mediated recovery of a P transporter gene is considered as a potential pathway to abolish the Pt-dependent growth, resulting in escape from the containment. Here, we assessed the risk of HGT driven escape using the Pt-dependent cyanobacterium Synechococcus elongatus PCC 7942. Transformation experiments revealed that the Pt-dependent strain could regain phosphate transporter genes from the S. elongatus PCC 7942 wild-type genome and from the genome of the closely related strain, S. elongatus UTEX 2973. Transformed S. elongatus PCC 7942 became viable in a phosphate-containing medium. Meanwhile, transformation of the Synechocystis sp. PCC 6803 genome or environmental DNA did not yield escape strains, suggesting that only genetic material derived from phylogenetically-close species confer high risk to generate escape. Eliminating a single gene necessary for natural competence from the Pt-dependent strain reduced the escape occurrence rate. These results demonstrate that natural competence could be a potential risk to destabilize Pt-dependence, and therefore inhibiting exogenous DNA uptake would be effective for enhancing the robustness of the gene disruption-dependent biocontainment. (c) 2023, The Society for Biotechnology, Japan. All rights reserved.

Phosphite dehydrogenase (PtxD) is a promising enzyme for NAD(P)H regeneration. To expand the usability of PtxD, we cloned, expressed, and analyzed PtxD from the marine cyanobacterium Cyanothece sp. ATCC 51142 (Ct-PtxD). Ct-PtxD exhibited maximum activity at pH 9.0°C and 50°C and high stability over a wide pH range of 6.0–10.0. Compared to previously reported PtxDs, Ct-PtxD showed increased resistance to salt ions such as Na + , K + , and NH 4 + . It also exhibited high tolerance to organic solvents such as ethanol, dimethylformamide, and methanol when bound to its preferred cofactor, NAD + . Remarkably, these organic solvents enhanced the Ct-PtxD activity while inhibiting the PtxD activity of Ralstonia sp. 4506 (Rs-PtxD) at concentrations ranging from 10% to 30%. Molecular electrostatic potential analysis showed that the NAD + -binding site of Ct-PtxD was rich in positively charged residues, which may attract the negatively charged pyrophosphate group of NAD + under high-salt conditions. Amino acid composition analysis revealed that Ct-PtxD contained fewer hydrophobic amino acids than other PtxD enzymes, which reduced the hydrophobicity and increased the hydration of protein surface under low water activity. We also demonstrated that the NADH regeneration system using Ct-PtxD is useful for the coupled chiral conversion of trimethylpyruvic acid into L -tert-leucine using leucine dehydrogenase under high ammonium conditions, which is less supported by the Rs-PtxD enzyme. These results imply that Ct-PtxD might be a potential candidate for NAD(P)H regeneration in industrial applications under the reaction conditions containing salt and organic solvent.

Objectives This study aimed to investigate whether chromatography using an ExoPUA column, an affinity column for phospholipid membranes, could potentially serve as an efficient, rapid, scalable, and reproducible method for purifying small extracellular vesicles (sEVs). Results We used the ExoPUA column connected to a fast-performance liquid chromatography system. One-step chromatographic purification of sEVs from culture supernatant using the ExoPUA protocol resulted in an 82 ± 16-fold increase in purity with a yield of 38 ± 5% of sEVs. The purified sEVs contained CD9, CD63, TSG101, and miRNA (miR-21), but not the endoplasmic reticulum protein Calnexin. Transmission electron microscopy indicated that the purified sEVs were intact. The purification performance of the ExoPUA protocol showed superior results in terms of yield compared to that of the differential ultracentrifugation method, the most commonly used method for purifying sEVs in laboratories, and purity compared to that of the DEAE chromatography protocol. Conclusion The sEVs were effectively purified in the bind-elute mode and the ExoPUA column can be refreshed and sterilized with sodium hydroxide (NaOH), having high potential for multiple sEV purification in a scalable and industrial manner.

Microalgae are promising cell factories for producing value-added products. Large-scale microalgal cultivation suffers from invasion by contaminating microorganisms. Since most contaminating organisms cannot utilize phosphite as a unique phosphorus source, phosphite-utilizing ability may provide a growth advantage against contaminating organisms and solve this problem. Studies showed that microorganisms, typically unable to metabolize phosphite, can utilize phosphite by expressing exogenous phosphite dehydrogenase. Here, we constructed Cyanidioschyzon merolae strains introduced with the phosphite dehydrogenase gene, ptxD, from Ralstonia sp. 4506. The ptxD-introduced strains grew in a phosphite-dependent manner, with the phosphite-related growth rate almost matching that with phosphate as sole phosphorus source.

Asbestos contamination in common materials, including powdered talc used in manufacturing cosmetics, pharma-ceuticals, and baby powder, is of great public concern. The use of polarized light microscopy (PLM) for asbestos detection is limited because it cannot detect fibers with diameters in the nano range. Furthermore, the use of electron microscopy (EM), the preferred method for determining asbestos contamination in talc, is hindered by a rather complicated and laborious process. Fluorescence microscopy (FM) is a simple analytical tool in mod-ern life sciences, especially given its superior sensitivity and selectivity; however, it is rarely used for inorganic materials. Previously, we demonstrated that airborne asbestos fibers were fluorescently visualized under FM us -ing fluorescently labeled asbestos-binding proteins. Here, we evaluated the FM method's feasibility in detecting fine asbestos fibers in powdered talc. The limit of detection of the fiber diameter of asbestos was approximately 0.06 & mu;m using FM. The FM method's sensitivity surpassed that of PLM and approached that of EM, and thus, it can detect nano-scale fibers. Notably, the FM method enabled the selective visualization of asbestos fibers, including those hidden beneath numerous talc particles. A point counting method using FM images enabled a semi-quantitative analysis of 0.01-1.0% (w/w) asbestos contamination in talc.

Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide.