Nonribosomal Peptide Synthetase Specific Genome Amplification Using Rolling Circle Amplification for Targeted Gene Sequencing.

Next-generation sequencing has transformed the acquisition of vast amounts of genomic information, including the rapid identification of target gene sequences in metagenomic databases. However, dominant species can sometimes hinder the detection of rare bacterial species. Therefore, a highly sensitive amplification technique that can selectively amplify bacterial genomes containing target genes of interest was developed in this study. The rolling circle amplification (RCA) method can initiate amplification from a single locus using a specific single primer to amplify a specific whole genome. A mixed cell suspension was prepared using Pseudomonas fluorescens ATCC17400 (targeting nonribosomal peptide synthetase [NRPS]) and Escherichia coli (non-target), and a specific primer designed for the NRPS was used for the RCA reaction. The resulting RCA product (RCP) amplified only the Pseudomonas genome. The NRPS was successfully amplified using RCP as a template from even five cells, indicating that the single-priming RCA technique can specifically enrich the target genome using gene-specific primers. Ultimately, this specific genome RCA technique was applied to metagenomes extracted from sponge-associated bacteria, and NRPS sequences were successfully obtained from an unknown sponge-associated bacterium. Therefore, this method could be effective for accessing species-specific sequences of NRPS in unknown bacteria, including viable but non-culturable bacteria.

Characterization of Biogenic PbS Quantum Dots.

Heavy metals in a polluted environment are toxic to life. However, some microorganisms can remove or immobilize heavy metals through biomineralization. These bacteria also form minerals with compositions similar to those of semiconductors. Here, this bioprocess was used to fabricate semiconductors with low energy consumption and cost. Bacteria that form lead sulfide (PbS) nanoparticles were screened, and the crystallinity and semiconductor properties of the resulting nanoparticles were characterized. Bacterial consortia that formed PbS nanoparticles were obtained. Extracellular particle size ranged from 3.9 to 5.5 nm, and lattice fringes were observed. The lattice fringes and electron diffraction spectra corresponded to crystalline PbS. The X-ray diffraction (XRD) patterns of bacterial PbS exhibited clear diffraction peaks. The experimental and theoretical data of the diffraction angles on each crystal plane of polycrystalline PbS were in good agreement. Synchrotron XRD measurements showed no crystalline impurity-derived peaks. Thus, bacterial biomineralization can form ultrafine crystalline PbS nanoparticles. Optical absorption and current-voltage measurements of PbS were obtained to characterize the semiconductor properties; the results showed semiconductor quantum dot behavior. Moreover, the current increased under light irradiation when PbS nanoparticles were used. These results suggest that biogenic PbS has band gaps and exhibits the general fundamental characteristics of a semiconductor.

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria.

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 104 clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.

Dysgonomonas alginatilytica sp. nov., an alginate-degrading bacterium isolated from a microbial consortium.

Gram-stain-negative, facultatively anaerobic, non-motile, non-spore-forming, rod-shaped bacterium, designated strain HUA-2T, was isolated from an alginate-degrading microbial consortium. Strain HUA-2T was related to Dysgonomonas capnocytophagoides JCM 16697T, Dysgonomonas macrotermitis JCM 19375T and Dysgonomonas mossii CCUG 43457T with 95.1 %, 94.1 % and 92.1 % 16S rRNA gene sequence similarity, respectively. The optimal growth temperature and pH for strain HUA-2T were 35 °C and pH 8.0, respectively. Enzyme production, major fermentation products from glucose, and the major cellular fatty acids were different from those of D. capnocytophagoides CCUG 17966T or other members of the genus Dysgonomonas. Therefore, strain HUA-2T is proposed to represent a novel species of the genus Dysgonomonas, for which we propose the name Dysgonomonas alginatilytica sp. nov. The type strain is HUA-2T ( = DSM 100214T = HUT 8134T).

Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid.

Thraustochytrid production of polyunsaturated fatty acids and xanthophylls have been generally sourced from crop-derived substrates, making the exploration of alternative feedstocks attractive since they promise increased sustainability and lower production costs. In this study, a distinct two-stage fermentation system was conceptualized for the first time, using the brown seaweed sugar mannitol as substrate for the intermediary biocatalyst Gluconobacter oxydans, an acetic acid bacterium, along with the marine thraustochytrid Aurantiochytrium sp. to produce the value-added lipids and xanthophylls. Jar fermenter culture resulted in seaweed mannitol conversion to fructose with an efficiency of 83 % by G. oxydans and, after bacteriostasis with sea salts, production of astaxanthin and docosahexaenoic acid by Aurantiochytrium sp. KH105. Astaxanthin productivity was high at 3.60 mg/L/day. This new system, therefore, widens possibilities of obtaining more varieties of industrially valuable products including foods, cosmetics, pharmaceuticals, and biofuel precursor lipids from seaweed fermentation upon the use of suitable thraustochytrid strains.

Development of a Transformation System for Nitratireductor sp.

Nitratireductor sp. OM-1 can accumulate butenoic acid, which is a short-chain unsaturated carboxylic acid utilized for chemical products. So far, we have predicted the thioesterase gene, te, as a candidate gene for butenoic acid biosynthesis, based on comparative transcriptome analysis. To confirm the function of te, the gene transfer system in Nitratireductor sp. OM-1 was required. Thus, in this study, we used electroporation as a transformation system and pRK415, a broad host range plasmid, and optimized the conditions. As a result, a maximum transformation efficiency of 7.9 × 104 colonies/µg DNA was obtained at 22.5 kV/cm. Moreover, an expression vector, pRK415-te, was constructed by insertion of te, which was successfully transferred into strain OM-1, using electroporation. The recombinant OM-1 strain produced butenoic acid at 26.7 mg/g of dried cell weight, which was a 254% increase compared to transformants harboring an empty vector. This is the first report of a gene transfer system for Nitratireductor sp., which showed that the te gene was responsible for butenoic acid production.

Whole genome sequence of Desulfovibrio magneticus strain RS-1 revealed common gene clusters in magnetotactic bacteria.

Magnetotactic bacteria are ubiquitous microorganisms that synthesize intracellular magnetite particles (magnetosomes) by accumulating Fe ions from aquatic environments. Recent molecular studies, including comprehensive proteomic, transcriptomic, and genomic analyses, have considerably improved our hypotheses of the magnetosome-formation mechanism. However, most of these studies have been conducted using pure-cultured bacterial strains of alpha-proteobacteria. Here, we report the whole-genome sequence of Desulfovibrio magneticus strain RS-1, the only isolate of magnetotactic microorganisms classified under delta-proteobacteria. Comparative genomics of the RS-1 and four alpha-proteobacterial strains revealed the presence of three separate gene regions (nuo and mamAB-like gene clusters, and gene region of a cryptic plasmid) conserved in all magnetotactic bacteria. The nuo gene cluster, encoding NADH dehydrogenase (complex I), was also common to the genomes of three iron-reducing bacteria exhibiting uncontrolled extracellular and/or intracellular magnetite synthesis. A cryptic plasmid, pDMC1, encodes three homologous genes that exhibit high similarities with those of other magnetotactic bacterial strains. In addition, the mamAB-like gene cluster, encoding the key components for magnetosome formation such as iron transport and magnetosome alignment, was conserved only in the genomes of magnetotactic bacteria as a similar genomic island-like structure. Our findings suggest the presence of core genetic components for magnetosome biosynthesis; these genes may have been acquired into the magnetotactic bacterial genomes by multiple gene-transfer events during proteobacterial evolution.

Visualization of Gene Reciprocity among Lactic Acid Bacteria in Yogurt by RNase H-Assisted Rolling Circle Amplification-Fluorescence In Situ Hybridization.

Recently, we developed an in situ mRNA detection method termed RNase H-assisted rolling circle amplification-fluorescence in situ hybridization (RHa-RCA-FISH), which can detect even short mRNA in a bacterial cell. However, because this FISH method is sensitive to the sample condition, it is necessary to find a suitable cell permeabilization and collection protocol. Here, we demonstrate its further applicability for detecting intrinsic mRNA expression using lactic acid bacteria (LAB) as a model consortium. Our results show that this method can visualize functional gene expression in LAB cells and can be used for monitoring the temporal transition of gene expression. In addition, we also confirmed that data obtained from bulk analyses such as RNA-seq or microarray do not always correspond to gene expression in individual cells. RHa-RCA-FISH will be a powerful tool to compensate for insufficient data from metatranscriptome analyses while clarifying the carriers of function in microbial consortia. By extending this technique to capture spatiotemporal microbial gene expression at the single-cell level, it will be able to characterize microbial interactions in phytoplankton-bacteria interactions.

Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.

Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.

Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification.

Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell's function in microbiota. In this report, we developed novel fluorescence in situ hybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.

Discovery of a Novel Gene Conferring Tellurite Tolerance Through Tellurite Reduction to Escherichia coli Transformant in Marine Sediment Metagenomic Library.

Metagenomic library construction using a marine sediment-enrichment was employed in order to recover tellurium from tellurite, a tellurium oxyanion, dissolved in water and then functional screening was performed to discover a novel gene related to tellurite reduction. Transmission electron microscopy (TEM) revealed the formation of intracellular Te crystals in Escherichia coli cells transformed with a specific DNA fragment from the marine sediment metagenome. The metagenome fragment was composed of 691 bp and showed low homology to known proteins. Phylogenetic analysis suggested that the metagenome fragment was related to Pseudomonas stutzeri. Cloning and expression of an open reading frame (ORF) on the metagenome fragment validated the role of the fragment in conferring tellurite resistance and tellurite-reducing activity to E. coli host cells. E. coli transformant containing the ORF1 showed resistance to 1 mM Na2TeO3. The optimal tellurite-reducing activity of cells containing the ORF1 was recorded at 37 °C and pH 7.0.

Metabolite Profile Analysis of Aurantiochytrium limacinum SR21 Grown on Acetate-based Medium for Lipid Fermentation.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.

Complete genome sequence of Nitratireductor sp. strain OM-1: A lipid-producing bacterium with potential use in wastewater treatment.

Reducing CO2 emissions is necessary to alleviate rising global temperature. Renewable sources of energy are becoming an increasingly important substitute for fossil fuels. An important step in this direction is the isolation of novel, technologically relevant microorganisms. Nitratireductor sp. strain OM-1 can convert volatile short-chain fatty acids in wastewater into 2-butenoic acid and its ester and can accumulate intracellularly esterified compounds up to 50% of its dried cell weight under nitrogen-depleted conditions. It is believed that a novel fatty acid biosynthesis pathway including an esterifying enzyme is encoded in its genome. In this study, we report the whole-genome sequence (4.8 Mb) of OM-1, which comprises a chromosome (3,977,827 bp) and a megaplasmid (857,937 bp). This sequence information provides insight into the genome organization and biochemical pathways of OM-1. In addition, we identified lipid biosynthesis pathways in OM-1, paving the way to a better understanding of its biochemical characterization.

Development of a cell surface display system in a magnetotactic bacterium, "Magnetospirillum magneticum" AMB-1.

Bacterial cell surface display is a widely used technology for bioadsorption and for the development of a variety of screening systems. Magnetotactic bacteria are unique species of bacteria due to the presence of magnetic nanoparticles within them. These intracellular, nanosized (50 to 100 nm) magnetic nanoparticles enable the cells to migrate and be manipulated by magnetic force. In this work, using this unique characteristic and based on whole-genomic and comprehensive proteomic analyses of these bacteria, a cell surface display system has been developed by expressing hexahistidine residues within the outer coiled loop of the membrane-specific protein (Msp1) of the "Magnetospirillum magneticum" (proposed name) AMB-1 bacterium. The optimal display site of the hexahistidine residues was successfully identified via secondary structure prediction, immunofluorescence microscopy, and heavy metal binding assay. The established AMB-1 transformant showed high immunofluorescence response, high Cd(2+) binding, and high recovery efficiency in comparison to those of the negative control when manipulated by magnetic force.

RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection.

RNA-primed rolling circle amplification (RPRCA) is a useful laboratory method for RNA detection; however, the detection of RNA is limited by the lack of information on 3'-terminal sequences. We uncovered that conventional RPRCA using pre-circularized probes could potentially detect the internal sequence of target RNA molecules in combination with RNase H. However, the specificity for mRNA detection was low, presumably due to non-specific hybridization of non-target RNA with the circular probe. To overcome this technical problem, we developed a method for detecting a sequence of interest in target RNA molecules via RNase H-assisted RPRCA using padlocked probes. When padlock probes are hybridized to the target RNA molecule, they are converted to the circular form by SplintR ligase. Subsequently, RNase H creates nick sites only in the hybridized RNA sequence, and single-stranded DNA is finally synthesized from the nick site by phi29 DNA polymerase. This method could specifically detect at least 10 fmol of the target RNA molecule without reverse transcription. Moreover, this method detected GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without background DNA amplification. Therefore, this method can potentially detect almost all types of RNA molecules without reverse transcription and reveal full-length sequence information.

Efficient conversion of mannitol derived from brown seaweed to fructose for fermentation with a thraustochytrid.

Macroalgae are a promising biomass feedstock for energy and valuable chemicals. Mannitol and alginate are the major carbohydrates found in the microalga Laminaria japonica (Konbu). To convert mannitol to fructose for its utilization as a carbon source in mannitol non-assimilating bacteria, a psychrophile-based simple biocatalyst (PSCat) was constructed using a psychrophile as a host by expressing mesophilic enzymes, including mannitol 2-dehydrogenase for mannitol oxidation, and NADH oxidase and alkyl hydroxyperoxide reductase for NAD+ regeneration. PSCat was treated at 40 °C to inactivate the psychrophilic enzymes responsible for byproduct formation and to increase the membrane permeability of the substrate. PSCat efficiently converted mannitol to fructose with high conversion yield without additional input of NAD+. Konbu extract containing mannitol was converted to fructose with hydroperoxide scavenging, inhibiting the mannitol dehydrogenase activity. Auranthiochytrium sp. could grow well in the presence of fructose converted by PSCat. Thus, PSCat is a potential carbohydrate converter for mannitol non-assimilating microorganism.

Isolation of High Carotenoid-producing Aurantiochytrium sp. Mutants and Improvement of Astaxanthin Productivity Using Metabolic Information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

Cytoplasmic ATPase involved in ferrous ion uptake from magnetotactic bacterium Magnetospirillum magneticum AMB-1.

A non-magnetic mutant of Magnetospirillum magneticum AMB-1 (NMA61), harboring a defective gene located in ORF4 (gene ID: amb4111) was generated by transposon mutagenesis. Biochemical characterization of the gene product of ORF4 revealed that it was localized in the cytoplasm and displayed ATPase activity. The ability of NMA61 to take up iron was severely compromised. Ferrous ion concentration in the medium decreased more with the wild-type than with NMA61, while the iron content in the cytoplasmic fraction of NMA61 was much lower than the wild-type strain. This cytoplasmic ATPase is essential for iron trafficking within M. magneticum AMB-1.

Improved methanization and microbial diversity during batch mode cultivation with repetition of substrate addition using defined organic matter and marine sediment inoculum at seawater salinity.

The activation of microbes, which are needed to initiate continuous methane production, can be accomplished by fed-batch methanization. In the present study, marine sediment inoculum was activated by batch mode methanization with repetition of substrate addition using defined organic matter from sugar, protein, or fat at seawater salinity to investigate the potential for application of the activation method to various types of saline waste and microbial community compositions. All substrates had methane potentials close to the theoretical value except for bovine serum albumin (BSA) whose methane potential was lower, but the maximum methane potential reached the value during repeated methanization. Beta diversity analysis revealed that substrate (especially BSA)-fed and non-fed cultures had distinct microbial community compositions. Bacterial members depended on substrate. Thus, marine sediment inocula activated via the methanization method can be used to effectively treat various types of saline waste.

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed.

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.