Kusaya shows a high preservability due to the microorganism-derived antibiotics contained in kusaya gravy, which is important for kusaya manufacturing. However, the antimicrobial compounds and its producing bacteria, as well as the antimicrobial activity of the kusaya gravy itself, have remained unknown. In this study, we isolated antibiotic-producing bacteria of the genus Streptomyces from kusaya gravy from Hachijojima and found that they produced antibacterial substances against various fungi and bacteria. In addition, we demonstrated that kusaya gravy itself shows antimicrobial activity, which was consistent with that of the isolates. This is the first report to directly indicate that kusaya gravy contains microorganism-derived antibiotics, which are assumed to be produced by actinomycetes.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Polyamide 4 (PA4) is expected to solve the issue of marine plastic pollution due to its excellent mechanical properties and biodegradability. In this study, to reveal the mechanism of PA4 biodegradation in the marine environment, we isolated 5 strains of PA4-degrading bacteria belonging to Aliiglaciecola, Dasania, and Pseudophaeobacter from a marine environment. The isolated 5 strains are novel PA4-degrading bacteria that are phylogenetically distinct from those isolated in previous studies. In addition, we compared the PA4-degrading activities and structures of the PA4-degrading enzymes secreted by the 5 strains and PA4-degrading strains isolated in our previous study. The PA4-degrading activity in the supernatant of the cultivation solutions differed among the strains. Native-PAGE and zymography using a polyacrylamide gel containing a PA4 emulsion demonstrated that PA4-degrading enzymes are classified into no less than three types of structures. These results suggested that marine PA4-degrading bacteria have multiple PA4-degrading enzymes. Our findings will contribute to a better understanding of the microbial degradation of PA4 in the marine environment.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3'-phosphoadenosine-5'-phosphate (PAP) and leaves 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Our group previously acquired a yeast 3',5'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YND displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YND to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

Fusarium meridionale is one of the pathogens causing maize ear rot, it produce bioactive secondary metabolites may threaten humans food safty, however, the production mechanism of the secondary metabolites and their interaction with maize ear remains poorly understood. To facilitate related studies, we sequenced and assembled the genome of F. meridionale strain JX18-4. The size of F. meridionale JX18-4 genome is 37.11 Mbp, include four nuclear chromosome contigs that consists of 11920 coding genes and one mitochondrial contig. 95.64% gene synteny collinearity was found between the assembly and the reference genomes F. graminearum strain PH-1. Compared to the sequences of seconary matabolism gene clusters sequences reported previously, the stain JX18-4 was predicted potential producing 8 clusters, including nivalenol, zearalenone, aurofusarin, fusarielin, fusaristatin A, fusarin, fusarubin and butenolide. This study aims to reveal the molecular mechanism of secondary metabolites producing, and the genomic information of JX18-4 will provide resources for the study of biological control mechanisms and plant-microbe interactions.

Protein trafficking to vacuoles in plants and fungi, and to lysosomes in animals, is essential for the maintenance of cellular homeostasis. In Saccharomyces cerevisiae, the vacuolar protein sorting (VPS) pathway has been well studied by using vacuolar carboxypeptidase Y (CPY) as a model, and many VPS genes have been identified. By contrast, the vacuolar protein trafficking pathway in Schizosaccharomyces pombe remains poorly understood. In this study, we identified a novel VPS gene (SPBC1709.03) in S. pombe that is broadly conserved in fungi, but not in S. cerevisiae. Owing to its DUF3844 domain of unknown function, the gene was named vps3844. Disruption mutants of vps3844 had defects in both CPY sorting and incorporation of FM4-64 dye into the vacuolar membrane. Partial deletion analysis of the Vps3844 protein revealed that, within the DUF3844 domain, the region comprising amino acids 354 to 380 is important for protein trafficking to the vacuole. Our findings represent the first report of a VPS gene involved in vacuolar transport that is conserved in fungi, particularly S. pombe, but lacks representation in S. cerevisiae.

For centuries, quinoline alkaloids from the tree bark of Cinchona ledgeriana (C. ledgeriana) have been used in the treatment of malaria. However, unsustainable harvesting and poor growth conditions greatly limit its use as raw materials. Since plant endophytes are known to contribute to the physiology of the host and its metabolism for survival, this study showed the potential of endophytes isolated from C. ledgeriana roots in promoting the germination of Catharathus roseus (C. roseus) seedlings and the biosynthesis of quinoline alkaloid. In this present study, we found that the Enterobacteriaceae family comprised the majority of the bacterial community, with Klebsiella pneumoniae being the most abundant species at the C. ledgeriana roots. Characterization of culturable bacterial endophytes from the C. ledgeriana roots showed that all the isolates displayed plant growth-promoting factors and antifungal activities. Interestingly, chromatographic analyses led to the identification of the quinoline alkaloids producing Achromobacter xylosoxidans (A. xylosoxidans) A1. Moreover, the co-cultures of A. xylosoxidans A1, Cytobacillus solani (C. solani) A3, and Klebsiella aerogenes A6 increased the fresh and dry weight of the C. roseus seedlings. These results suggest that these bacterial endophytes may enhance quinine and quinidine production as well as the growth of the plant host.

To enhance the value of surimi, efforts have been made to develop a fermentation method with lactic acid bacteria (LAB) to proteolyze fish protein. However, fermenting unheated surimi poses a spoilage risk due to its high bacterial content. Surimi heat treatment can prevent spoilage, but gel formation induced by heating introduces another technical issue: it hinders uniform fermentation. Thus, this study aims to observe the proteolysis and enhance the functionality of seafood product through lactic acid fermentation of kamaboko, a heated surimi. Upon analyzing the kamaboko fermented with Lactobacillus helveticus JCM1004, we observed that LAB produced protease, resulting in the degradation of myosin heavy chain and actin during fermentation. Lactic acid fermentation significantly augmented the peptide content of kamaboko, subsequently elevating the angiotensin Ⅰ-converting enzyme (ACE) inhibitory activity in 200-fold diluted extract of fermented kamaboko to approximately 70% and higher. Notably, our investigation revealed that proteolysis was confined to the surface of kamaboko, as evidenced by SDS-PAGE analysis. This observation implies that the surface area of kamaboko influences the ACE inhibitory activity. Through a comparative analysis of various bacterial strains, we demonstrated that the increase in ACE inhibitory activity is contingent on the protease generated by LAB. These results suggest that LAB-mediated proteolysis of fish proteins liberates bioactive peptides, thereby manifesting in the ACE inhibitory activity. In summary, this study underscores that the fermentation of kamaboko employing proteolytic LAB holds promise in the development of novel functional seafood products.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 β-1,3-glucanase.

The culture filtrates of the predominant bacterial strains isolated from soil samples have been shown to increase the microbial colony counts on agar plates used for the isolation of uncultured bacteria. One of the factors in the culture filtrates responsible for this increase was identified to be superoxide dismutase (SOD). The generation of reactive oxygen species (O, HO, and ・OH) was detected from conventional laboratory agar media. The use of agar media supplemented with radical scavengers (SOD, catalase, ascorbic acid, or rutin) effectively increased the colony counts and kinds of microbial strains that grew from soil samples. Taxonomical studies on these isolates revealed new taxa for phylum Actinomycetota; one family, three genera, and nine species were newly described. One of the strains, Patulibacter minatonensis KV-614 belonging to the new family Patulibacteraceae, was isolated on agar medium supplemented with SOD. P. minatonensis KV-614 represents a novel lineage within the phylum Actinomycetota. A polymerase chain reaction (PCR) study using specific primers for the detection of strains related to the genus Patulibacter, order Solirubrobacterales, showed a high distribution frequency, with detection in over 70% of the soil samples tested. These data suggest that the use of radical scavengers may facilitate the isolation of some hitherto-uncultivated microorganisms widely distributed in soil.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The K value and k of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s respectively. The K value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

In Streptomyces pristinaespiralis, AfsKRS system has differential regulation for PI and PII component biosynthesis of pristinamycin, but it is unknown whether S-adenosylmethionine (SAM) plays an important role in the AfsK-AfsR-AfsS signal transduction cascade during pristinamycin production. The possible target of exogenous SAM in the AfsKRS system and the biological role of SAM during the production of PI and PII were investigated using three mutantsΔafsK,ΔafsR andΔafsS defective in signal cascade pathway of AfsKRS. It was found that external SAM had a significant activation of PI production (1.85-fold increase) but had no obvious effect on PII production in the original strain F618 with the normal response of AfsKRS regulation. Addition of SAM resulted in a similar increase in pristinamycin yield in the mutant with defective afsK or afsR, but induced more crucial activation of PI biosynthesis than PII biosynthesis both in ΔafsK (1.65-fold and 1.15-fold increase respectively) and ΔafsR (1.27-fold and 1.09-fold increase respectively). Exogenous SAM only significantly enhanced PII production in ΔafsS (1.1-fold increase). These results could provide valuable insights into the regulatory function of the AfsKRS system in S. pristinaespiralis.

Aureispira marina is a marine bacterium with gliding motility isolated from the southern coastline of Thailand. It contained ceramide as a major cellular lipid composed of saturated or unsaturated branched chain 2-hydroxy-fatty acid and sphingosine. The structure of unsaturated 2-hydroxy-fatty acid was investigated in our previous study, but the geometric configuration of the double bond remained unclear. In the present study, 14-methyl-∆-pentadecenol (∆-iso-C-ol) was prepared from D-2-hydroxy-15-methyl-∆-hexadecenoic acid (D-2-OH-∆-iso-C) of the ceramide component, and analyzed by H and C NMR in comparison with ∆-trans-hexadecenol (∆-trans-n-C-ol) derived from commercially available D-sphingosine. From the coupling constants of protons in the double bond and the chemical shift value of allylic carbon, the configuration of the double bond was determined as trans. Since the structure of 2-hydroxy-fatty acids was clarified, cellular fatty acids of A. marina and A. maritima, another species of the genus Aureispira, were reexamined, and the description on the cellular fatty acid composition of the genus Aureispira in the previous papers (Hosoya et al., 2006, Int. J. System. Evol. Microbiol., 56, 2931-2935; Hosoya et al., 2007, Int. J. System. Evol. Microbiol., 57, 1948-1951) lacking the description of 2-hydroxy-fatty acids was emended.

Rapid sand filters (RSFs) are employed in a drinking water treatment to remove undesirable elements such as suspended solids and dissolved metal ions. At a closed uranium (U) mine site, two sets of tandemly linked paired RSF systems (RSF1-RSF2 and RSF1-RSF3) were utilized to remove iron and manganese from mine water. In this study, a 16S rRNA-based amplicon sequencing survey was conducted to investigate the core microbes within the RSF system treating the mine water. In RSF1, two operational taxonomic units (OTUs) related to methanotrophic bacteria, Methylobacter tundripaludum (relative abundance: 18.1%) and Methylovulum psychrotolerans (11.5%), were the most and second most dominant species, respectively, alongside iron-oxidizing bacteria. The presence of these OUTs in RSF1 can be attributed to the microbial community in the inlet mine water, as the three most abundant OTUs in the mine water also dominated RSF1. Conversely, in both RSF2 and RSF3, Nevskia sp., previously isolated from the Ytterby mine manganese oxide producing ecosystem, became dominant, although known manganese-oxidizing bacterial OTUs were not detected. In contrast, a unique OTU related to Rhodanobacter sp. was the third most abundant (8.0%) in RSF1, possibly due to selective pressure from the radionuclide-contaminated environment during RSF operation, as this genus is known to be abundant at nuclear legacy waste sites. Understanding the key bacterial taxa in RSF system for mine water treatment could enhance the effectiveness of RSF processes in treating mine water from closed U mines.

Naphthalene is a persistent environmental pollutant for its potential teratogenic, carcinogenic and mutagenic effects. In this study, 10 strains of bacteria capable of degrading naphthalene were isolated from crude-oil contaminated soil. Among them, Pseudomonas plecoglossicida 2P exhibited prominent growth with 1000 mg/L naphthalene as the sole carbon source and degraded 94.15% of naphthalene in 36 h. Whole genome sequencing analysis showed that P. plecoglossicida 2P had a total of 22 genes related to naphthalene degradation, of which 8 genes were related to the salicylic acid pathway only, 5 genes were related to the phthalic acid pathway only, 8 genes were common in both the salicylic acid and phthalic acid pathways, and 1 gene was related to the gentisic acid pathway. P. plecoglossicida 2P was applied in a two-phase partition bioreactor (TPPB) to degrade naphthalene in wastewater. The optimal operating conditions of the reactor were obtained through response surface optimization: initial naphthalene concentration (C) =1600 mg/L, bacterial liquid concentration (OD) = 1.3, and polymer-to-wastewater mass ratio (PWR) = 2%. Under these conditions, the naphthalene degradation rate was 98.36% at 24 h. The degradation kinetics were fitted using the Haldane equation with a high coefficient of determination (R=0.94). The present study laid foundations for naphthalene degradation mechanism of genus Pseudomonas and its potential application in TPPB.

Most cyanobacterial genomes possess more than two copies of genes encoding cyAbrBs (cyanobacterial AbrB-like proteins) having an AbrB-like DNA-binding domain at their C-terminal region. Accumulating data suggest that a wide variety of metabolic and physiologic processes are regulated by cyAbrBs. In this study, we investigated the function of the essential gene cyabrB1 (sll0359) in Synechocystis sp. PCC 6803 by using CRISPR interference technology. The conditional knockdown of cyabrB1 caused increases of cyAbrB2 transcript and protein levels. However, the effect of cyabrB1 knockdown on global gene expression profile was quite limited compared to the previously reported profound effect of knockout of cyabrB2. Among 24 up-regulated genes, 16 genes were members of the divergently transcribed icfG and sll1783 operons related to carbon metabolism. The results of this and previous studies indicate the different contributions of two cyAbrBs to transcriptional regulation of genes related to carbon, hydrogen and nitrogen metabolism. Possession of a pair of cyAbrBs has been highly conserved during the course of evolution of the cyanobacterial phylum, suggesting physiological significance of transcriptional regulation attained by their interaction.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of better SAM biosensing systems.

We have successfully isolated two novel compounds, 24R005A (1, CHO) and 24R005B (2, CHClO), from Streptomyces sp. 24R005, using fish (anchovy) powder as a medium. In this study, we evaluated the use of fish (anchovy) powder as a fermentation material for producing bioactive compounds. Spectroscopic analyses revealed that the two compounds share a common skeletal structure. However, each compound contains unique branched side chains. Furthermore, compounds 1 and 2 exhibit moderate radical-scavenging activity for 1,1-diphenyl-2-picrylhydrazyl (DPPH), with ED values of 200 and 130 μM, respectively.

Zn-deficiency, a global health challenge affects one-third of the world population. Zn-biofertilizer offer an efficient and cost-effective remedy. As Zn-biofertilizer can improve plant growth and grain's Zn-content ensuring improved dietary Zn-supply. This study sought to understand how silver and TiO nanoparticles in the rhizosphere affect the activity of Zn-solubilization bacteria (ZSB) and plant growth. Two ZSB strains Bacillus sp. D-7 and Pseudomonas sp. D-117 with excellent Zn-solubilization efficiency of 254 and 260%, respectively were isolated and characterized using polyphasic characterization including 16S rRNA gene sequencing to formulate an effective Zn-biofertilizer. The plant growth promoting activity of this biofertilizer in Mung bean was checked in the presence and absence of various doses of TiO and Ag-NPs and was compared with plant grown without biofertilizer. The change in rate of seed germination, vegetative growth (shoot and root length, fresh and dry weight), photosynthetic pigment and Zn-content was checked. Lower doses of nanomaterials (50 and 100 mg kg⁻¹ soil) slightly promoted the plant growth compared to control. While, higher doses (200 and 400 mg kg⁻¹ soil) inhibited the growth. A maximum decrease of shoot length, root length, fresh-weight, and dry-weight of 57.1, 53.9, 53.1, and 10.4% respectively was observed with 400 mg kg⁻¹ of Ag-NPs. However, in the presence of ZSB, the decrease at the same Ag-NP concentration was 41.6, 31.5, 27.4, and 6.6, respectively. These results strongly suggest that Zn-solubilizing bacteria improve resilience to nanoparticles toxicity and helps in Zn fortification in Mung bean even under nanomaterial stress.