We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD2K2, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD2K2 component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD2K2 formation, effectively increased nitrogenase activity.

As the first step toward understanding how NADPH levels are regulated in Bacillus subtilis, we sought to obtain mutant strains with enhanced NADPH levels. Our previous study demonstrated that in a strain of B. subtilis expressing bacterial luciferase derived from Photorhabdus luminescens, artificially enhancing NADPH levels enhanced luciferase luminescence in the colonies. In this study, from a library of ethyl methanesulfonate-treated mutants, those with enhanced luciferase luminescence in colonies were isolated, and five isolates were further selected by luminescence in microplate culture. Finally, we measured intracellular NADPH levels of them and found that all the five strains had significantly enhanced NADPH levels compared to the parental strain. In addition, four strains significantly increased total NADP(H) levels. These results demonstrate the effectiveness of our strategy as a methodology for obtaining mutant strains useful for elucidating the mechanisms for regulation of NADPH levels in B. subtilis.

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 OTUs 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.

We have successfully isolated two novel compounds, 24R005A (1, C13H14O4) and 24R005B (2, C13H13ClO4), 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 ED50 values of 200 and 130 μM, respectively.

Phthalates esters (PAEs) are a kind of polymeric material additives widely been added into plastics to improve products' flexibility. It can easily cause environmental pollution which are hazards to public health. In this study, we isolated an efficient PAEs degrading strain, Janthinobacterium sp. E1, and determined its degradation effect of di-2-ethylhexyl phthalate (DEHP) under stress conditions. Strain E1 showed an obvious advantage in pollutants degradation under various environmental stress conditions. Degradation halo clearly occurred around the colony of strain E1 on agar plate supplemented with triglyceride. Strain E1's esterase is a constitutively expressed intracellular enzyme. The esterase purified from strain E1 showed a higher catalytic effect on short-chain PAEs than long-chain PAEs. The input of DEHP, DBP (dibutyl phthalate) and DMP (dimethyl phthalate) into the tested soil did not change the species composition of soil prokaryotic community, but altered the dominant species in specific environmental conditions. And the community diversity and richness decreased to a certain extent. However, the diversity and richness of the microbial community were improved after the contaminated soil was treated with the strain E1. Our results also suggested that strain E1 exhibited a tremendous potential in environmental bioremediation in the real environment, which provides a new insight into the elimination of the pollutants contamination in the urban environment.

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.

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.

Disc immuno-immobilization is a simple method for typing flagellar antigens from Salmonella enterica. In this study, we successfully adapted this method for Escherichia coli. All eleven strains tested were determined their antigens within 14 h from inoculation. This method improves the efficiency and speed, highlighting its usefulness in clinical laboratories.

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 YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D 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.

Genome modification would be useful for developing breeding techniques for haploid Zygosaccharomyces rouxii and natural hybrid allodiploid Zygosaccharomyces sp. yeast strains used in miso and soy sauce production. In this study, genome editing using CRISPR-Cas9 was attempted in Zygosaccharomyces sp. strains. Based on techniques in Saccharomyces cerevisiae, the Cas9 gene and guide RNA (gRNA) were expressed from the same plasmid. Targeting of the ZygoLEU2 gene of haploid Z. rouxii strain DA2 led to of a single-nucleotide insertion in the ORF, resulting in termination of translation at 10 amino acids. This single-base insertion was 3-bp upstream of the protospacer-associated motif (PAM) sequence, suggesting that it occurred during the repair process following the Cas9-induced double-strand break. The transformant was auxotrophic for leucine, verifying that genome editing using CRISPR-Cas9 had occurred. Application of the CRISPR-Cas9 system to allodiploid Zygosaccharomyces sp. strains, which have T- and P-subgenomes, resulted in transformants with base insertions or deletions upstream of the PAM sequence, or insertions of different subgenome sequences. Leucine-auxotrophic transformants were obtained in which the ORF of the ZygoLEU2 gene in both subgenomes were mutated. In some genome-edited strains, a significant region of one subgenome chromosome was missing. Lastly, we applied CRISPR-Cas9 to the gene encoding Hog1, a protein kinase involved in adaptation to high-salt and high-osmolarity conditions. Mutation of the HOG1 genes of both the T- and P-subgenomes by CRISPR-Cas9 significantly reduced growth in high salt and high osmolarity conditions.