{"title":"Secretory Expression of a Multifunctional Nuclease Nuc A in Bacillus licheniformis 2709.","authors":"Huimin Guo, Kefen Wang, Tongtong Zhang, Honglei Fang, Wei Hui, Huitu Zhang","doi":"10.2323/jgam.2025.05.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.05.001","url":null,"abstract":"<p><p>Serratia nuclease Nuc A is a non-specific nucleotide hydrolase that has been widely used in large-scale protein purification or eliminating nucleic acid contamination from purified proteins. To enhance the enzyme production, the Serratia nuclease gene was synthesized and expressed in Bacillus licheniformis 2709, a robust strain capable of secreting native and heterologous proteins selectively or non-selectively. To further increase the secretory expression level of the enzyme, different strong promoters and signal peptides were fused with the mature Nuc A-encoding gene at various genetic loci. The highest expression level of Nuc A was observed under the control of regulatory elements P<sub>aprE</sub>, which occur naturally in B. licheniformis 2709 for the alkaline protease (AprE) expression. Through maximizing the number of copies of P<sub>aprE-nucA</sub> expression cassette at different integration sites, the yield of nuclease Nuc A reached approximately 31954 U/mL after 60 hours of cultivation in shake flasks. The specific activity of the recombinant nuclease reached 1.58×107 U/mg, which is about 9 times higher than that expressed in Escherichia coli strain. Additionally, the recombinant Nuc A exhibited high catalytic activities in the pH range of 7-10. Furthermore, it was resistant to 0.2% SDS, 1.0 mM PMSF, and 0.4% Triton X-100. After 8 M Urea treatment, residual activity is measured. The high expression levels and positive characteristics of recombinant Nuc A provide an effective solution for large-scale production and industrial application of the nuclease.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144174138","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"PCR bias in 16S rRNA genes caused by GC content leads to insufficient detection of some abundant species in amplicon sequencing analyses of thermophilic microbial communities.","authors":"Shigeru Kawai, Katsumi Matsuura, Shawn Erin McGlynn","doi":"10.2323/jgam.2025.04.003","DOIUrl":"https://doi.org/10.2323/jgam.2025.04.003","url":null,"abstract":"<p><p>Amplicon sequencing is a widely used method for surveying biological diversity. However, the technique is disturbed by PCR bias leading to errors in community composition analyses. In this study, microbial community composition was evaluated in twenty-eight locations of hot spring water with temperatures between 87-48°C at Nakabusa Hot Springs, Japan, using amplicon sequencing analysis with the V4 region of the 16S rRNA gene. In discrepancy with the greenish color and the absorption spectra of the microbial samples, the relative abundance of amplicon sequence variants (ASVs) in the major photosynthetic organisms, Chloroflexus spp., were scarce in any sample when using the annealing temperature of 50°C in amplicon PCR. Changing the annealing temperature to 68ºC significantly improved the detection efficiency of Chloroflexus ASVs, and the obtained numbers were consistent with the presence of the photosynthetic pigments. The abundance of many other microbial ASVs was also dependent on the annealing temperature. The log ratio in the abundance of major ASVs between two annealing temperatures was correlated with the GC content of the 16S rRNA gene, suggesting that even some other major ASVs in the community are seriously affected by PCR bias due to the GC content. Combined usage of results from two different annealing temperatures, rather than a result using a single annealing temperature, seems to be a better way to obtain community structure information with less PCR bias in thermophilic organisms of high 16S rRNA GC content.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144142698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"CRISPR-Cas9 genome editing of miso and soy source yeast Zygosaccharomyces sp.","authors":"Tomoo Ogata, Kotori Koide, Shiori Kudou, Miu Suto, Kotaro Uehara, Teruya Kaneko","doi":"10.2323/jgam.2025.04.002","DOIUrl":"https://doi.org/10.2323/jgam.2025.04.002","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Identification of Escherichia coli flagellar antigen by disc immuno-immobilization.","authors":"Shiho Oyama, Masatoshi Fujihara","doi":"10.2323/jgam.2025.04.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.04.001","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143999619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Asmaa Ali Ahmed, Akiko Hida, Takahisa Tajima, Junichi Kato
{"title":"Identification and characterization of a methyl-accepting chemotaxis protein in Ralstonia pseudosolanacearum using chemically undefined materials.","authors":"Asmaa Ali Ahmed, Akiko Hida, Takahisa Tajima, Junichi Kato","doi":"10.2323/jgam.2025.03.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.03.001","url":null,"abstract":"<p><p>Ralstonia pseudosolanacearum is a plant-pathogenic bacterium that causes bacterial wilt in economically important crops. Chemotaxis is required for full virulence in R. pseudosolanacearum. R. pseudosolanacearum Ps29 possesses 20 methyl-accepting chemotaxis proteins (MCPs) and 2 MCP-like chemoreceptors. To understand the role of chemotaxis in plant infection, we are characterizing the functions of these 20 MCPs. Out of 20 MCPs, 8 MCPs have been characterized. To characterize the remaining MCPs, we deleted the 8 genes encoding characterized MCPs in R. pseudosolanacearum Ps29 to construct R. pseudosolanacearum D8. R. pseudosolanacearum D8 was examined for chemotactic responses to several chemically undefined materials including vegetable juices and tryptic soy broth (TSB) to find attractants. R. pseudosolanacearum D8 showed strong responses to green pepper and carrot juices and TSB. We constructed a mutant library of R. pseudosolanacearum D8 by deleting each of the MCP genes. Chemotaxis assays to TSB revealed that an MCP which we named McpD was responsible for sensing an attractant(s) in TSB. Because amino acids are the major constituents of TSB, we measured chemotactic responses of R. pseudosolanacearum D8 to 20 proteinogenic amino acids and found Asp and Glu as the major attractants of McpD and Cys as the minor attractant. R. pseudosolanacearum Ps29 can utilize Asp and Glu as sole carbon and nitrogen sources, suggesting that the role of McpD-mediated chemotaxis is finding growth substrates.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143639722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Production of p-anisaldehyde via whole-cell transformation using recombinant E. coli expressing trans-anethole oxygenase.","authors":"Zhikai Zhang, Qian Lin","doi":"10.2323/jgam.2025.02.001","DOIUrl":"https://doi.org/10.2323/jgam.2025.02.001","url":null,"abstract":"<p><p>p-Anisaldehyde, a fragrance and flavour with important roles in food, cosmetics, and drug industries, is currently synthesized through chemical methods. Production of p-anisaldehyde by chemical oxidation of trans-anethole in industry gives rise to excessive by-products and adverse environmental impacts, whereas biological process would address such problems. Here, we presented a process of biotransformation of trans-anethole for production of p-anisaldehyde. The tao gene encoding for trans-anethole oxygenase (TAO) from Paraburkholderia sp. MR185 was fused with a solubilization tag GST and ProS2, respectively. GST did not exhibit solubility enhancement effect, whereas fusion with ProS2 significantly improved TAO's soluble expression in E. coli and the fusion protein ProS2-TAO-Sil3K accounted for more than 40% of total soluble proteins. ProS2-TAO-Sil3K was purified by simple silica affinity and its activity did not require addition of NADH, NADPH, and FAD. Metal ions Co<sup>2+</sup>, Zn<sup>2+</sup>, Ni<sup>2+</sup>, and Cu<sup>2+</sup> displayed significant inhibition effect on TAO activity, and addition of Fe<sup>2+</sup> improved enzyme activity by 32.6%. After induction, engineered E. coli cells were used as whole-cell biocatalyst for transformation of trans-anethole, and the final concentration of p-anisaldehyde reached 10.18 mM (1.38 g/L), with the volumetric productivity of 0.11 g/L/h and conversion rate of 67.9%. These results reveal that the biosynthesis of p-anisaldehyde has a great potential in practice.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Determination of double bond configuration of 2-hydroxy-fatty acids and emendation of cellular fatty acid composition of Aureispira marina and Aureispira maritima.","authors":"Fuka Iriyama, Hirokazu Iida, Kazuyoshi Kawahara","doi":"10.2323/jgam.2024.09.001","DOIUrl":"10.2323/jgam.2024.09.001","url":null,"abstract":"<p><p>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-∆<sup>2</sup>-pentadecenol (∆<sup>2</sup>-iso-C<sub>16:1</sub>-ol) was prepared from D-2-hydroxy-15-methyl-∆<sup>3</sup>-hexadecenoic acid (D-2-OH-∆<sup>3</sup>-iso-C<sub>17:1</sub>) of the ceramide component, and analyzed by <sup>1</sup>H and <sup>13</sup>C NMR in comparison with ∆<sup>2</sup>-trans-hexadecenol (∆<sup>2</sup>-trans-n-C<sub>16:1</sub>-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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A DUF3844 domain-containing protein is required for vacuolar protein sorting in Schizosaccharomyces pombe.","authors":"Tomoaki Inagawa, Kazuma Ohkubo, Masahiro Watanabe, Tomotake Morita, Yujiro Higuchi, Hiromi Maekawa, Kaoru Takegawa","doi":"10.2323/jgam.2024.10.001","DOIUrl":"10.2323/jgam.2024.10.001","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142545832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Fauzi Akhbar Anugrah, I Nyoman Pugeg Aryantha, Rahmi Masita, Siti Zubaidah, Nur Izzati Mohd Noh
{"title":"Isolation of Bacterial Endophytes Associated with Cinchona ledgeriana Moens. and Their Potential in Plant-growth Promotion, Antifungal and Quinoline Alkaloids Production.","authors":"Fauzi Akhbar Anugrah, I Nyoman Pugeg Aryantha, Rahmi Masita, Siti Zubaidah, Nur Izzati Mohd Noh","doi":"10.2323/jgam.2024.09.002","DOIUrl":"10.2323/jgam.2024.09.002","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142501746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Microbial community analysis of sand filters used to treat mine water from a closed uranium mine.","authors":"Hiroshi Habe, Tomohiro Inaba, Tomo Aoyagi, Hidenobu Aizawa, Yuya Sato, Tomoyuki Hori, Keiko Yamaji, Yoshiyuki Ohara, Kenjin Fukuyama, Takuro Nishimura","doi":"10.2323/jgam.2024.08.001","DOIUrl":"10.2323/jgam.2024.08.001","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142289014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}