Journal of Microbiology

Journal Articles

Enhancing Seed Germination of Cremastra appendiculata: Screening and Identification of Four New Symbiotic Fungi in the Psathyrellaceae Family.: Zhangneng Pan, Jing Wang, Shanshan He, Haiyang Zhao, Xinyue Dong, Tao Feng, Yanyan Meng, Xiaojun Li; J. Microbiol. 2024;62(8):671-682. Published online June 28, 2024; DOI: https://doi.org/10.1007/s12275-024-00148-7

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Abstract: Several coprinoid fungi have been identified as promotors of Cremastra appendiculata seed germination, while others appear ineffective. This study aimed to discern which genera within the Psathyrellaceae family exhibit this capability and to identify the most effective coprinoid fungi for the cultivation of C. appendiculata. We collected 21 coprinoid fungi from diverse sources and symbiotically cultured them with C. appendiculata seeds. 9 fungi were found to induce seed germination and support seed development, specifically within the genera Coprinellus, Tulosesus, and Candolleomyces. In contrast, fungi that failed to promote germination predominantly belonged to the genera Coprinopsis and Parasola. Notably, four fungi-Coprinellus xanthothrix, Coprinellus pseudodisseminatus, Psathyrella singeri, and Psathyrella candolleana-were documented for the first time as capable of enhancing C. appendiculata seed germination. Strain 218LXJ-10, identified as Coprinellus radians, demonstrated the most significant effect and has been implemented in large-scale production, underscoring its considerable practical value. These findings contribute vital scientific insights for the conservation and sustainable use of C. appendiculata resources.

Crystal Structures of Plk1 Polo‑Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2‑Derived Peptide: Sujin Jung , Hye Seon Lee , Ho-Chul Shin , Joon Sig Choi , Seung Jun Kim , Bonsu Ku; J. Microbiol. 2023;61(8):755-764. Published online September 8, 2023; DOI: https://doi.org/10.1007/s12275-023-00071-3

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Abstract: Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4–E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S–S–pT–P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria: Uisang Yu , Jiha Kim , Seohui Park , Kyungyun Cho; J. Microbiol. 2023;61(6):627-632. Published online June 14, 2023; DOI: https://doi.org/10.1007/s12275-023-00056-2

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Abstract: Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

Effects of Continuous Straw Returning on Soil Functional Microorganisms and Microbial Communities: Yunpeng Guan , Meikang Wu , Songhao Che , Shuai Yuan , Xue Yang , Siyuan Li , Ping Tian , Lei Wu , Meiying Yang , Zhihai Wu; J. Microbiol. 2023;61(1):49-62. Published online January 26, 2023; DOI: https://doi.org/10.1007/s12275-022-00004-6

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9 Citations

Abstract: This study examined the changes in soil enzymatic activity, microbial carbon source metabolic diversity, and straw decomposition rates in paddy fields treated with 1, 2, or 3 years of straw returning (SR1–SR3). The soil’s ability to decompose straw and cellulolytic bacteria increased with the number of treatment years (1: 31.9% vs. 2: 43.9% vs. 3: 51.9%, P < 0.05). The numbers of Azotobacter, Nitrobacteria, cellulolytic bacteria, and inorganic phosphate bacteria increased progressively with the numbers of straw returning years. Cellulolytic bacteria and inorganic phosphate bacteria were significantly positively correlated with the decomposition rate (r = 0.783 and r = 0.375, P < 0.05). Based on 16S sequencing results, straw returning improved the microbial diversity of paddy soils by increasing unclassified bacteria and keeping dominant soil microorganism populations unchanged. The relative importance of individual microbial taxa was compared using random forest models. Proteobacteria, ammoniating bacteria, and potassium dissolving bacteria contributed to peroxidase activity. The significant contributors to phosphate monoesterase were Acidobacteriota, Desulfobacterota, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria. Proteobacteria, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria contributed to urease activity. Desulfobacterota, ammoniating bacteria, cellulolytic bacteria, and potassium-dissolving bacteria contributed to the neutral invertase activity. In conclusion, soil microbial community structure and function were affected within 2 years of straw returning, which was driven by the combined effects of soil organic carbon, available nitrogen, available potassium, and pH. With elapsing straw returning years, soil properties interacted with soil microbial communities, and a healthier soil micro-ecological environment would form.

Differences in the methanogen community between the nearshore and offshore sediments of the South Yellow Sea: Ye Chen , Yu Zhen , Jili Wan , Siqi Li , Jiayin Liu , Guodong Zhang , Tiezhu Mi; J. Microbiol. 2022;60(8):814-822. Published online July 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2022-2

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Abstract: The differences in methanogen abundance and community composition were investigated between nearshore and offshore sediments in the South Yellow Sea (SYS). Shannon, Simpson, and Chao1 indices revealed a higher diversity of methanogens in the nearshore sediments than in the offshore sediments. The Mann–Whitney U test demonstrated that the relative abundance of Methanococcoides was significantly higher in the offshore sediments, while the relative abundances of Methanogenium, Methanosarcina, Methanosaeta, Methanolinea, and Methanomassiliicoccus were significantly higher in the nearshore sediments (P < 0.05). The abundance of the mcrA gene in the nearshore sediments was significantly higher than that in the offshore sediments. Furthermore, a similar vertical distribution of the methanogen and sulfatereducing bacteria (SRB) abundances was observed in the SYS sediments, implying there is potential cooperation between these two functional microbes in this environment. Finally, total organic carbon (TOC) was significantly correlated with methanogen community composition.

Helicobacter pylori-mediated gastric pathogenesis is attenuated by treatment of 2-deoxyglucose and metformin: Hanfu Su , Eun-Jung Bak , Aeryun Kim , Kavinda Tissera , Jeong-Heon Cha , Sungil Jang; J. Microbiol. 2022;60(8):849-858. Published online June 22, 2022; DOI: https://doi.org/10.1007/s12275-022-2130-z

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Abstract: Helicobacter pylori infection causes chronic inflammation in the stomach, which is linked to the development of gastric cancer. The anti-inflammatory and anticancer effects of a glycolysis inhibitor 2-deoxyglucose (2DG) and an antidiabetic medication metformin (Met) have gotten attention. Using a Mongolian gerbil animal model, we investigated H. pylorimediated gastric pathogenesis and how this pathogenesis is influenced by 2DG and Met. Five-week-old male gerbils were infected with H. pylori strain 7.13. After 2 weeks of infection, gerbils were fed 2DG-containing food (0.03% w/w), Met-containing water (0.5% w/v), or both (Combi) for 2 (short-term) or 10 weeks (long-term). Gastric pathogenesis and host response to H. pylori infection were examined by macroscopic and histopathologic analysis of gerbils’ stomach. As a result, indicators of gastric pathogenesis by H. pylori infection including infiltration of polymorphonuclear neutrophils and lymphocytes, intestinal metaplasia, atrophy, and proliferation of gastric epithelial cells were attenuated by short-term administration of 2DG, Met, or Combi. When the infection was sustained for long-term, gastric pathogenesis in drug-treated gerbils was equivalent to that in untreated gerbils, with the exception that the infiltration of neutrophil was reduced by 2DG. Colonization of H. pylori in stomach was unaffected by both short- and long-term treatments. Our findings demonstrate that the progression of gastric pathogenesis induced by H. pylori infection can be attenuated by the shortterm individual or combinational treatment of 2DG and Met, implying that 2DG or Met could be considered as a treatment option for gastric diseases in the early stages of infection.

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae: Hye Yun Moon† , Gyu Hun Sim† , Hyeon Jin Kim , Keunpil Kim , Hyun Ah Kang; J. Microbiol. 2022;60(1):18-30. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1580-7

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Abstract: We evaluated the Cre-lox and CRISPR-Cas9 systems as markerrecycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4- based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

Function of Rhs proteins in porcine extraintestinal pathogenic Escherichia coli PCN033: Wenjia Lu , Jia Tan , Hao Lu , Gaoyan Wang , Wenqi Dong , Chenchen Wang , Xiaodan Li , Chen Tan; J. Microbiol. 2021;59(9):854-860. Published online August 12, 2021; DOI: https://doi.org/10.1007/s12275-021-1189-2

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Abstract: Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen that places severe burdens on public health and animal husbandry. There are many pathogenic factors in E. coli. The type VI secretion system (T6SS) is a nano-microbial weapon that can assemble quickly and inject toxic effectors into recipient cells when danger is encountered. T6SSs are encoded in the genomes of approximately 25% of sequenced Gram-negative bacteria. When these bacteria come into contact with eukaryotic cells or prokaryotic microbes, the T6SS assembles and secretes associated effectors. In the porcine ExPEC strain PCN033, we identified four classic rearrangement hotspot (Rhs) genes. We determined the functions of the four Rhs proteins through mutant construction and protein expression. Animal infection experiments showed that the Δrhs-1CT, Δrhs-2CT, Δrhs-3CT, and Δrhs-4CT caused a significant decrease in the multiplication ability of PCN033 in vivo. Cell infection experiments showed that the Rhs protein is involved in anti-phagocytosis activities and bacterial adhesion and invasion abilities. The results of this study demonstrated that rhs1, rhs3, and rh4 plays an important role in the interaction between PCN033 and host cell. Rhs2 has contribution to cell and mice infection. This study helps to elucidate the pathogenic mechanism governing PCN033 and may help to establish a foundation for further research seeking to identify potential T6SS effectors.

Different distribution patterns of microorganisms between aquaculture pond sediment and water: Lili Dai , Chengqing Liu , Liang Peng , Chaofeng Song , Xiaoli Li , Ling Tao; J. Microbiol. 2021;59(4):376-388. Published online February 25, 2021; DOI: https://doi.org/10.1007/s12275-021-0635-5

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23 Citations

Abstract: Aquatic microorganisms in the sediment and water column are closely related; however, their distribution patterns between these two habitats still remain largely unknown. In this study, we compared sediment and water microeukaryotic and bacterial microorganisms in aquaculture ponds from different areas in China, and analyzed the influencing environmental factors as well as the inter-taxa relationships. We found that bacteria were significantly more abundant than fungi in both sediment and water, and the bacterial richness and diversity in sediment were higher than in water in all the sampling areas, but no significant differences were found between the two habitats for microeukaryotes. Bacterial taxa could be clearly separated through cluster analysis between the sediment and water, while eukaryotic taxa at all classification levels could not. Spirochaetea, Deltaproteobacteria, Nitrospirae, Ignavibacteriae, Firmicutes, Chloroflexi, and Lentimicrobiaceae were more abundantly distributed in sediment, while Betaproteobacteria, Alphaproteobacter, Cyanobacteria, Roseiflexaceae, Dinghuibacter, Cryomorphaceae, and Actinobacteria were more abundant in water samples. For eukaryotes, only Cryptomonadales were found to be distributed differently between the two habitats. Microorganisms in sediment were mainly correlated with enzymes related to organic matter decomposition, while water temperature, pH, dissolved oxygen, and nutrient levels all showed significant correlation with the microbial communities in pond water. Intensive interspecific relationships were also found among eukaryotes and bacteria. Together, our results indicated that eukaryotic microorganisms are distributed less differently between sediment and water in aquaculture ponds compared to bacteria. This study provides valuable data for evaluating microbial distributions in aquatic environments, which may also be of practical use in aquaculture pond management.

Review

The osmotic stress response operon betIBA is under the functional regulation of BetI and the quorum-sensing regulator AnoR in Acinetobacter nosocomialis: Bindu Subhadra , Surya Surendran , Bo Ra Lim , Jong Sung Yim , Dong Ho Kim , Kyungho Woo , Hwa-Jung Kim , Man Hwan Oh , Chul Hee Choi; J. Microbiol. 2020;58(6):519-529. Published online May 27, 2020; DOI: https://doi.org/10.1007/s12275-020-0186-1

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Abstract: Adaptation to changing environmental conditions is crucial for the survival of microorganisms. Bacteria have evolved various mechanisms to cope with osmotic stress. Here, we report the identification and functional characterization of the osmotic stress response operon, betIBA, in Acinetobacter nosocomialis. The betIBA operon encodes enzymes that are important for the conversion of choline to the osmoprotectant, glycine betaine. The betIBA operon is polycistronic and is under the regulation of the first gene, betI, of the same operon. A bioinformatics analysis revealed the presence of a BetI-binding motif upstream of the betIBA operon, and electrophoretic mobility shift assays confirmed the specific binding of BetI. An mRNA expression analysis revealed that expression of betI, betB, and betA genes is elevated in a betIeletion mutant compared with the wild type, confirming that the autorepressor BetI represses the betIBA operon in A. nosocomialis. We further found that the betIBA operon is under the transcriptional control of the quorum-sensing (QS) regulator, AnoR in, A. nosocomialis. A subsequent analysis of the impact of BetI on expression of the QS genes, anoR and anoI, demonstrated that BetI acts as a repressor of anoR and anoI. In addition, it was noticed that the osmotic stress response regulator, OmpR might play an important role in controlling the expression of betIBA operon in A. nosocomialis. Collectively, these data demonstrate that QS and osmotic stress-response systems are correlated in A. nosocomialis and that the expression of genes in both systems is finely tuned by various feedback loops depending on osmolarity conditions.

Introductory Journal Article

[Editorial]Omics-based microbiome analysis in microbial ecology: from sequences to information: Jang-Cheon Cho; J. Microbiol. 2021;59(3):229-232.; DOI: https://doi.org/10.1007/s12275-021-0698-3

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Abstract: Microbial ecology is the study of microorganisms present in nature. It particularly focuses on microbial interactions with any biota and with surrounding environments. Microbial ecology is entering its golden age with innovative multi-omics
methods
triggered by next-generation sequencing technologies. However, the extraction of ecologically relevant information from ever-increasing omics data remains one of the most challenging tasks in microbial ecology. This special issue includes 11 review articles that provide an overview of the state of the art of omics-based approaches in the field of microbial ecology, with particular emphasis on the interpretation of omics data, environmental pollution tracking, interactions in microbiomes, and viral ecology.

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