Journal of Microbiology

Volume 60(7); July 2022

Journal Articles

[Protocol] Development of DNA aptamers specific for small therapeutic peptides using a modified SELEX method: Jaemin Lee , Minkyung Ryu , Dayeong Bae , Hong-Man Kim , Seong-il Eyun , Jeehyeon Bae , Kangseok Lee; J. Microbiol. 2022;60(7):659-667. Published online June 22, 2022; DOI: https://doi.org/10.1007/s12275-022-2235-4

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Abstract: Aptamers are short single-stranded DNA or RNA oligonucleotides capable of binding with high affinity and specificity to target molecules. Because of their durability and ease of synthesis, aptamers are used in a wide range of biomedical fields, including the diagnosis of diseases and targeted delivery of therapeutic agents. The aptamers were selected using a process called systematic evolution of ligands by exponential enrichment (SELEX), which has been improved for various research purposes since its development in 1990. In this protocol, we describe a modified SELEX method that rapidly produces high aptamer screening yields using two types of magnetic beads. Using this method, we isolated an aptamer that specifically binds to an antimicrobial peptide. We suggest that by conjugating a small therapeutic-specific aptamer to a gold nanoparticle-based delivery system, which enhances the stability and intracellular delivery of peptides, aptamers selected by our method can be used for the development of therapeutic agents utilizing small therapeutic peptides.

Description of Corynebacterium poyangense sp. nov., isolated from the feces of the greater white-fronted geese (Anser albifrons): Qian Liu , Guoying Fan , Kui Wu , Xiangning Bai , Xi Yang , Wentao Song , Shengen Chen , Yanwen Xiong , Haiying Chen; J. Microbiol. 2022;60(7):668-677. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-2089-9

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Abstract: Two novel Gram-positive, non-spore-forming, facultatively anaerobic, non-motile, and short rods to coccoid strains were isolated from the feces of the greater white-fronted geese (Anser albifrons) at Poyang Lake. The 16S rRNA gene sequences of strains 4H37-19T and 3HC-13 shared highest identity to that of Corynebacterium uropygiale Iso10T (97.8%). Phylogenetic and phylogenomic analyses indicated that strains 4H37-19T and 3HC-13 formed an independent clade within genus Corynebacterium and clustered with Corynebacterium uropygiale Iso10T. The average nucleotide identity and digital DNA-DNA hybridization value between strains 4H37-19T and 3HC-13 and members within genus Corynebacterium were all below 95% and 70%, respectively. The genomic G + C content of strains 4H37-19T and 3HC-13 was 52.5%. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine, and phosphatidyl inositol mannosides (PIM) were the major polar lipids, with C18:1ω9c, C16:0, and C18:0 as the major fatty acids, and MK-8 (H4), MK-8(H2), and MK-9(H2) as the predominant respiratory quinones. The major whole cell sugar was arabinose, and the cell wall included mycolic acids. The cell wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The polyphasic taxonomic data shows that these two strains represent a novel species of the genus Corynebacterium, for which the name Corynebacterium poyangense sp. nov. is proposed. The type strain of Corynebacterium poyangense is 4H37-19T (=GDMCC 1.1738T = KACC 21671T).

Brachybacterium kimchii sp. nov. and Brachybacterium halotolerans subsp. kimchii subsp. nov., isolated from the Korean fermented vegetables, kimchi, and description of Brachybacterium halotolerans subsp. halotolerans subsp. nov.: Yujin Kim , Yeon Bee Kim , Juseok Kim , Joon Yong Kim , Tae Woong Whon , Won-Hyong Chung , Eun-Ji Song , Young-Do Nam , Se Hee Lee , Seong Woon Roh; J. Microbiol. 2022;60(7):678-688. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-1581-6

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Abstract: Two Gram-stain-positive, oxidase-negative, catalase-positive, and coccus-shaped bacterial strains, designated CBA3104T and CBA3105T, were isolated from kimchi. Strain CBA3104T and CBA3105T grew at 10–35°C (optimum, 25°C and 30°C, respectively), at pH 6.0–8.5 (optimum, pH 6.5), and in the presence of 0–15% (w/v) NaCl (optimum, 5%). A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CBA3104T formed a distinct phylogenetic lineage within the genus Brachybacterium whereas strain CBA3105T was closely positioned with Brachybacterium halotolerans MASK1Z-5T. The 16S rRNA gene sequence similarity between strains CBA3104T and CBA3105T was 99.9%, but ANI and dDDH values between strains CBA3104T and CBA3105T were 93.61% and 51.5%, respectively. Strain CBA3104T showed lower ANI and dDDH values than species delineation against three closely related strains and type species of the genus Brachybacterium, however, strain CBA3105T showed 96.63% ANI value and 69.6% dDDH value with Brachybacterium halotolerans MASK1Z-5T. Among biochemical analysis results, strain CBA3104T could uniquely utilize bromo-succinic acid whereas only strain CBA3105T was positive for alkaline phosphatase and α-fucosidase among two novel strains, closely related strains, and type species of the genus Brachybacterium. Compared with strain CBA3105T and Brachybacterium halotolerans JCM 34339T, strain CBA3105T was differentially positive for acid production of D-arabinose, D-adonitol, and potassium 5-ketogluconate and enzyme activity of β-glucuronidase. Both strains contained menaquinone-7 as the dominant quinone. The cell-wall peptidoglycan of two novel strains contained meso-diaminopimelic acid. The major fatty acids of strains CBA3104T and CBA3105T were anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The major polar lipids of both strains were phosphatidylglycerol and diphosphatidylglycerol. Strain CBA3104T possessed a uniquely higher abundance of tRNA (97 tRNAs) than four Brachybacterium strains used for comparative taxonomic analysis (54–62 tRNAs). Both the CBA3104T and CBA3105T strain harbored various oxidoreductase, transferase, hydrolase, and lyase as strain-specific functional genes compared to closely related strains and Brachybacterium type species. The results of biochemical/physiological, chemotaxonomic, and genomic analyses demonstrated that strains CBA3104T and CBA3105T represent a novel species of the genus Brachybacterium and a novel subspecies of B. halotolerans, respectively, for which the names Brachybacterium kimchii sp. nov. and B. halotolerans subsp. kimchii subsp. nov. are proposed. The type strains of the novel species and the novel subspecies are CBA3104T (= KCCM 43417T = JCM 34759T) and CBA3105T (= KCCM 43418T = JCM 34760T), respectively.

Whole-genome sequencing analysis of Shiga toxin-producing Escherichia coli O22:H8 isolated from cattle prediction pathogenesis and colonization factors and position in STEC universe phylogeny: Wanderson Marques Da Silva , Mariano Larzabal , Flavia Figueira Aburjaile , Nahuel Riviere , Luisina Martorelli , James Bono , Ariel Amadio , Angel Cataldi; J. Microbiol. 2022;60(7):689-704. Published online June 22, 2022; DOI: https://doi.org/10.1007/s12275-022-1616-z

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Abstract: Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo. Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2dact, a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen.

Yeast polyubiquitin unit regulates synaptonemal complex formation and recombination during meiosis: Min-Kyung Jo , Kiwon Rhee , Keun Pil Kim , Soogil Hong; J. Microbiol. 2022;60(7):705-714. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-2204-y

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Abstract: Ubiquitin is highly conserved in most eukaryotes and involved in diverse physiological processes, including cell division, protein quality control, and protein degradation mediated by the ubiquitin-proteasome system after heat shock, glucose-starvation, and oxidative stress. However, the role of the ubiquitin gene UBI4, which contains five consecutive head-to-tail ubiquitin repeats, in meiosis has not been investigated. In this study, we show that the Saccharomyces cerevisiae polyubiquitin precursor gene, UBI4, is required to promote synaptonemal complex (SC) formation and suppress excess doublestrand break formation. Moreover, the proportion of Zip1 polycomplexes, which indicate abnormal SC formation, in cells with a mutation in UBI4 (i.e., ubi4Δ cells) is higher than that of wild-type cells, implying that the UBI4 plays an important role in the early meiotic prophase I. Interestingly, although ubi4Δ cells rarely form full-length SCs in the pachytene stage of prophase I, the Zip3 foci are still seen, as in wild-type cells. Moreover, ubi4Δ cells proficiently form crossover and noncrossover products with a slight delay compared to wild-type cells, suggesting that UBI4 is dispensable in SCcoupled recombination. Our findings demonstrate that UBI4 exhibits dual functions that are associated with both positive and negative roles in SC formation and recombination during meiosis.

Isolation of a novel Lactiplantibacillus plantarum strain resistant to nitrite stress and its transcriptome analysis: Chae Young Kwon , Kyoung Jin Choi , Dongeun Yong , Ji-Eun Kim , Sang Sun Yoon; J. Microbiol. 2022;60(7):715-726. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-2221-x

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Abstract: Nitric oxide (NO) is a reactive nitrogen species (RNS) that plays a vital role in regulating inflammatory processes. Under abnormal conditions, excessive NO levels can promote the oxidation of cellular components, which may cause or exacerbate diseases such as hypertension, cardiovascular dysfunction, and inflammatory bowel disease (IBD). Previous studies have shown that reducing NO levels in the lumen can attenuate the clinical symptoms of IBD. Thus, we aimed to identify bacteria that can reduce RNS and that can be used as valuable probiotics. In this study, we isolated bacteria resistant to nitrite stress from human feces and used 16S and whole-genome sequencing to identify them as Lactiplantibacillus plantarum LP7 (LP7). The ability to survive at high nitrite levels and to decrease them was greater in the LP7 strain than in the reference strain L. plantarum ATCC14917 (ATCC- 14917). To characterize the LP7 genome in more detail, we performed a comparative genome analysis. However, the unique genes that directly confer the ability to withstand nitrite stress were not present in the LP7 genome. Furthermore, we performed transcriptomic analysis of LP7 and ATCC14917 cells treated with nitrite. We found that the expression levels of genes involved in the cell division process were induced in LP7, which showed a more regular rod-shape than ATCC- 14917. This could explain why LP7 can survive better than ATCC14917 under nitrite stress. Based on its ability to survive better in nitrite stress and decrease nitrite concentration, we suggest that LP7 could be a valuable probiotic strain.

Mutational analysis on stable expression and LasB inhibition of LasB propeptide in Pseudomonas aeruginosa: Youngsun Shin , Xi-Hui Li , Cheol Seung Lee , Joon-Hee Lee; J. Microbiol. 2022;60(7):727-734. Published online May 25, 2022; DOI: https://doi.org/10.1007/s12275-022-1671-5

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Abstract: Three major proteases, elastase B (LasB), protease IV (PIV), and elastase A (LasA) expressed in Pseudomonas aeruginosa play important roles in infections and pathogeneses. These are activated by a proteolytic cascade initiated by the activation of LasB. In this study, we investigated whether LasB could be inhibited using its propeptide (LasBpp). Although LasA and PIV were inhibited by their propeptides, LasB was not inhibited by purified LasBpp because LasB degraded LasBpp. To address this problem, mutant LasBpp variants were constructed to obtain a mutant LasBpp resistant to LasB degradation. A C-terminal deletion series of LasBpp was tested in vivo, and two positive candidates, T2 and T2-1, were selected. However, both caused growth retardation and were unstably expressed in vivo. Since deleting the C-terminal end of LasBpp significantly affected its stable expression, substitution mutations were introduced at the two amino acids near the truncation site of T2-1. The resulting mutants, LasBppE172D, LasBppG173A, and LasBppE172DG173A, significantly diminished LasB activity when overexpressed in vivo and were stably expressed in MW1, a quorum sensing mutant that does not produce LasB. In vitro analysis showed that purified LasBppE172DG173A inhibited LasB activity to a small extent. Summarizing, Cterminal modification of LasBpp profoundly affected the stable expression of LasBpp, and little enhanced the ability of LasBpp to resist degradation by LasB.

Extracellular vesicles derived from Lactobacillus plantarum restore chemosensitivity through the PDK2-mediated glucose metabolic pathway in 5-FU-resistant colorectal cancer cells: JaeJin An , Eun-Mi Ha; J. Microbiol. 2022;60(7):735-745. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-2201-1

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Abstract: Metabolic abnormalities are one of the main hallmarks of cancer and are associated with chemoresistance. Therefore, targeting the metabolic reprogramming of cancer cells has the potential to overcome chemoresistance. Probiotic-derived extracellular vesicles (EVs) play important roles in biological function and intracellular communication. However, the inhibitory effect of Lactobacillus plantarum-derived EVs (LpEVs) on colorectal cancer (CRC) cells has not yet been elucidated. This study clearly revealed that increased glycolysis in 5-fluorouracil (5-FU)-resistant CRC cells (CRC/5FUR) is directly related to chemoresistance and that the metabolic shift reversed by LpEVs inhibits cancer cell proliferation and eventually leads to apoptosis. Pyruvate dehydrogenase kinase 2 (PDK2), one of the crucial enzymes for enhancing glycolysis, was upregulated in CRC/5FUR cells. In our study, LpEVs sensitized CRC/5FUR cells to 5-FU by attenuating PDK2 expression in p53-p21-dependent metabolic signaling, thereby circumventing 5-FU resistance. We demonstrated the effect of cellular responses to 5-FU by modifying the PDK2 expression level in both 5-FU-sensitive parental CRC and 5- FU resistant CRC cell lines. Finally, we revealed that the PDK2 signaling pathway can potentially be targeted using LpEVs treatment to overcome chemoresistant CRC, thereby providing a potential strategy for CRC treatment by intervening in tumor metabolism.

Crystal structure of the phage-encoded N-acetyltransferase in complex with acetyl-CoA, revealing a novel dimeric arrangement: Nayeon Ki , Inseong Jo , Yongseong Hyun , Jinwook Lee , Nam-Chul Ha , Hyun-Myung Oh; J. Microbiol. 2022;60(7):746-755. Published online July 4, 2022; DOI: https://doi.org/10.1007/s12275-022-2030-2

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Abstract: Bacteriophages employ diverse mechanisms to facilitate the proliferation of bacteriophages. The Salmonella-infecting phage SPN3US contains a putative N-acetyltransferase, which is widely found in bacteriophages. However, due to low sequence similarity to the N-acetyltransferases from bacteria and eukaryotic cells, the structure and function of phage-encoded acetyltransferases are mainly unknown. This study determines the crystal structure of the putative N-acetyltransferase of SPN3US in complex with acetyl-CoA. The crystal structure showed a novel homodimeric arrangement stabilized by exchanging the C-terminal α-helix within the dimer. The following biochemical analyses suggested that the phageencoded acetyltransferase might have a very narrow substrate specificity. Further studies are required to reveal the biochemical activity, which would help elucidate the interaction between the phage and host bacteria in controlling pathogenic bacteria.

Predicting quorum sensing peptides using stacked generalization ensemble with gradient boosting based feature selection: Muthusaravanan Sivaramakrishnan , Rahul Suresh , Kannapiran Ponraj; J. Microbiol. 2022;60(7):756-765. Published online June 22, 2022; DOI: https://doi.org/10.1007/s12275-022-2044-9

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Abstract: Bacteria exist in natural environments for most of their life as complex, heterogeneous, and multicellular aggregates. Under these circumstances, critical cell functions are controlled by several signaling molecules known as quorum sensing (QS) molecules. In Gram-positive bacteria, peptides are deployed as QS molecules. The development of antibodies against such QS molecules has been identified as a promising therapeutic intervention for bacterial control. Hence, the identification of QS peptides has received considerable attention. Availability of a fast and reliable predictive model to effectively identify QS peptides can help the existing high throughput experiments. In this study, a stacked generalization ensemble model with Gradient Boosting Machine (GBM)-based feature selection, namely EnsembleQS was developed to predict QS peptides with high accuracy. On selected GBM features (791D), the EnsembleQS outperformed finely tuned baseline classifiers and demonstrated robust performance, indicating the superiority of the model. The accuracy of EnsembleQS is 4% higher than those resulting from ensemble model on hybrid dataset. When evaluating an independent data set of 40 QS peptides, the EnsembleQS model showed an accuracy of 93.4% with Matthew’s Correlation Coefficient (MCC) and area under the ROC curve (AUC) values 􀁇􀁇of 0.91 and 0.951, respectively. These
results
suggest that EnsembleQS will be a useful computational framework for predicting QS peptides and will efficiently support proteomics research. The source code and all datasets used in this study are publicly available at https:// github.com/proteinexplorers/EnsembleQS.

Published Erratum

[Erratum] A split face study on the effect of an anti-acne product containing fermentation products of Enterococcus faecalis CBT SL-5 on skin microbiome modification and acne improvement: Hye Sung Han , Sun Hye Shin , Bo-Yun Choi , Nayeon Koo , Sanghyun Lim , Dooheon Son , Myung Jun Chung , Kui Young Park , Woo Jun Sul; J. Microbiol. 2022;60(7):766-766.; DOI: https://doi.org/10.1007/s12275-022-1682-2

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