Journal of Microbiology

Volume 58(4); April 2020

Journal Articles

[Protocol] Detecting Salmonella Type II flagella production by transmission electron microscopy and immunocytochemistry: Yoontak Han , Eun-Jin Lee; J. Microbiol. 2020;58(4):245-251. Published online November 23, 2019; DOI: https://doi.org/10.1007/s12275-020-9297-y

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Abstract: The bacterial flagellum is an appendage structure that provides a means for motility to promote survival in fluctuating environments. For the intracellular pathogen Salmonella enterica serovar Typhimurium to survive within macrophages, flagellar gene expression must be tightly regulated, and thus, is controlled at multiple levels, including DNA recombination, transcription, post-transcription, protein synthesis, and assembly within host cells. To understand the contribution of flagella to Salmonella pathogenesis within the host, it is critical to detect flagella production within macrophages via microscopy. In this paper, we describe two methods for detecting bacterial flagella by microscopy both in vitro and in vivo infection models.

Paradesulfovibrio onnuriensis gen. nov., sp. nov., a chemolithoautotrophic sulfate-reducing bacterium isolated from the Onnuri vent field of the Indian Ocean and reclassification of Desulfovibrio senegalensis as Paradesulfovibrio senegalensis comb. nov.: Yun Jae Kim , Jhung-Ahn Yang , Jae Kyu Lim , Mi-Jeong Park , Sung-Hyun Yang , Hyun Sook Lee , Sung Gyun Kang , Jung-Hyun Lee , Kae Kyoung Kwon; J. Microbiol. 2020;58(4):252-259. Published online February 27, 2020; DOI: https://doi.org/10.1007/s12275-020-9376-0

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Abstract: An anaerobic, rod-shaped, mesophilic, chemolithoautotrophic, sulfate-reducing bacterial strain IOR2T was isolated from a newly found deep-sea hydrothermal vent (OVF, Onnuri Vent Field) area in the central Indian Ocean ridge (11°24􍿁88􍿂􀁇 S 66°25􍿁42􍿂􀁇E, 2021 m water depth). The 16S rRNA gene sequence analysis revealed that the strain IOR2T was most closely related to Desulfovibrio senegalensis BLaC1T (96.7%). However, it showed low similarity with the members of the family Desulfovibrionaceae, such as Desulfovibrio tunisiensis RB22T (94.0%), D. brasiliensis LVform1T (93.9%), D. halophilus DSM 5663T (93.7%), and Pseudodesulfovibrio aespoeensis Aspo-2T (93.2%). The strain IOR2T could grow at 23– 42°C (optimum 37°C), pH 5.0–8.0 (optimum pH 7.0) and with 0.5–6.5% (optimum 3.0%) NaCl. The strain could use lactate, pyruvate, H2, and glycerol as electron donors and sulfate, thiosulfate, and sulfite as electron acceptors. The major fatty acids of the strain IOR2T were iso-C15:0, iso-C17:0, anteiso- C15:0, and summed feature 9 (C16:0 methyl/iso-C17:1ω9c). Both the strains IOR2T and BLaC1T could grow with CO2 and H2 as the sole sources of carbon and energy, respectively. Genomic evidence for the Wood-Ljungdahl pathway in both the strains reflects chemolithoautotrophic growth. The DNA G + C content of the strain IOR2T and BLaC1T was 58.1–60.5 mol%. Based on the results of the phylogenetic and physiologic studies, Paradesulfovibrio onnuriensis gen. nov., sp. nov. with the type strain IOR2T (= KCTC 15845T = MCCC 1K04559T) was proposed to be a member of the family Desulfovibrionaceae. We have also proposed the reclassification of D. senegalensis as Paradesulfovibrio senegalensis comb. nov.

Thermococcus indicus sp. nov., a Fe(III)-reducing hyperthermophilic archaeon isolated from the Onnuri Vent Field of the Central Indian Ocean ridge: Jae Kyu Lim , Yun Jae Kim , Jhung-Ahn Yang , Teddy Namirimu , Sung-Hyun Yang , Mi-Jeong Park , Yong Min Kwon , Hyun Sook Lee , Sung Gyun Kang , Jung-Hyun Lee , Kae Kyoung Kwon; J. Microbiol. 2020;58(4):260-267. Published online April 1, 2020; DOI: https://doi.org/10.1007/s12275-020-9424-9

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Abstract: A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2–1.8 μm in diameter, and motile by flagella. Growth was at 70–82°C (optimum 80°C), pH 5.4–8.0 (optimum pH 6.0) with 2–4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Light affects picocyanobacterial grazing and growth response of the mixotrophic flagellate Poterioochromonas malhamensis: Thomas Weisse , Michael Moser; J. Microbiol. 2020;58(4):268-278. Published online January 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9567-8

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Abstract: We measured the grazing and growth response of the mixotrophic chrysomonad flagellate Poterioochromonas malhamensis on four closely related picocyanobacterial strains isolated from subalpine lakes in central Europe. The picocyanobacteria represented different pigment types (phycoerythrin- rich, PE, and phycocyanin-rich, PC) and phylogenetic clusters. The grazing experiments were conducted with laboratory cultures acclimated to 10 μmol photon/m2/sec (low light, LL) and 100 μmol photon/m2/sec (moderate light, ML), either in the dark or at four different irradiances ranging from low (6 μmol photon/m2/sec) to high (1,500 μmol photon/m2/ sec) light intensity. Poterioochromonas malhamensis preferred the larger, green PC-rich picocyanobacteria to the smaller, red PE-rich picocyanobacterial, and heterotrophic bacteria. The feeding and growth rates of P. malhamensis were sensitive to the actual light conditions during the experiments; the flagellate performed relatively better in the dark and at LL conditions than at high light intensity. In summary, our
results
found strain-specific ingestion and growth rates of the flagellate; an effect of the preculturing conditions, and, unexpectedly, a direct adverse effect of high light levels. We conclude that this flagellate may avoid exposure to high surface light intensities commonly encountered in temperate lakes during the summer.

Distribution characteristics of fungal communities with depth in paddy fields of three soil types in China: Xu Li , Huanhuan Wang , Xiang Li , Xinyu Li , Huiwen Zhang; J. Microbiol. 2020;58(4):279-287. Published online February 27, 2020; DOI: https://doi.org/10.1007/s12275-020-9409-8

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Abstract: Little is known about the distribution of fungal communities with soil depth on relatively large scales. In this study, typical paddy soils in three regions (Hailun, Changshu, and Yingtan) from north to south China were selected to investigate the vertical distribution (0-100 cm) of the fungal community by Illumina MiSeq sequencing, and to identify the main factors influencing the fungal community distribution. The results indicated that the structure of the soil fungal community changed significantly with region and soil depth. Soil fungal taxa such as Zygomycota, Glomeromycota, Saccharomycete, Kazachstania, Mortierella, Massariosphaeria, Hypholoma, and Zopfiella were enriched at depths of 0–20 cm, whereas Dothideomycetes, Microbotryomycetes, Tremellomycetes, Sporobolomyces, Cryptococcus, Rhodotorula, Fusarium, and Pyrenochaetopsis had high relative abundances at 80–100 cm. Variance partitioning analysis indicated that the geographic distance contributed more to the fungal community variation than environmental variables on a large scale. In addition, soil total carbon and nitrogen contents were the main environmental factors driving the vertical distribution of the fungal community in paddy soils.

Optimization of bacterial sporulation using economic nutrient for self-healing concrete: Youngung Ryu , Ki-Eun Lee , In-Tae Cha , Woojun Park; J. Microbiol. 2020;58(4):288-296. Published online February 27, 2020; DOI: https://doi.org/10.1007/s12275-020-9580-y

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Abstract: The use of heat- and alkali-resistant bacteria is essential for the biological repair of damaged concrete. Lysinibacillus boronitolerans YS11 was isolated from the rhizosphere of Miscanthus sacchariflorus. The increased pH in the urea-minus condition during the growth of the YS11 strain promoted calcium carbonate (CaCO3) formation. To identify the optimum medium that promoted the growth of the YS11 strain, a Plackett- Burman design was conducted for the screening process. Consequently, malt powder, rice bran, (NH4)2SO4, and corn syrup were chosen to enhance YS11 growth. The optimization of these four useful factors was carried out using a central composite design. To obtain higher survivability in mortar, the sporulation process is essential, and additional factors such as Mn2+, Fe2+, and Ca2+ were found to contribute to sporulation. A mixture of L. boronitolerans YS11 spore powder, cement, paste, sand, yeast extract, calcium lactate, and water showed a healing effect on a 0.3 mm mortar crack in 7 days. Furthermore, calcium carbonate precipitation was observed over the crack surface. Thus, we confirmed that mortar treated with YS11 spore powder was effective in healing micro-cracks in concrete.

Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis: Ozan Ertekin , Meltem Kutnu , Aslı Aras Ta&# , Mustafa Demir , Ayten Yazgan Karata&# , Gülay Özcengiz; J. Microbiol. 2020;58(4):297-313. Published online January 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9064-0

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Abstract: Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA􍿁::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLCMS/ MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment- specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoCregulated proteins.

Lactobacillus plantarum lipoteichoic acid disrupts mature Enterococcus faecalis biofilm: A Reum Kim , Minji Kang , Yeon-Jee Yoo , Cheol-Heui Yun , Hiran Perinpanayagam , Kee-Yeon Kum , Seung Hyun Han; J. Microbiol. 2020;58(4):314-319. Published online January 28, 2020; DOI: https://doi.org/10.1007/s12275-020-9518-4

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Abstract: Apical periodontitis is caused by biofilm-mediated root canal infection. Early phase oral bacterial biofilms are inhibited by Lactobacillus plantarum lipoteichoic acid (Lp.LTA). However, mature biofilms that develop over 3 weeks are more resistant to traditional endodontic medicaments. Therefore, this study examined the effectiveness of Lp.LTA on disrupting mature Enterococcus faecalis biofilms, and on enhancing the effects of endodontic medicaments. LTA was purified from L. plantarum through butanol extraction followed by hydrophobic and ion-exchange chromatography. E. faecalis biofilms were formed over 3 weeks on glass bottom dishes and in dentin blocks obtained from human single-rooted premolars. These mature biofilms were treated with or without Lp.LTA for 1 h, followed by additional treatment with either chlorhexidine digluconate (CHX), calcium hydroxide (CH), or triple antibiotics for 24 h. Biofilms on glass were live/dead stained and quantified by ZEN through confocal laser microscopy. Biofilms in dentin were fixed, sputter coated and analyzed by ImageJ with scanning electron microscopy. Preformed E. faecalis mature biofilms on the culture dishes were dose-dependently disrupted by Lp.LTA. Lp.LTA potentiated the effects of CHX or CH on the disruption of mature biofilm. Interestingly, CHX-induced disruption of preformed E. faecalis mature biofilms was synergistically enhanced only when pretreated with Lp.LTA. Furthermore, in the dentin block model, Lp.LTA alone reduced E. faecalis mature biofilm and pre-treatment with Lp.LTA promoted the anti-biofilm activity of CHX. Lp.LTA could be an anti-biofilm or supplementary agent that can be effective for E. faecalis-biofilminduced diseases.

Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus: Na Geng , Kangping Liu , Jianwei Lu , Yuliang Xu , Xiaozhou Wang , Run Wang , Jianzhu Liu , Yongxia Liu , Bo Han; J. Microbiol. 2020;58(4):320-329. Published online February 26, 2020; DOI: https://doi.org/10.1007/s12275-020-9182-8

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Abstract: Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The
results
showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.

Streptococcus pneumoniae aminopeptidase N contributes to bacterial virulence and elicits a strong innate immune response through MAPK and PI3K/AKT signaling: Ling Wang , Xuemei Zhang , Guangying Wu , Yuhong Qi , Jinghui Zhang , Jing Yang , Hong Wang , Wenchun Xu; J. Microbiol. 2020;58(4):330-339. Published online February 27, 2020; DOI: https://doi.org/10.1007/s12275-020-9538-0

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Abstract: Streptococcus pneumoniae is a Gram-positive pathogen with high morbidity and mortality globally but some of its pathogenesis remains unknown. Previous research has provided evidence that aminopeptidase N (PepN) is most likely a virulence factor of S. pneumoniae. However, its role in S. pneumoniae virulence and its interaction with the host remains to be confirmed. We generated a pepN gene deficient mutant strain and found that its virulence for mice was significantly attenuated as were in vitro adhesion and invasion of host cells. The PepN protein could induce a strong innate immune response in vivo and in vitro and induced secretion of IL-6 and TNF-α by primary peritoneal macrophages via the rapid phosphorylation of MAPK and PI3K/AKT signaling pathways and this was confirmed using specific pathway inhibitors. In conclusion, PepN is a novel virulence factor that is essential for the virulence of S. pneumoniae and induces host innate immunity via MAPK and PI3K/AKT signaling.

Published Erratums

[ERRATUM] The discovery of potent immunostimulatory CpG-ODNs widely distributed in bacterial genomes: Juan Liu , Yan Wei , Yongling Lu , Yangyuling Li , Qian Chen , Yan Li; J. Microbiol. 2020;58(4):340-340.; DOI: https://doi.org/10.1007/s12275-020-9724-0

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[ERRATUM]Hahyoungchilella caricis gen. nov., sp. nov., isolated from a rhizosphere mudflat of a halophyte (Carex scabrifolia), transfer of Thioclava arenosa Thongphrom et al. 2017 to Pseudothioclava as Pseudothioclava arenosa gen. nov., comb. nov. and proposal of Thioclava electrotropha Chang et al.: Young-Ju Kim , Soon Dong Lee; J. Microbiol. 2020;58(4):341-341.; DOI: https://doi.org/10.1007/s12275-020-9725-z

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Abstract: Hahyoungchilella caricis gen. nov., sp. nov., isolated from a rhizosphere mudflat of a halophyte (Carex scabrifolia), transfer of Thioclava arenosa Thongphrom et al. 2017 to Pseudothioclava as Pseudothioclava arenosa gen. nov., comb. nov. and proposal of Thioclava electrotropha Chang et al. 2018 as a later heterosynonym of Thioclava sediminum

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