Journal of Microbiology

Volume 53(1); January 2015

Review

Minireview] Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects: Tsugunori Notomi , Yasuyoshi Mori , Norihiro Tomita , Hidetoshi Kanda; J. Microbiol. 2015;53(1):1-5. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4656-9

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613 Citations

Abstract: Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

Research Support, U.S. Gov't, Non-P.H.S.

Description of Pseudomonas asuensis sp. nov. from biological soil crusts in the Colorado plateau, United States of America: Gundlapally Sathyanarayana Reddy , # , Ferran Garcia-Pichel; J. Microbiol. 2015;53(1):6-13. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4462-4

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Abstract: A Gram-negative, aerobic, non spore-forming, non-motile, rod-shaped, yellow pigmented bacterium CP155-2T was isolated from a biological soil crusts sample collected in the Colorado plateau, USA and subjected to polyphasic taxonomic characterization. Strain CP155-2T contained summed feature 3 (C16:1ω5c/C16:1ω7c) and C18:1ω7c as major fatty acids and diphosphatidylglycerol (DPG) along with phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) as major polar lipids. Based on these characteristics CP155-2T was assigned to the genus Pseudomonas. Phylogenetic analysis based on 16S rRNA gene sequence further confirmed the affiliation of CP155-2T to the genus Pseudomonas and showed a 16S rRNA gene sequence similarity of less than 98.7% with already described species of the genus. Pseudomonas luteola, Pseudomonas zeshuii, and Pseudomonas duriflava were identified as the closest species of the genus Pseudomonas with 16S rRNA gene sequence similarities of 98.7%, 98.6%, and 96.9%, respectively. The values for DNA–DNA relatedness between CP155-2T and Pseudomonas luteola and Pseudomonas zeshuii were 23% and 14% respectively a value below the 70% threshold value, indicating that strain CP155-2T belongs to a novel taxon of the genus Pseudomonas lineage. The novel taxon status was strengthened by a number of phenotypic differences wherein CP155-2T was positive for oxidase, negative for gelatin hydrolysis, could utilize D-cellobiose, D-raffinose, L-rhamnose, D-sorbitol but not L-aspartic acid and L-glutamic acid. Based on the collective differences strain CP155-2T exhibited, it was identified as a novel species and the name Pseudomonas asuensis sp. nov. was proposed. The type strain of Pseudomonas asuensis sp. nov. is CP155- 2T (DSM 17866T =ATCC BAA-1264T =JCM13501T =KCTC 32484T).

Journal Article

Genetic diversity of Clavispora lusitaniae isolated from Agave fourcroydes Lem, as revealed by DNA fingerprinting: Daisy Pérez-Brito , Anuar Magaña-Alvarez , Patricia Lappe-Oliveras , Alberto Cortes-Velazquez , Claudia Torres-Calzada , Teófilo Herrera-Suarez , Alfonso Larqué-Saavedra , Raul Tapia-Tussell; J. Microbiol. 2015;53(1):14-20. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4373-4

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Abstract: This study characterized Clavispora lusitaniae strains isolated from different stages of the processing and early fermentation of a henequen (Agave fourcroydes) spirit produced in Yucatan, Mexico using a molecular technique. Sixteen strains identified based on morphological features, obtained from different substrates, were typed molecularly. Nine different versions of the divergent D1/D2 domain of the large-subunit ribosomal DNA sequence were identified among the C. lusitaniae strains. The greatest degree of polymorphism was found in the 90-bp structural motif of the D2 domain. The MSP-PCR technique was able to differentiate 100% of the isolates. This study provides significant insight into the genetic diversity of the mycobiota present during the henequen fermentation process, especially that of C. lusitaniae, for which only a few studies in plants have been published. The applied MSP-PCR markers were very efficient in revealing polymorphisms between isolates of this species.

Research Support, Non-U.S. Gov'ts

Characterization of NpgA, a 4'-phosphopantetheinyl transferase of Aspergillus nidulans, and evidence of its involvement in fungal growth and formation of conidia and cleistothecia for development: Jung-Mi Kim , Ha-Yeon Song , Hyo-Jin Choi , Kum-Kang So , Dae-Hyuk Kim , Keon-Sang Chae , Dong-Min Han , Kwang-Yeop Jahng; J. Microbiol. 2015;53(1):21-31. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4657-8

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Abstract: The null pigmentation mutant (npgA1) in Aspergillus nidulans
results
in a phenotype with colorless organs, decreased branching growth, delayed of asexual spore development, and aberrant cell wall structure. The npgA gene was isolated from A. nidulans to investigate these pleiomorphic phenomena of npgA1 mutant. Sequencing analysis of the complementing gene indicated that it contained a 4􍿁-phosphopantetheinyl transferase (PPTase) superfamily domain. Enzymatic assay of the PPTase, encoded by the npgA gene, was implemented in vivo and in vitro. Loss-of-function of LYS5, which encoded a PPTase in Saccharomyces cerevisiae, was functionally complemented by NpgA, and Escherichia coli-derived NpgA revealed phosphopantetheinylation activity with the elaboration of 3􍿁5􍿁-ADP. Deletion of the npgA gene caused perfectly a lethal phenotype and the absence of asexual/sexual sporulation and secondary metabolites such as pigments in A. nidulans. However, a cross feeding effect with A. nidulans wild type allowed recovery from deletion defects, and phased-culture filtrate from the wild type were used to verify that the npgA gene was essential for formation of metabolites needed for development as well as growth. In addition, forced expression of npgA promoted the formation of conidia and cleistothecia as well as growth. These results indicate that the npgA gene is involved in the phosphopantetheinylation required for primary biological processes such as growth, asexual/sexual development, and the synthesis of secondary metabolites in A. nidulans.

Crystal structure of the bacterial type VI secretion system component TssL from Vibrio cholerae: Jeong Ho Chang , Yeon-Gil Kim; J. Microbiol. 2015;53(1):32-37. Published online December 4, 2014; DOI: https://doi.org/10.1007/s12275-015-4539-0

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Abstract: The type VI secretion system (T6SS), commonly found in Gram-negative bacteria, is responsible for exporting effector proteins. The T6SS has been reported to be cytotoxic to host cells. While the components and assembly of the T6SS complex have been largely assessed, structural data on T6SS components from virulent bacteria is remarkably insufficient. Here, we report the crystal structure of Vibrio cholerae TssL (VcTssL), a core component of T6SS. In spite of a relatively low sequence identity, the overall structure of VcTssL is largely similar to those from other bacterial homologs except for several differences found in local structural elements. A unique feature attributed to the C-terminal fragment of Vc- TssL is a crystallographic artifact. This incidental feature of VcTssL may provide insights into screening of molecular partners for the cytoplasmic domain of TssL. Additionally, our results may help in the design of molecular probes for a detailed understanding of the functional relationship between TssL and other T6SS components.

Performance of a real-time PCR assay for the rapid identification of Mycobacterium species: Hye-young Wang , Hyunjung Kim , Sunghyun Kim , Do-kyoon Kim , Sang-Nae Cho , Hyeyoung Lee; J. Microbiol. 2015;53(1):38-46. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4495-8

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Abstract: Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM species are prevalent in the environment and because they have fastidious properties. In the present study, we evaluated 279 clinical isolates grown in liquid culture provided by The Catholic University of Korea, St. Vincent’s Hospital using real-time PCR based on mycobacterial rpoB gene sequences. The positive rate of real-time PCR assay accurately discriminated 100% (195/195) and 100% (84/84) between MTB and NTM species. Comparison of isolates identified using the MolecuTech REBA Myco-ID? and Real Myco-ID? were completely concordant except for two samples. Two cases that were identified as mixed infection (M. intracellulare-M. massiliense and M. avium-M. massiliense co-infection) by PCRREBA assay were only detected using M. abscessus-specific probes by Real Myco-ID?. Among a total of 84 cases, the most frequently identified NTM species were M. intracellulare (n=38, 45.2%), M. avium (n=18, 23.7%), M. massiliense (n=10, 13.2%), M. fortuitum (n=5, 6%), M. abscessus (n=3, 3.9%), M. gordonae (n=3, 3.9%), M. kansasii (n=2, 2.4%), M. mucogenicum (n=2, 2.4%), and M. chelonae (n= 1, 1.2%). Real Myco-ID? is an efficient tool for the rapid detection of NTM species as well as MTB and sensitive and specific and comparable to conventional methods.

Oral administration of Lactobacillus plantarum lysates attenuates the development of atopic dermatitis lesions in mouse models: Hangeun Kim , Hye Rim Kim , Na-Ra Kim , Bong Jun Jeong , Jong Suk Lee , Soojin Jang , Dae Kyun Chung; J. Microbiol. 2015;53(1):47-52. Published online December 4, 2014; DOI: https://doi.org/10.1007/s12275-015-4483-z

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27 Citations

Abstract: Lactobacillus plantarum is a well-documented probiotic that has been used in clinical trials for the regulation of the immune system and treatment of gastrointestinal diseases. In this study, we evaluated the effects of L. plantarum cell lysates on the immune regulation through the in vitro and in vivo studies. L. plantarum lysates were prepared by sonication
method
, and we observed that the repetition of disruption step increased indicator components within the bacterial lysates. Indicator components might affect TNF-α production. L. plantarum lysates did not induce TNF-α production, while LPS-induced TNF-α production was dramatically inhibited in a sonication-dependent manner in THP-1 cells. Oral administration of L. plantarum lysates effectively attenuated the horny layer formation and decreased epidermal thickening in NC/Nga mice skin. The damage to barrier function after the 8 weeks oral administration was reduced by L. plantarum lysates as compared to that in the atopic dermatitis (AD) mice. Further study revealed that L. plantarum lysates polarized Th1 response via induction of IL-12 and IFN-γ production and inhibition of IL-4 and IgE production in NC/Nga mice. Together, our results suggest that L. plantarum lysates are remarkable material for host homeostasis and it could be used for the treatment of inflammatory diseases.

Effect of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on killing Acinetobacter baumannii by colistin: Young Kyoung Park , Kwan Soo Ko; J. Microbiol. 2015;53(1):53-59. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4498-5

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47 Citations

Abstract: We investigated the effect of cyanide 3-chlorophenylhydrazone (CCCP) and other efflux pump inhibitors (EPIs) on the colistin susceptibility in Acinetobacter baumannii. While minimum inhibitory concentrations (MICs) of colistin in all colistin-resistant strains decreased significantly with 25 μM of CCCP and 2,4-dinitrophenol (DNP), phenyl-arginine-β- naphthylamide (PAβN), and reserpine did not decrease the colistin MICs. However, CCCP and DNP as well as PAβN and reserpine did not have a significant effect on the MICs of the other agents. Efflux pump gene expressions in colistinresistant strains were not increased compared with those in colistin-susceptible strains. When only 5X MIC of colistin (5 mg/L) was provided to a colistin-susceptible A. baumannii strain, the bacterial cell number was reduced by 9 h after exposure to colistin, but regrowth was observed. When CCCP was added to colistin, bacterial cells were completely killed after 24 to 48 h of incubation, which was not due to the toxicity of CCCP itself. Colistin resistance in A. baumannii may not be due to efflux pumps. Our present study suggests that bacterial cells with reduced metabolic activity by CCCP are more susceptible to colistin in A. baumannii. It may show the possibility that combined therapy with colistin and other antimicrobial agents could effective against A. baumannii infections.

Improved pipeline for reducing erroneous identification by 16S rRNA sequences using the Illumina MiSeq platform: Yoon-Seong Jeon , Sang-Cheol Park , Jeongmin Lim , Jongsik Chun , Bong-Soo Kim; J. Microbiol. 2015;53(1):60-69. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4601-y

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42 Citations

Abstract: The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3􍿁-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended
method
corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform.

Hypermethylation of the interferon regulatory factor 5 promoter in Epstein-Barr virus-associated gastric carcinoma: Seung Myung Dong , Hyun Gyu Lee , Sung-Gyu Cho , Seung-Hyun Kwon , Heejei Yoon , Hyun-Jin Kwon , Ji Hae Lee , Hyemi Kim , Pil-Gu Park , Hoguen Kim , S. Diane Hayward , Jeon Han Park , Jae Myun Lee; J. Microbiol. 2015;53(1):70-76. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-014-4654-3

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Abstract: Interferon regulatory factor-5 (IRF-5), a member of the mammalian IRF transcription factor family, is regulated by p53, type I interferon and virus infection. IRF-5 participates in virus-induced TLR-mediated innate immune responses and may play a role as a tumor suppressor. It was suppressed in various EBV-infected transformed cells, thus it is valuable to identify the suppression mechanism. We focused on a promoter CpG islands methylation, a kind of epigenetic regulation in EBV-associated Burkitt’s lymphomas (BLs) and gastric carcinomas. IRF-5 is not detected in most of EBV-infected BL cell lines due to hypermethylation of IRF-5 distal promoter (promoter-A), which was restored by a demethylating agent, 5-aza-2􍿁-deoxycytidine. Hypomethylation of CpG islands in promoter-A was observed only in EBV type III latent infected BL cell lines (LCL and Mutu III). Similarly, during EBV infection to Akata-4E3 cells, IRF-5 was observed at early time periods (2 days to 8 weeks), concomitant unmethylation of promoter-A, but suppressed in later infection periods as observed in latency I BL cell lines. Moreover, hypermethylation in IRF-5 promoter-A region was also observed in EBV-associated gastric carcinoma (EBVaGC) cell lines or primary gastric carcinoma tissues, which show type I latent infection. In summary, IRF-5 is suppressed by hypermethylation of its promoter-A in most of EBV-infected transformed cells, especially BLs and EBVaGC. EBV-induced carcinogenesis takes an advantage of proliferative effects of TLR signaling, while limiting IRF-5 mediated negative effects in the establishment of EBVaGCs.

Journal Article

Extended stability of cyclin D1 contributes to limited cell cycle arrest at G1-phase in BHK-21 cells with Japanese encephalitis virus persistent infection: Ji Young Kim , Soo Young Park , Hey Rhyoung Lyoo , Eung Seo Koo , Man Su Kim , Yong Seok Jeong; J. Microbiol. 2015;53(1):77-83. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4661-z

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Abstract: There is increasing evidence that many RNA viruses manipulate cell cycle control to achieve favorable cellular environments for their efficient replication during infection. Although virus-induced G0/G1 arrest often delays early apoptosis temporarily, a prolonged replication of the infected virus leads host cells to eventual death. In contrast, most mammalian cells with RNA virus persistent infection often escape cytolysis in the presence of productive viral replication. In this study, we demonstrated that the extended endurance of cyclin D1 was clearly associated with the suppression of glycogen synthase kinase-3β (GSK-3β) expression in BHK-21 cells that are persistently infected with Japanese encephalitis virus (JEV). The G0/G1 arrest of these cells turned much loose compared to the normal BHK-21 cells with JEV acute infection. After cycloheximide treatment, cyclin D1 in the persistently infected cells lasted several hours longer than those in acutely infected cells. Furthermore, both p21Cip1 and p27Kip1, positive regulators for cyclin D1 accumulation in the nucleus, were suppressed in their expression, which contrasts with those in JEV acute infection. Inhibition of the GSK-3β by lithium chloride treatment rescued a significant number of cells from cytolysis in JEV acute infection, which coincided with the levels of cyclin D1 that escaped from proteolysis. Therefore, the limitation of G1/S arrest in the BHK-21 cells with JEV persistent infection is associated with the suppression of GSK-3β expression, resulting in the extended duration of cyclin D1.

Research Support, Non-U.S. Gov't

Note] Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics: Jae Yoon Hwang , Soo Hee Kim , Hye Ryeung Oh , Eunju Kwon , Doo Hyun Nam; J. Microbiol. 2015;53(1):84-89. Published online December 4, 2014; DOI: https://doi.org/10.1007/s12275-015-4340-0

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Abstract: Kitasatospora cheerisanensis KCTC 2395, producing bafilomycin antibiotics belonging to plecomacrolide group, was isolated from a soil sample at Mt. Jiri, Korea. The draft genome sequence contains 8.04 Mb with 73.6% G+C content and 7,810 open reading frames. All the genes for aerial mycelium and spore formations were confirmed in this draft genome. In phylogenetic analysis of MurE proteins (UDPN- acetylmuramyl-L-alanyl-D-glutamate:DAP ligase) in a conserved dcw (division of cell wall) locus, MurE proteins of Kitasatospora species were placed in a separate clade between MurEs of Streptomyces species incorporating LL-diaminopimelic acid (DAP) and MurEs of Saccharopolyspora erythraea as well as Mycobacterium tuberculosis ligating meso- DAP. From this finding, it was assumed that Kitasatospora MurEs exhibit the substrate specificity for both LL-DAP and meso-DAP. The bafilomycin biosynthetic gene cluster was located in the left subtelomeric region. In 71.3 kb-long gene cluster, 17 genes probably involved in the biosynthesis of bafilomycin derivatives were deduced, including 5 polyketide synthase (PKS) genes comprised of 12 PKS modules.

Published Erratum

Erratum] Protective Role of Gut Commensal Microbes against Intestinal Infections: My Young Yoon , Keehoon Lee , Sang Sun Yoon; J. Microbiol. 2015;53(1):90-90.; DOI: https://doi.org/10.1007/s12275-015-0705-7

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Abstract: In the article by Yoon et al. published in Journal of Microbiology 2014; 52, 983-989. First author name should be changed as Mi Young Yoon

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