Journal of Microbiology

Volume 56(4); April 2018

Review

MINIREVIEW] Rapid and robust MALDI-TOF MS techniques for microbial identification: a brief overview of their diverse applications: Kyoung-Soon Jang , Young Hwan Kim; J. Microbiol. 2018;56(4):209-216. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7457-0

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104 Citations

Abstract: Advances in mass spectrometry have enabled the investigation of various biological systems by directly analyzing diverse sets of biomolecules (i.e., proteins, lipids, and carbohydrates), thus making a significant impact on the life sciences field. Over the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely utilized as a rapid and reliable method for the identification of microorganisms. MALDI-TOF MS has come into widespread use despite its relatively low resolving power (full width at half maximum, FWHM: < 5,000) and its incompatibility with tandem MS analysis, features with which other high-resolution mass spectrometers are equipped. Microbial identification is achieved by searching databases containing mass spectra of peptides and proteins extracted from microorganisms of interest, using scoring algorithms to match analyzed spectra with reference spectra. In this paper, we give a brief overview of the diverse applications of rapid and robust MALDI-TOF MS-based techniques for microbial identification in a variety of fields, such as clinical diagnosis and environmental and food monitoring. We also describe the fundamental principles of MALDI-TOF MS. The general specifications of the two major MS-based microbial identification systems available in the global market (BioTyper® and VITEK® MS Plus) and the distribution of these instruments in Republic of Korea are also discussed. The current review provides an understanding of this emerging microbial identification and classification technology and will help bacteriologists and cell biologists take advantage of this powerful technique.

Journal Articles

Bacillus spongiae sp. nov., isolated from sponge of Jeju Island: Ga-Eun Lee , Wan-Taek Im , Jin-Sook Park; J. Microbiol. 2018;56(4):217-222. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7511-y

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Abstract: A Gram-reaction-positive, strictly aerobic, motile, endospore- forming, and rod-shaped bacterial strain designated 135PIL107-10T was isolated from a sponge on Jeju Island, and its taxonomic position was investigated using a polyphasic approach. Strain 135PIL107-10T grew at 20–37°C (optimum temperature, 25°C) and pH 6.0–10.0 (optimum pH, 6.0) on marine and R2A agars. Based on 16S rRNA gene phylogeny analysis, the novel strain formed a new branch within the genus Bacillus of the family Bacillaceae, and formed clusters with Bacillus thaohiensis NHI-38T (96.8%), Bacillus fengqiuensis NPK15T (96.7%), and Bacillus songklensis CAU 1033T (96.7%). Lower sequence similarities (97.0%) were found with the type strains of all other recognized members of the genus Bacillus (95.6–96.8% similarity). The G + C content of the genomic DNA was 43.6 mol%. The predominant respiratory quinone was menaquinone-7 and the major fatty acids were iso-C15:0 and iso-C17:1ω10c. The overall polar lipid patterns were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The isolate therefore represents a novel species, for which the name Bacillus spongiae sp. nov. is proposed, with the type strain 135PIL107-10T (= KACC 19275T = LMG 30080T).

Taxonomic description and genome sequence of Halobacillus marinus sp. nov., a novel strain isolated from Chilika Lake, India: Ananta N. Panda , Samir Ranjan Mishra , Lopamudra Ray , Surajit Das , Gurdeep Rastogi , Tapan Kumar Adhya , Mrutyunjay Suar , Vishakha Raina; J. Microbiol. 2018;56(4):223-230. Published online April 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7387-x

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Abstract: A moderately halophilic spore forming, motile, Gram-positive, rod-shaped bacterial strain designated as KGW1T was isolated from water sample of Chilika Lake and characterized taxonomically using polyphasic approach. The strain grew in the presence of 0–25% (w/v) NaCl in marine salt agar media, hydrolyzes casein, and gelatin and shows presence of alkaline proteases. The major cell wall menaquinone was MK7 and major cellular fatty acids were anteiso-C15:0 (44.89%), anteiso-C17:0 (6.18%), isoC15:0 (19.38%), and iso-C16:0 (7.39%). Several chemotaxonomic features conform the isolate be a member of genus Halobacillus. The isolate KGW1T contained A1γ meso-Dpm-direct type of peptidoglycan which is different from its phylogenetically closest neighbours. The 16S rRNA gene sequence based phylogenetic analysis also revealed the strain KGW1T was affiliated to the genus Halobacillus and sequence similarity between the isolated strain and the type strains of Halobacillus species were found closest to, H. dabanensis D-8 DSM 18199T (99.08%) and H. faecis IGA7-4 DSM 21559T (99.01%), H. trueperi SL-5 DSM 10404T (98.94%). The in silico DDH showed that the values in a range of 14.2–17.5% with the most closest strain H. dabanensis D-8 DSM 18199T and other type strains of the genus Halobacillus for which whole genome sequence is reported. DNA-DNA relatedness between strain KGW1T and the closest type strain Halobacillus trueperi DSM 10404T was 11.75% (± 1.15). The draft genome sequence includes 3,683,819 bases and comprises of 3898 predicted coding sequences with a G + C content of 46.98%. Thus, the significant distinctiveness supported by phenotypic and genotypic data with its closest neighbors and other closely related species confirm the strain KGW1T to be classified as a novel species within the genus Halobacillus, for which the name Halobacillus marinus sp. nov. is proposed. The type strain is KGW1T (= DSM 29522 = JCM 30443).

Hymenobacter terrigena sp. nov., isolated from soil: Jeong-Eun Ohn , Leonid N. Ten , Kyeung Il Park , Byung-Oh Kim , Jeung-Sul Han , Hee-Young Jung; J. Microbiol. 2018;56(4):231-237. Published online April 2, 2018; DOI: https://doi.org/10.1007/s12275-018-8029-z

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Abstract: A Gram-stain-negative, non-motile, non-spore-forming, rodshaped, aerobic bacterial strain, designated S1-2-2-5T, was isolated from the Jeollabuk-do province, Republic of Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-2-5T belonged to the family Cytophagaceae in phylum Bacteroidetes, and was most closely related to Hymenobacter terrae DG7AT (98.2%), Hymenobacter rubidus DG7BT (98.0%), Hymenobacter soli PB17T (97.7%), Hymenobacter daeguensis 16F3Y-2T (97.2%) and Hymenobacter saemangeumensis GSR0100T (97.0%). The G + C content of the genomic DNA of strain S1-2-2-5T was 59.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone, a fatty acid profile with summed feature 3 (C16:1 ω7c/C16:1 ω6c; 32.0%), C15:0 iso (19.0%), and C15:0 anteiso (15.0%) as the major components, and a polar lipid profile with phosphatidylethanolamine as the major component supported the affiliation of strain S1-2-2-5T to the genus Hymenobacter. The DNA-DNA relatedness between strain S1-2-2-5T and H. terrae KCTC 32554T, H. rubidus KCTC 32553T, H. soli KCTC 12607T, H. daeguensis KCTC 52537T, and H. saemangeumensis KACC 16452T were 49.5, 48.2, 34.1, 28.1, and 31.8% respectively, clearly showing that the isolate is not related to them at the species level. Strain S1-2-2-5T could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain S1-2-2-5T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter terrigena sp. nov. is proposed. The type strain is S1-2-2-5T (= KCTC 52737T = JCM 32195T).

Hydrogen sulfide inhibits the growth of Escherichia coli through oxidative damage: Liu-Hui Fu , Zeng-Zheng Wei , Kang-Di Hu , Lan-Ying Hu , Yan-Hong Li , Xiao-Yan Chen , Zhuo Han , Gai-Fang Yao , Hua Zhang; J. Microbiol. 2018;56(4):238-245. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7537-1

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Abstract: Many studies have shown that hydrogen sulfide (H2S) is both detrimental and beneficial to animals and plants, whereas its effect on bacteria is not fully understood. Here, we report that H2S, released by sodium hydrosulfide (NaHS), significantly inhibits the growth of Escherichia coli in a dose-dependent manner. Further studies have shown that H2S treatment stimulates the production of reactive oxygen species (ROS) and decreases glutathione (GSH) levels in E. coli, resulting in lipid peroxidation and DNA damage. H2S also inhibits the antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and induces the response of the SoxRS and OxyR regulons in E. coli. Moreover, pretreatment with the antioxidant ascorbic acid (AsA) could effectively prevent H2S-induced toxicity in E. coli. Taken together, our results indicate that H2S exhibits an antibacterial effect on E. coli through oxidative damage and suggest a possible application for H2S in water and food processing.

The crystal structure of methanol dehydrogenase, a quinoprotein from the marine methylotrophic bacterium Methylophaga aminisulfidivorans MPT: Thinh-Phat Cao , Jin Myung Choi , Si Wouk Kim , Sung Haeng Lee; J. Microbiol. 2018;56(4):246-254. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7483-y

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11 Citations

Abstract: The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDHMas), was determined at 1.7 Å resolution. The active form of MDHMas (or MDHIMas) is a heterotetrameric α2β2, where each β-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two β-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a β-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the β-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHIMas integrity in the sea water environment to provide a firm basis for complex formation with MxaJMas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

Proteome analysis reveals global response to deletion of mrflbA in Monascus ruber: Qingqing Yan , Zhouwei Zhang , Yishan Yang , Fusheng Chen , Yanchun Shao; J. Microbiol. 2018;56(4):255-263. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-7425-8

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Abstract: Monascus spp. are commonly used for a wide variety of applications in the food and pharmaceutical industries. In previous studies, the knock-out of mrflbA (a putative regulator of the G protein α subunit) in M. ruber led to autolysis of the mycelia, decreased pigmentation and lowered mycotoxin production. Therefore, we aimed to obtain a comprehensive overview of the underlying mechanism of mrflbA deletion at the proteome level. A two-dimensional gel electrophoresis analysis of mycelial proteins indicated that the abundance of 178 proteins was altered in the ΔmrflbA strain, 33 of which were identified with high confidence. The identified proteins are involved in a range of activities, including carbohydrate and amino acid metabolism, hyphal development and the oxidative stress response, protein modification, and the regulation of cell signaling. Consistent with these findings, the activity of antioxidative enzymes and chitinase was elevated in the supernatant of the ΔmrflbA strain. Furthermore, deletion of mrflbA resulted in the transcriptional reduction of secondary metabolites (pigment and mycotoxin). In short, the mutant phenotypes induced by the deletion of mrflbA were consistent with changes in the expression levels of associated proteins, providing direct evidence of the regulatory functions mediated by mrflbA in M. ruber.

Identification of a novel phospholipase D gene and effects of carbon sources on its expression in Bacillus cereus ZY12: Yu Zhao , Yinfeng Xu , Fang Yu , Chunzhi Zhang; J. Microbiol. 2018;56(4):264-271. Published online April 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7529-1

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Abstract: In the present study, a new strain, Bacillus cereus ZY12, producing phospholipase D (PLD) was identified. The expression of PLD in this strain was found to be induced by its substrate, phosphatidylcholine (PC), and completely silenced by other carbon sources, such as glucose, fructose, and maltose, which are generally used in microbial growth cultures, thus presenting a unique expression pattern different from other PLD-producing microorganisms. This study is the first to report on the ability of B. cereus to produce PLD, and successfully clone its PLD-coding gene and identify its function, extending the knowledge on PLD distribution and evolution in microorganisms.

A murine colitis model developed using a combination of dextran sulfate sodium and Citrobacter rodentium: Jin-Il Park , Sun-Min Seo , Jong-Hyung Park , Hee-Yeon Jeon , Jun-Young Kim , Seung-Hyun Ryu , Yang-Kyu Choi; J. Microbiol. 2018;56(4):272-279. Published online April 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7504-x

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Abstract: Adult mice were treated with dextran sulfate sodium (DSS) and infected with Citrobacter rodentium for developing a novel murine colitis model. C57BL/6N mice (7-week-old) were divided into four groups. Each group composed of control, dextran sodium sulfate-treated (DSS), C. rodentiuminfected (CT), and DSS-treated and C. rodentium-infected (DSS-CT) mice. The DSS group was administered 1% DSS in drinking water for 7 days. The CT group was supplied with normal drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The DSS-CT group was supplied with 1% DSS in drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The mice were sacrificed 10 days after the induction of C. rodentium infection. The DSS-CT group displayed significantly shorter colon length, higher spleen to body weight ratio, and higher histopathological score compared to the other three groups. The mRNA expression levels of tumor necrosis factor (TNF)-α and interferon (INF)-γ were significantly upregulated; however, those of interleukin (IL)-6 and IL-10 were significantly downregulated in the DSS-CT group than in the control group. These results demonstrated that a combination of low DSS concentration (1%) and C. rodentium infection could effectively induce inflammatory bowel disease (IBD) in mice. This may potentially be used as a novel IBD model, in which colitis is induced in mice by the combination of a chemical and a pathogen.

UBCG: Up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction: Seong-In Na , Yeong Ouk Kim , Seok-Hwan Yoon , Sung-min Ha , Inwoo Baek , Jongsik Chun; J. Microbiol. 2018;56(4):280-285. Published online February 28, 2018; DOI: https://doi.org/10.1007/s12275-018-8014-6

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965 Citations

Abstract: Genome-based phylogeny plays a central role in the future taxonomy and phylogenetics of Bacteria and Archaea by replacing 16S rRNA gene phylogeny. The concatenated core gene alignments are frequently used for such a purpose. The bacterial core genes are defined as single-copy, homologous genes that are present in most of the known bacterial species. There have been several studies describing such a gene set, but the number of species considered was rather small. Here we present the up-to-date bacterial core gene set, named UBCG, and software suites to accommodate necessary steps to generate and evaluate phylogenetic trees. The method was successfully used to infer phylogenomic relationship of Escherichia and related taxa and can be used for the set of genomes at any taxonomic ranks of Bacteria. The UBCG pipeline and file viewer are freely available at https://www.ezbiocloud.net/ tools/ubcg and https://www.ezbiocloud.net/tools/ubcg_viewer, respectively.

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