Journal of Microbiology

Volume 57(2); February 2019

Review

[MINIREVIEW] Alanine dehydrogenases in mycobacteria: Ji-A Jeong , Jeong-Il Oh; J. Microbiol. 2019;57(2):81-92. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8543-7

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Abstract: Since NAD(H)-dependent L-alanine dehydrogenase (EC 1.1.4.1; Ald) was identified as one of the major antigens present in culture filtrates of Mycobacterium tuberculosis, many studies on the enzyme have been conducted. Ald catalyzes the reversible conversion of pyruvate to alanine with concomitant oxidation of NADH to NAD+ and has a homohexameric quaternary structure. Expression of the ald genes was observed to be strongly upregulated in M. tuberculosis and Mycobacterium smegmatis grown in the presence of alanine. Furthermore, expression of the ald genes in some mycobacteria was observed to increase under respiration-inhibitory conditions such as oxygen-limiting and nutrient-starvation conditions. Upregulation of ald expression by alanine or under respiration-inhibitory conditions is mediated by AldR, a member of the Lrp/AsnC family of transcriptional regulators. Mycobacterial Alds were demonstrated to be the enzymes required for utilization of alanine as a nitrogen source and to help mycobacteria survive under respiration-inhibitory conditions by maintaining cellular NADH/NAD+ homeostasis. Several inhibitors of Ald have been developed, and their application in combination with respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline was recently suggested.

Journal Articles

[PROTOCOL] Determination of protein phosphorylation by polyacrylamide gel electrophoresis: Chang-Ro Lee , Young-Ha Park , Huitae Min , Yeon-Ran Kim , Yeong-Jae Seok; J. Microbiol. 2019;57(2):93-100. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-9021-y

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35 Citations

Abstract: Phosphorylation is the most important modification for protein regulation; it controls many signal transduction pathways in all organisms. While several tools to detect phosphorylated proteins have been developed to study a variety of basic cellular processes involving protein phosphorylation, these methods have several limitations. Many proteins exhibit a phosphorylation-dependent electrophoretic mobility shift (PDEMS) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular mechanism responsible for this phenomenon has been elucidated recently. The method for detecting phosphorylated proteins can be simplified by the application of the PDEMS. Herein, we present a novel simple method to detect protein phosphorylation, which is based on the construction of a variant protein displaying a PDEMS. The PDEMS of proteins is caused by the distribution of negatively charged amino acids around the phosphorylation site, i.e. an electrophoretic mobility shift (EMS)-related motif (ΘX1-3ΘX1-3Θ, where Θ corresponds to an acidic or phosphorylated amino acid and X represents any amino acid). The EMS-related motif can be constructed by the introduction of a negative charge by phosphorylation; it results in the decreased binding of SDS to the proteins, consequently inducing the retardation of the mobility of the protein during SDS-PAGE. Based on these molecular analyses of the PDEMS, a protein with the EMSrelated motif is designed and used to determine the in vivo phosphorylation state of the protein. This method may be used as a general strategy to easily measure the ratio of protein phosphorylation in cells.

Blue-Red LED wavelength shifting strategy for enhancing beta-carotene production from halotolerant microalga, Dunaliella salina: Sang-Il Han , Sok Kim , Changsu Lee , Yoon-E Choi; J. Microbiol. 2019;57(2):101-106. Published online September 28, 2018; DOI: https://doi.org/10.1007/s12275-019-8420-4

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Abstract: In the present study, to improve the photosynthetic betacarotene productivity of Dunaliella salina, a blue-red LED wavelength-shifting system (B-R system) was investigated. Dunaliella salina under the B-R system showed enhanced density and beta-carotene productivity compared to D. salina cultivated under single light-emitting diode light wavelengths (blue, white, and red light-emitting diode). Additionally, we developed blue light-adapted D. salina (ALE-D. salina) using an adaptive laboratory evolution (ALE) approach. In combination with the B-R system applied to ALE-D. salina (ALE B-R system), the beta-carotene concentration (33.94 ± 0.52 μM) was enhanced by 19.7% compared to that observed for the non-ALE-treated wild-type of D. salina (intact D. salina) under the B-R system (28.34 ± 0.24 μM).

Paraburkholderia dokdonella sp. nov., isolated from a plant from the genus Campanula: Man-Young Jung , Myung-Suk Kang , Ki-Eun Lee , Eun-Young Lee , Soo-Je Park; J. Microbiol. 2019;57(2):107-112. Published online November 19, 2018; DOI: https://doi.org/10.1007/s12275-019-8500-5

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Abstract: The novel Gram-stain-negative, rod-shaped, aerobic bacterial strain DCR-13T was isolated from a native plant belonging to the genus Campanula on Dokdo, an island in the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence indicated that this strain is closely related to Paraburkholderia peleae PP52-1T (98.43% 16S rRNA gene sequence similarity), Paraburkholderia oxyphila NBRC 105797T (98.42%), Paraburkholderia sacchari IPT 101T (98.28%), Paraburkholderia mimosarum NBRC 106338T (97.80%), Paraburkholderia denitrificans KIS30-44T (97.46%), and Paraburkholderia paradise WAT (97.45%). This analysis of the 16S rRNA gene sequence also suggested that DCR-13T and the six closely related strains formed a clade within the genus Paraburkholderia, but that DCR-13T was clearly separated from the established species. DCR-13T had ubiquinone 8 as its predominant respiratory quinone, and its genomic DNA G + C content was 63.9 mol%. The isolated strain grew at a pH of 6.0–8.0 (with an optimal pH of 6.5), 0–4% w/v NaCl (with an optimal level of 0%), and a temperature of 18–42°C (with an optimal temperature of 30°C). The predominant fatty acids were C16:0, summed feature 8 (C18:1 ω7c/C18:1 ω6c), C17:0 cyclo, C19:0 cyclo ω8c, summed feature 3 (C16:1 ω6c/C16:1 ω7c) and summed feature 2 (C12:0 aldehyde), and the major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. On the basis of polyphasic evidence, it is proposed that strain DCR-13T (= KCTC 62811T = LMG 30889T) represents the type strain of a novel species, Paraburkholderia dokdonella sp. nov.

Rotavirus-mediated alteration of gut microbiota and its correlation with physiological characteristics in neonatal calves: Ja-Young Jang , Suhee Kim , Min-Sung Kwon , Jieun Lee , Do-Hyeon Yu , Ru-Hui Song , Hak-Jong Choi , Jinho Park; J. Microbiol. 2019;57(2):113-121. Published online November 19, 2018; DOI: https://doi.org/10.1007/s12275-019-8549-1

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34 Citations

Abstract: Diarrhea is a fatal disease to neonatal calves, and rotavirus is the main pathogen associated with neonatal calf diarrhea. Although previous studies have reported that the gut microbiota is changed in calves during diarrhea, less is known about whether rotavirus infection alters the structure of the gut microbiota. Here, we characterized fecal microbial communities and identified possible relationships between the gut microbiota profiles and physiological parameters. Five fecal specimens of rotavirus-infected calves from 1 to 30 days after birth and five fecal specimens of age-matched healthy calves were used for the microbial community analysis using the Illumina MiSeq sequencer. Rotavirus infection was associated with reduced rotavirus infection significantly reduced the richness and diversity of the bacterial community. Weighted unique fraction metric analysis exhibited significant differences in community membership and structure between healthy and rotavirus-infected calves. Based on relative abundance analysis and linear discriminant analysis effect size, we found that the representative genera from Lactobacillus, Subdoligranulum, Blautia, and Bacteroides were closely related to healthy calves, while the genera Escherichia and Clostridium were closely affiliated to rotavirus-infected calves. Furthermore, canonical correlation analysis and Pearson correlation coefficient results revealed that the increased relative abundances of Lactobacillus, Subdoligranulum, and Bacteroides were correlated with normal levels of physiological characteristics such as white blood cells, blood urea nitrogen, serum amyloid protein A, and glucose concentration in serum. These
results
suggest that rotavirus infection alters the structure of the gut microbiota, correlating changes in physiological

Chryseolinea soli sp. nov., isolated from soil: Shin Ae Lee , Yiseul Kim , Mee-Kyung Sang , Jaekyeong Song , Soon-Wo Kwon , Hang-Yeon Weon; J. Microbiol. 2019;57(2):122-126. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8562-4

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Abstract: A yellow-colored bacterium with gliding motility, strain KIS68-18T, was isolated from a soil sample at Bijin Island in Tongyeong city, Republic of Korea. The cells were strictly aerobic, Gram-staining-negative, non-spore-forming, and rod-shaped. The strain grew at the range of 10–35°C (optimum, 25–30°C), pH 5.5–8.0 (optimum, 6.0–7.5), and 0–0.5% (w/v) NaCl. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KIS68-18T was closely related to Chryseolinea serpens DSM 24574T (98.9%) and had low sequence similarities (below 92.6%) with other members of the family ‘Cytophagaceae’ in the phylum Bacteroidetes. The major respiratory quinone system was MK-7 and the predominant cellular fatty acids were C16:1 ω5c (38.8%), iso-C15:0 (18.5%), and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c, 10.6%). The polar lipids consisted of phosphatidylethanolamine, one unidentified phospholipid, three unidentified aminophospholipids, two unidentified aminolipids, and five unidentified lipids. The DNA G + C content was 50.9%. Based on the phylogenetic, physiological, and chemotaxonomic data, stain KIS68-18T represents a novel species of the genus Chryseolinea, for which the name Chryseolinea soli sp. nov. is proposed. The type strain of Chryseolinea soli is KIS68-18T (= KACC 17327T = NBRC 113100T).

Transcriptome analysis of differential gene expression in Dichomitus squalens during interspecific mycelial interactions and the potential link with laccase induction: Zixuan Zhong , Nannan Li , Binghui He , Yasuo Igarashi , Feng Luo; J. Microbiol. 2019;57(2):127-137. Published online September 13, 2018; DOI: https://doi.org/10.1007/s12275-019-8398-y

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Abstract: Interspecific mycelial interactions between white rot fungi are always accompanied by an increased production of laccase. In this study, the potential of the white rot fungus Dichomitus squalens to enhance laccase production during interactions with two other white rot fungi, Trametes versicolor or Pleurotus ostreatus, was assessed. To probe the mechanism of laccase induction and the role that laccase plays during combative interaction, we analyzed the differential gene expression profile of the laccase induction response to stressful conditions during fungal interaction. We further confirmed the expression patterns of 16 selected genes by qRT-PCR analysis. We noted that many differentially expressed genes (DEGs) encoded proteins that were involved in xenobiotic detoxification and reactive oxygen species (ROS) generation or reduction, including aldo/keto reductase, glutathione S-transferases, cytochrome P450 enzymes, alcohol oxidases and dehydrogenase, manganese peroxidase and laccase. Furthermore, many DEG-encoded proteins were involved in antagonistic mechanisms of nutrient acquisition and antifungal properties, including glycoside hydrolase, glucanase, chitinase and terpenoid synthases. DEG analyses effectively revealed that laccase induction was likely caused by protective responses to oxidative stress and nutrient competition during interspecific fungal interactions.

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1: Seong Hyuk Lee , Hwan Youn , Sung Gyun Kang , Hyun Sook Lee; J. Microbiol. 2019;57(2):138-142. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8592-y

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Abstract: Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

Competition/antagonism associations of biofilm formation among Staphylococcus epidermidis Agr groups I, II, and III: Sergio Martínez-García , César I. Ortiz-García , Marisa Cruz-Aguilar , Juan Carlos Zenteno , José Martin Murrieta-Coxca , Sonia Mayra Pérez-Tapia , Sandra Rodríguez-Martínez , Mario E. Cancino-Diaz , Juan C. Cancino-Diaz; J. Microbiol. 2019;57(2):143-153. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8322-5

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Abstract: Staphylococci have quorum-sensing (QS) systems that enable cell-to-cell communication, as well as the regulation of numerous colonization and virulence factors. The accessory gene regulator (Agr) operon is one of the Staphylococcus genus QS systems. Three groups (I, II, and III) are present in Staphylococcus epidermidis Agr operon. To date, it is unknown whether Agr groups can interact symbiotically during biofilm development. This study analyzed a symbiotic association among Agr groups during biofilm formation in clinical and commensal isolates. Different combinations among Agr group isolates was used to study biofilm formation in vitro and in vivo (using a mouse catheter-infection model). The analysis of Agr groups were also performed from samples of human skin (head, armpits, and nostrils). Different predominant coexistence was found within biofilms, suggesting symbiosis type. In vitro, Agr I had a competition with Agr II and Agr III. Agr II had a competition with Agr III, and Agr II was an antagonist to Agr I and III when the three strains were combined. In vivo, Agr II had a competition to Agr I, but Agr I and II were antagonists to Agr III. The associations found in vitro and in vivo were also found in different sites of the skin. Besides, other associations were observed: Agr III antagonized Agr I and II, and Agr III competed with Agr I and Agr II. These results suggest that, in S. epidermidis, a symbiotic association of competition and antagonism occurs among different Agr groups during biofilm formation.

Gentic overexpression increases production of hypocrellin A in Shiraia bambusicola S4201: Dan Li , Ning Zhao , Bing-Jing Guo , Xi Lin , Shuang-Lin Chen , Shu-Zhen Yan; J. Microbiol. 2019;57(2):154-162. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8259-8

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Abstract: Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.

Efficacy of A/H1N1/2009 split inactivated influenza A vaccine (GC1115) in mice and ferrets: Hae Jung Han , Min-Suk Song , Su-Jin Park , Han Yeul Byun , Norbert John C. Robles , Suk-Hoon Ha , Young Ki Choi; J. Microbiol. 2019;57(2):163-169. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8504-1

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Abstract: To evaluate the efficacy of a non-adjuvant A/H1N1/2009 influenza A vaccine (GC1115), we demonstrated the immunogenicity and protective efficacy of GC1115 in mouse and ferret models. The immunogenicity of GC1115 was confirmed after intramuscular administration of 1.875, 3.75, 7.5, and 15 μg hemagglutinin antigen (HA) in mice and 7.5, 15, and 30 μg HA in ferrets at 3-week intervals. A single immunization with GC1115 at HA doses > 7.5 μg induced detectable seroconversion in most mice, and all mice given a second dose exhibited high antibody responses in a dose-dependent manner. The mice in the mock (PBS) and 1.875 μg HA immunized groups succumbed by 13 days following A/California/ 04/09 infection, while all mice in groups given more than 3.75 μg HA were protected from lethal challenge with the A/California/04/09 virus. In ferrets, although immunization with even a single dose of 15 or 30 μg of HA induced detectable HI antibodies, all ferrets given two doses of vaccine seroconverted and exhibited HI titers greater than 80 units. Following challenge with A/California/04/09, the mock (PBS) immunized ferrets showed influenza-like clinical symptoms, such as increased numbers of coughs, elevated body temperature, and body weight loss, for 7 days, while GC1115- immunized ferrets showed attenuated clinical symptoms only for short time period (3–4 days). Further, GC1115-immunized ferrets displayed significantly lower viral titers in the upper respiratory tract (nasal cavity) than the mock vaccinated group in a dose-dependent manner. Taken together, this study demonstrates the immunogenicity and protective efficacy of GC1115 as a non-adjuvanted vaccine.

Lytic KFS-SE2 phage as a novel bio-receptor for Salmonella Enteritidis detection: In Young Choi , Cheonghoon Lee , Won Keun Song , Sung Jae Jang , Mi-Kyung Park; J. Microbiol. 2019;57(2):170-179. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8610-0

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Abstract: Since Salmonella Enteritidis is one of the major foodborne pathogens, on-site applicable rapid detection methods have been required for its control. The purpose of this study was to isolate and purify S. Enteritidis-specific phage (KFS-SE2 phage) from an eel farm and to investigate its feasibility as a novel, efficient, and reliable bio-receptor for its employment. KFS-SE2 phage was successfully isolated at a high concentration of (2.31 ± 0.43) × 1011 PFU/ml, and consisted of an icosahedral head of 65.44 ± 10.08 nm with a non-contractile tail of 135.21 ± 12.41 nm. The morphological and phylogenetic analysis confirmed that it belongs to the Pis4avirus genus in the family of Siphoviridae. KFS-SE2 genome consisted of 48,608 bp with 45.7% of GC content. Genome analysis represented KFS-SE2 to have distinctive characteristics as a novel phage. Comparative analysis of KFS-SE2 phage with closely related strains confirmed its novelty by the presence of unique proteins. KFS-SE2 phage exhibited excellent specificity to S. Enteritidis and was stable under the temperature range of 4 to 50°C and pH of 3 to 11 (P < 0.05). The latent time was determined to be 20 min. Overall, a new lytic KFS-SE2 phage was successfully isolated from the environment at a high concentration and the excellent feasibility of KFS-SE2 phage was demonstrated as a new bio-receptor for S. Enteritidis detection.

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