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- Volume 57(12); December 2019
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Journal Articles
- [Protocol]Construction of a multicopy genomic DNA library and its application for suppression analysis
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Hongbaek Cho
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J. Microbiol. 2019;57(12):1041-1047. Published online November 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-9417-8
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Abstract
- Suppression analysis is used for the identification of new genes
and genetic interactions when there is a notable phenotype
available for genetic selection or screening. A random genomic
DNA library constructed on a multi-copy plasmid is a
useful tool for suppression analysis when one expects that
an overdose of a few genes will suppress the phenotype. These
libraries have been successfully used to determine the function
of a gene by revealing genes whose functions are related
to the gene of interest. They have also been used to identify
the targets of chemical or biological agents by increasing the
number of unaffected target gene products in a cell. In this
article, I will discuss important considerations for constructing
multicopy genomic DNA libraries. The protocol provided
in this paper should be a useful guide for constructing
genomic DNA libraries in many bacterial species for which
multi-copy plasmids are available.
- 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
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Young-Ju Kim , Soon Dong Lee
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J. Microbiol. 2019;57(12):1048-1055. Published online September 25, 2019
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DOI: https://doi.org/10.1007/s12275-019-9260-y
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Abstract
- A Gram-stain-negative, strictly aerobic, marine bacterium,
designated GH2-2T, was isolated from a rhizosphere mudflat
of a halophyte (Carex scabrifolia) in Gangwha Island,
the Republic of Korea. The cells of the organism were oxidase-
positive, catalase-positive, flagellated, short rods that
grew at 10–40°C, pH 4–10, and 0–13% (w/v) NaCl. The predominant
ubiquinone was Q-10. The major polar lipids were
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol.
The major fatty acid is C18:1. Phylogenetic
analysis based on 16S rRNA gene sequences revealed that the
novel isolate formed an independent lineage at the base of
the radiation encompassing members of the genus Thioclava,
except for Thioclava arenosa. The closest relatives were T.
nitratireducens (96.03% sequence similarity) and T. dalianensis
(95.97%). The genome size and DNA G+C content
were 3.77 Mbp and 59.6 mol%, respectively. Phylogenomic
analysis supported phylogenetic distinctness based on 16S
rRNA gene sequences. Average nucleotide identity values
were 73.6–74.0% between the novel strain and members of
the genus Thioclava. On the basis of data obtained from a
polyphasic approach, the strain GH2-2T (= KCTC 62124T =
DSM 105743T) represents a novel species of a new genus for
which the name Hahyoungchilella caricis gen. nov., sp. nov. is
proposed. Moreover, the transfer of Thioclava arenosa Thongphrom
et al. 2017 to Pseudothioclava gen. nov. as Pseudothioclava
arenosa comb. nov. is also proposed. Finally, Thioclava
electrotropha Chang et al. 2018 is proposed to be a later
heterosynonym of Thioclava sediminum Liu et al. 2017.
- Soft sweep development of resistance in Escherichia coli under fluoroquinolone stress
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Xianxing Xie , Ruichen Lv , Chao Yang , Yajun Song , Yanfeng Yan , Yujun Cui , Ruifu Yang
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J. Microbiol. 2019;57(12):1056-1064. Published online September 25, 2019
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DOI: https://doi.org/10.1007/s12275-019-9177-5
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Abstract
- We employed a stepwise selection model for investigating the
dynamics of antibiotic-resistant variants in Escherichia coli
K-12 treated with increasing concentrations of ciprofloxacin
(CIP). Firstly, we used Sanger sequencing to screen the variations
in the fluoquinolone target genes, then, employed Illumina
NGS sequencing for amplicons targeted regions with
variations. The results demonstrated that variations G81C in
gyrA and K276N and K277L in parC are standing resistance
variations (SRVs), while S83A and S83L in gyrA and G78C
in parC were emerging resistance variations (ERVs). The variants
containing SRVs and/or ERVs were selected successively
based on their sensitivities to CIP. Variant strain 1, containing
substitution G81C in gyrA, was immediately selected
following ciprofloxacin exposure, with obvious increases in
the parC SRV, and parC and gyrA ERV allele frequencies.
Variant strain 2, containing the SRVs, then dominated the
population following a 20× increase in ciprofloxacin concentration,
with other associated allele frequencies also elevated.
Variant strains 3 and 4, containing ERVs in gyrA and parC,
respectively, were then selected at 40× and 160× antibiotic
concentrations. Two variants, strains 5 and 6, generated in
the selection procedure, were lost because of higher fitness
costs or a lower level of resistance compared with variants 3
and 4. For the second induction, all variations/indels were
already present as SRVs and selected out step by step at different
passages. Whatever the first induction or second induction,
our results confirmed the soft selective sweep hypothesis
and provided critical information for guiding clinical
treatment of pathogens containing SRVs.
- Flavobacterium zhairuonensis sp. nov., a gliding bacterium isolated from marine sediment of the East China Sea
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Sanjit Chandra Debnath , Ahmed Mohammed Abdo Miyah , Can Chen , Huan Sheng , Xue-Wei Xu , Yue-Hong Wu , Dao-Qiong Zheng , Jin-Zhong Xu , Ya-Nan Di , Pin-Mei Wang , Li Shen
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J. Microbiol. 2019;57(12):1065-1072. Published online September 27, 2019
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DOI: https://doi.org/10.1007/s12275-019-9194-4
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Abstract
- A yellow pigmented, Gram-stain-negative, aerobic bacterium
designated A5.7T was studied to evaluate the taxonomic position
following the modern polyphasic approach. The strain
was isolated from sediments near Zhairuo Island, which is
situated in the East China Sea. Cells were non-spore forming
rods without flagella but showed motility by gliding. Growth
was observed at 15–35°C (optimum 28°C), pH 6.0–9.0 (optimum
pH 6.5) and 0–2% (w/v) NaCl (optimum 0–0.5%) in
LB broth. The major respiratory quinone of A5.7T was menaquinone
6. The major polar lipid of A5.7T was phosphatidylethanolamine
and the predominant fatty acids (> 5%) were
iso-C15:0, iso-C17:0 3-OH, C15:1 ω6c, iso-C15:0 3-OH, iso-C15:1 G,
summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed
feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl). Phylogenetic
analysis based on 16S rRNA gene sequences showed that the
isolate belongs to the genus Flavobacterium and shares the
highest sequence similarities with Flavobacterium sharifuzzamanii
A7.6T (98.5%), Flavobacterium tistrianum GB 56.1T
(98.3%), Flavobacterium nitrogenifigens NXU-44T (97.8%),
Flavobacterium anhuiense D3T (97.6%), Flavobacterium ginsenosidimutans
THG 01T (97.6%), and Flavobacterium foetidum
CJ42T (97.6%). Digital DNA-DNA hybridization and
average nucleotide identity values between the strain and its
closest phylogenetic neighbors showed the ranges from 19.6
to 34.1% and 73.7 to 87.9%, respectively. Therefore, based
on polyphasic characteristics, strain A5.7T represents a novel
species of the genus Flavobacterium for which the name Flavobacterium
zhairuonensis sp. nov. is proposed. The type
strain is A5.7T (= KCTC 62406T = MCCC 1K03494T).
- Anaerotignum faecicola sp. nov., isolated from human faeces
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Seung-Hyeon Choi , Ji-Sun Kim , Jam-Eon Park , Keun Chul Lee , Mi Kyung Eom , Byeong Seob Oh , Seung Yeob Yu , Se Won Kang , Kook-Il Han , Min Kuk Suh , Dong Ho Lee , Hyuk Yoon , Byung-Yong Kim , Je Hee Lee , Ju Huck Lee , Jung-Sook Lee , Seung-Hwan Park
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J. Microbiol. 2019;57(12):1073-1078. Published online November 4, 2019
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DOI: https://doi.org/10.1007/s12275-019-9268-3
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Abstract
- A strictly anaerobic bacterium, designated as strain KGMB-
03357T, was isolated from the faeces of a healthy Korean selected
by Bundang Seoul National University based on health
status. Cells of strain KGMB03357T are Gram-stain-positive,
non-motile, non-spore-forming, and observed as straight or
curved rods. The isolate grew at 10–45°C (optimum temperature
of 40°C) and a pH range of 5.1–10.5 (optimum pH of
6.8). Analysis of phylogenetic trees based on the 16S rRNA
gene sequences revealed that strain KGMB03357T forms a
lineage within the genus Anaerotignum, and is most closely
related to Anaerotignum lactatifermentans G17T (= KCTC
15066T, 96.1%), Anaerotignum propionicum DSM 1682T (=
KCTC 5582T, 94.9%), Anaerotignum neopropionicum DSM
03847T (= KCTC 15564T, 94.9%), and Anaerotignum aminivorans
SH021T (= KCTC 15705T, 94.8%). The ANI values
between strain KGMB 03357T and members of the genus
Anaerotignum were 73.3–71.0%, which are below the ANI
criterion for interspecies identity. The DNA G + C content
based on the whole-genome sequence is 47.3 mol%. The major
cellular fatty acids of strain KGMB03357T are C16:0, C18:0,
C18:1 cis 9, and anteiso-C15:0. Strain KGMB03357T contains
meso-diaminopimelic acid as the diagnostic amino acid in
the cell wall peptidoglycan. Based on the phenotypic, phylogenetic,
and genomic properties, strain KGMB 03357T represents
a novel species of the genus Anaerotignum, for which
the name Anaerotignum faecicola sp. nov. is proposed. The
type strain is KGMB03357T (= KCTC 15736T = DSM 107953T).
- Flavobacterium humi sp. nov., a flexirubin-type pigment producing bacterium, isolated from soil
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Inhyup Kim , Jiyoun Kim , Geeta Chhetri , Taegun Seo
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J. Microbiol. 2019;57(12):1079-1085. Published online November 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-9350-x
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Abstract
- A yellow pigmented, Gram-stain-negative, strictly aerobic,
rod-shaped, motile by means of gliding, catalase and oxidase
positive bacterium, designated strain DS2-AT, was isolated
from soil. Growth was observed at 4–32°C (optimum, 28°C),
pH 6–9 (optimum, 7.0), and with 0–0.25% (w/v) NaCl (optimum,
0%). Phylogenetic analysis of 16S rRNA gene sequence
revealed that strain DS2-AT belonged to the genus Flavobacterium
and was most closely related to Flavobacterium
aquatile LMG 4008T (96.4%), Flavobacterium terrae DSM
18829T (95.6%), Flavobacterium vireti THG-SM1T (95.5%),
Flavobacterium inkyongense IMCC27201T (95.4%), Flavobacterium
brevivitae TTM-43T (95.2%), and Flavobacterium
cucumis DSM 18830T (95.2%). Strain DS2-AT produces flexirubin-
type pigments. The major fatty acids were iso-C15:0,
iso-C17:0 3-OH, and iso-C15:0 3-OH. The major respiratory
quinone was identified as menaquinone-6. The major polar
lipid was found to be phosphatidylethanolamine. The average
nucleotide identity values between strain DS2-AT and selected
taxa, F. aquatile LMG 4008T, F. terrae DSM 18829T,
and F. cucumis DSM 18830T, were 72, 72.7, and 71.6%, respectively.
The draft genome of strain DS2-AT has a number
of 14 contigs, scaffold N50 of 476,310 bp and a total size of
3,563,867 bp. Additionally, strain DS2-AT contains 3,127 of
gene, 41 of tRNA, 6 of rRNA, and 3 of ncRNA. The DNA G
+ C content of stain DS2-AT was 40.7 mol%. Based on phylogenetic
and phenotypic analyses, strain DS2-AT is considered
as a novel species of the genus Flavobacterium, for which
the name Flavobacterium humi sp. nov., (type strain DS2-AT
= KACC 19715T = JCM 32786T) has been proposed.
- Partial characteristics of hemolytic factors secreted from airborne Aspergillus and Penicillium, and an enhancement of hemolysis by Aspergillus micronesiensis CAMP-like factor via Staphylococcus aureus-sphingomyelinase
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Sumonrat Kaveemongkonrat , Kwanjit Duangsonk , Jos Houbraken , Phimchat Suwannaphong , Nongnuch Vanittanakom Vanittanakom , Malee Mekaprateep
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J. Microbiol. 2019;57(12):1086-1094. Published online November 4, 2019
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DOI: https://doi.org/10.1007/s12275-019-9133-4
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Abstract
- One of the advantages for initial survival of inhaled fungal
spores in the respiratory tract is the ability for iron acquisition
via hemolytic factor-production. To examine the ability
of indoor Aspergillus and Penicillium affecting hemolysis,
the secreted factors during the growth of thirteen strains from
eight species were characterized in vitro for their hemolytic
activity (HA) and CAMP-like reaction. The hemolytic index
of HA on human blood agar of Aspergillus micronesiensis,
Aspergillus wentii, Aspergillus westerdijkiae, Penicillium citrinum,
Penicillium copticola, Penicillium paxilli, Penicillium
steckii, and Penicillium sumatrense were 1.72 ± 0.34, 1.61 ±
0.41, 1.69 ± 0.16, 1.58 ± 0.46, 3.10 ± 0.51, 1.22 ± 0.19, 2.55 ±
0.22, and 1.90 ± 0.14, respectively. The secreted factors of
an Aspergillus wentii showed high HA when grown in undernourished
broth at 25°C at an exponential phase and were
heat sensitive. Its secreted proteins have an estimated relative
molecular weight over 50 kDa. Whereas, the factors of
Penicillium steckii were secreted in a similar condition at a
late exponential phase but showed low HA and heat tolerance.
In a CAMP-like test with sheep blood, the synergistic hemolytic
reactions between most tested mold strains and Staphylococcus
aureus were identified. Moreover, the enhancement
of α-hemolysis of Staphylococcus aureus could occur through
the interaction of Staphylococcus aureus-sphingomyelinase
and CAMP-like factors secreted from Aspergillus micronesiensis.
Further studies on the characterization of purified hemolytic-
and CAMP-like-factors secreted from Aspergillus
wentii and Aspergillus micronesiensis may lead to more understanding
of their involvement of hemolysis
- H2 Metabolism revealed by metagenomic analysis of subglacial sediment from East Antarctica
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Zhifeng Yang , Yu Zhang , Yongxin Lv , Wenkai Yan , Xiang Xiao , Bo Sun , Hongmei Ma
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J. Microbiol. 2019;57(12):1095-1104. Published online November 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-9366-2
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Abstract
- Subglacial ecosystems harbor diverse chemoautotrophic microbial
communities in areas with limited organic carbon,
and lithological H2 produced during glacial erosion has been
considered an important energy source in these ecosystems.
To verify the H2-utilizing potential there and to identify the
related energy-converting metabolic mechanisms of these
communities, we performed metagenomic analysis on subglacial
sediment samples from East Antarctica with and without
H2 supplementation. Genes coding for several [NiFe]-
hydrogenases were identified in raw sediment and were enriched
after H2 incubation. All genes in the dissimilatory
nitrate reduction and denitrification pathways were detected
in the subglacial community, and the genes coding for these
pathways became enriched after H2 was supplied. Similarly,
genes transcribing key enzymes in the Calvin cycle were detected
in raw sediment and were also enriched. Moreover,
key genes involved in H2 oxidization, nitrate reduction, oxidative
phosphorylation, and the Calvin cycle were identified
within one metagenome-assembled genome belonging to a
Polaromonas sp. As suggested by our results, the microbial
community in the subglacial environment we investigated
consisted of chemoautotrophic populations supported by H2
oxidation. These results further confirm the importance of
H2 in the cryosphere.
- Vibrio parahaemolyticus cqsA controls production of quorum sensing signal molecule 3-hydroxyundecan-4-one and regulatessensing signal molecule 3-hydroxyundecan-4-one and regulates colony morphology
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Kui Wu , Yangyun Zheng , Qingping Wu , Haiying Chen , Songzhe Fu , Biao Kan , Yongyan Long , Xiansheng Ni , Junling Tu
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J. Microbiol. 2019;57(12):1105-1114. Published online November 4, 2019
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DOI: https://doi.org/10.1007/s12275-019-9379-x
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Abstract
- In order to adapt to different environments, Vibrio parahaemolyticus
employed a complicated quorum sensing system to
orchestrate gene expression and diverse colony morphology
patterns. In this study, the function of the putative quorum
sensing signal synthase gene cqsA (VPA0711 in V. parahaemolyticus
strain RIMD2210633 genome) was investigated.
The cloning and expression of V. parahaemolyticus cqsA in
Escherichia coli system induced the production of a new quorum
sensing signal that was found in its culture supernatant.
The signal was purified by high performance liquid chromatography
methods
and determined to be 3-hydroxyundecan-
4-one by indirect and direct mass spectra assays. The deletion
of cqsA in RIMD2210633 changed V. parahaemolyticus
colony morphology from the classical ‘fried-egg’ shape (thick
and opaque in the center, while thin and translucent in the
edge) of the wild-type colony to a ‘pancake’ shape (no significant
difference between the centre and the edge) of the cqsAdeleted
colony. This morphological change could be restored
by complementary experiment with cqsA gene or the signal
extract. In addition, the expression of opaR, a well-known
quorum sensing regulatory gene, could be up-regulated by
cqsA deletion. Our results suggested that V. parahaemolyticus
used cqsA to produce 3-hydroxyundecan-4-one signal
and thereby regulated colony morphology and other quorum
sensing-associated behaviors.
- A histone deacetylase, MoHOS2 regulates asexual development and virulence in the rice blast fungus
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Jongjune Lee , Jae-Joon Lee , Junhyun Jeon
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J. Microbiol. 2019;57(12):1115-1125. Published online November 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-9363-5
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Abstract
- Histone acetylation/deacetylation represent a general and
efficient epigenetic mechanism through which fungal cells control
gene expression. Here we report developmental requirement
of MoHOS2-mediated histone deacetylation (HDAC)
for the rice blast fungus, Magnaporthe oryzae. Structural similarity
and nuclear localization indicated that MoHOS2 is an
ortholog of Saccharomyces cerevisiae Hos2, which is a member
of class I histone deacetylases and subunit of Set3 complex.
Deletion of MoHOS2 led to 25% reduction in HDAC
activity, compared to the wild-type, confirming that it is a
bona-fide HDAC. Lack of MoHOS2 caused decrease in radial
growth and impinged dramatically on asexual sporulation.
Such reduction in HDAC activity and phenotypic defects of
ΔMohos2 were recapitulated by a single amino acid change
in conserved motif that is known to be important for HDAC
activity. Expression analysis revealed up-regulation of MoHOS2
and concomitant down-regulation of some of the key genes
involved in asexual reproduction under sporulation-promoting
condition. In addition, the deletion mutant exhibited defect
in appressorium formation from both germ tube tip and
hyphae. As a result, ΔMohos2 was not able to cause disease
symptoms. Wound-inoculation showed that the mutant is
compromised in its ability to grow inside host plants as well.
We found that some of ROS detoxifying genes and known
effector genes are de-regulated in the mutant. Taken together,
our data suggest that MoHOS2-dependent histone deacetylation
is pivotal for proper timing and induction of transcription
of the genes that coordinate developmental changes
and host infection in M. oryzae.
- Methyltransferase of a cell culture-adapted hepatitis E inhibits the MDA5 receptor signaling pathway
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Jinjong Myoung , Jeong Yoon Lee , Kang Sang Min
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J. Microbiol. 2019;57(12):1126-1131. Published online November 22, 2019
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DOI: https://doi.org/10.1007/s12275-019-9478-8
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Abstract
- Hepatitis E virus (HEV) is a causative agent of acute hepatitis
and jaundice. The number of human infections is approximated
to be over 20 million cases per year. The transmission
is mainly via the fecal-oral route and contaminated water and
food are considered to be a major source of infection. As a
mouse model is not available, a recent development of a cell
culture-adapted HEV strain (47832c) is considered as a very
important tools for molecular analysis of HEV pathogenesis
in cells. Previously, we demonstrated that HEV-encoded methyltransferase
(MeT) encoded by the 47832c strain inhibits
MDA5- and RIG-I-mediated activation of interferon β (IFN-β)
promoter. Here, we report that MeT impairs the phosphorylation
and activation of interferon regulatory factor 3 and the
p65 subunit of NF-κB in a dose-dependent manner. In addition,
the MeT encoded by the 47832c, but not that of HEV
clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1),
displays the inhibitory effect. A deeper understanding of MeTmediated
suppression of IFN-β expression would provide
basis of the cell culture adaptation of HEV.
Published Erratum
- Erratum]Flavobacterium parvum sp. nov., isolated from soil polluted by sewer water
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Hyun Seo Lee , Woon Mo Hwang , Keunsoo Kang , Tae-Young Ahn
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J. Microbiol. 2019;57(12):1132-1132.
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DOI: https://doi.org/10.1007/s12275-019-0720-1
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Abstract
- In the article by Lee et al. published in Journal of Microbiology 2018; 56, 542–548, The KACC 19447T on 26th line of 4th paragraph
in the section of ‘Description of Flavobacterium parvum sp. nov.’ on page 546 should be corrected in KACC 19448T.
The sentence should have read: The type strain, HS916T (= KACC 19448T, = JCM 32368T), was isolated from soil polluted
by sewer water in Cheonan, South Korea.
We apologize for any inconvenience that this may have caused.
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