2-piperidone is a crucial industrial raw material of high-value nylon-5 and nylon-6,5. Currently, a major bottleneck in the biosynthesis of 2-piperidone is the identification of highly efficient 2-piperidone synthases. In this study, we aimed to identify specific strains among 51 human gut bacterial strains capable of producing 2-piperidone and to elucidate its synthetic mechanism. Our findings revealed that four gut bacterial strains, namely Collinsella aerofaciens LFYP39, Collinsella intestinalis LFYP54, Clostridium bolteae LFYP116, and Clostridium hathewayi LFYP18, could produce 2-piperidone from 5-aminovaleric acid (5AVA).
Additionally, we observed that 2-piperidone could be synthesized from proline through cross-feeding between Clostridium difficile LFYP43 and one of the four 2-piperidone producing strains, respectively. To identify the enzyme responsible for catalyzing the conversion of 5AVA to 2-piperidone, we utilized a gain-of-function library and identified avaC (5-aminovaleric acid cyclase) in C.
intestinalis LFYP54. Moreover, homologous genes of avaC were validated in the other three bacterial strains. Notably, avaC were found to be widely distributed among environmental bacteria. Overall, our research delineated the gut bacterial strains and genes involved in 2-piperidone production, holding promise for enhancing the efficiency of industrial biosynthesis of this compound.
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This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer.
In the post-genomic era, phylogenomics is a powerful and routinely-used tool to discover evolutionary relationships between
microorganisms. Inferring phylogenomic trees by concatenating core gene sequences into a supermatrix is the standard
method . The previously released up-to-date bacterial core gene (UBCG) tool provides a pipeline to infer phylogenomic trees
using single-copy core genes for the Bacteria domain. In this study, we established up-to-date archaeal core gene (UACG),
comprising 128 genes suitable for inferring archaeal phylogenomic trees. To test the gene set, we selected the Haloarcula
genus and scrutinized its phylogeny. The phylogeny inferred using the UACG tool was consistent with the orthoANIu
dendrogram, whereas the 16S rRNA gene phylogeny showed high intragenomic heterogeneity resulting in phylogenetic
discrepancies. The software tool using the UACG set is available at https:// www. ezbio cloud. net/ tools/ uacg.
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Update on the proposed minimal standards for the use of genome data for the taxonomy of prokaryotes Raúl Riesco, Martha E. Trujillo
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Catalases are key antioxidant enzymes in aerobic organisms.
Myxococcus xanthus expresses two monofunctional catalases,
small-subunit Cat1 and large-subunit Cat2. The Km of
H2O2 for recombinant Cat1 and Cat2 were 14.0 and 9.0 mM,
respectively, and the catalytic efficiency of Cat2 (kcat/Km =
500 sec-1 mM-1) was 4-fold higher than that of Cat1. The activity
ratio of Cat1 to Cat2 in the exponential growth phase
of M. xanthus was 1 to 3–4. A Cat1-deficient strain was constructed,
whereas a Cat2-deficient strain could not be produced.
In H2O2-supplemented medium, the cat1 mutant exhibited
marked growth retardation and a longer generation
time than the wild-type (wt) strain. After 2 h of incubation
in 0.5 mM H2O2-supplemented medium, the catalase activity
of the wt strain significantly increased (by 64-fold), but that
of the cat1 mutant strain did not. Under starvation-induced
developmental conditions, catalase activity was induced by
approximately 200-fold in both wt and cat1 strains, although
in the mutant the activity increase as well as spore formation
occurred one day later, indicating that the induction of catalase
activity during starvation was due to Cat2. In wt starved
cells, catalase activity was not induced by H2O2. These results
suggest that Cat2 is the primary housekeeping catalase
during M. xanthus growth and starvation-induced development,
whereas Cat1 may have a complementary role, being
responsible for the rapid degradation of H2O2 in proliferating
vegetative cells subjected to oxidative stress.
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The genome is highly organized hierarchically by the function
of structural maintenance of chromosomes (SMC) complex
proteins such as condensin and cohesin from bacteria
to humans. Although the roles of SMC complex proteins have
been well characterized, their specialized roles in nuclear processes
remain unclear. Condensin and cohesin have distinct
binding sites and mediate long-range and short-range genomic
associations, respectively, to form cell cycle-specific
genome organization. Condensin can be recruited to highly
expressed genes as well as dispersed repeat genetic elements,
such as Pol III-transcribed genes, LTR retrotransposon, and
rDNA repeat. In particular, mitotic transcription factors Ace2
and Ams2 recruit condensin to their target genes, forming
centromeric clustering during mitosis. Condensin is potentially
involved in various chromosomal processes such as the
mobility of chromosomes, chromosome territories, DNA reannealing,
and transcription factories. The current knowledge
of condensin in fission yeast summarized in this review can
help us understand how condensin mediates genome organization
and participates in chromosomal processes in other
organisms.
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(OMVs) are significant and highly functional. The proteins
and other biomolecules identified within OMVs provide
new insights into the possible functions of OMVs in bacteria.
OMVs are rich in proteins, nucleic acids, toxins and
virulence factors that play a critical role in bacteria-host interactions.
In this review, we discuss some proteins with multifunctional
features from bacterial OMVs and their role
involving the mechanisms of bacterial survival and defence.
Proteins with moonlighting activities in OMVs are discussed
based on their functions in bacteria. OMVs harbour many
other proteins that are important, such as proteins involved
in virulence, defence, and competition. Overall, OMVs are a
power-packed aid for bacteria, harbouring many defensive
and moonlighting proteins and acting as a survival kit in case of an emergency or as a defence weapon. In summary,
OMVs can be defined as bug-out bags for bacterial defence
and, therefore, survival.
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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.
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In the present study, a laccase gene (BaLc) from a lignin degrading
bacterium, Bacillus atrophaeus, has been cloned
and expressed in Escherichia coli. The optimal catalytic activity
of the protein was achieved at 5.5 pH and 35°C temperature,
measured by oxidation of ABTS. The Km and Vmax
values were determined as 1.42 mM and 4.16 μmole/min, respectively.
To achieve the enzyme recovery, the biocatalyst
(BaLc) was covalently attached onto the functionalized iron
magnetic-nanoparticles. The nanoparticles were characterized
by zeta-potential and FTIR analyses. The immobilized BaLc
enzyme was physico-kinetically characterized, exhibiting retention
of 60% of the residual activity after ten reaction cycles
of ABTS oxidation. The immobilized biocatalyst system was
tested for its biotechnological exploitability in plant juice
processing, achieving 41–58% of phenol reduction, 41–58%
decolorization, 50–59% turbidity reduction in the extracts of
banana pseudo-stem and sweet sorghum stalk, and apple fruit
juice. This is the first study to demonstrate the use of nanoparticle-
laccase conjugate in juice clarification. The findings
suggest that B. atrophaus laccase is a potential catalytic tool
for plant juice bioprocessing activities.
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in ecological studies, as well as in the classification, typing,
and phylogenetic analysis of prokaryotes. These techniques
are mainly aimed at whole genome comparisons and PCRderived
experiments, including amplifying the 16S rRNA
and other various housekeeping genes used in taxonomy,
as well as MLST (multilocus sequence typing) and MLSA
(multilocus sequence analysis) of different taxonomic bacterial
groups. The gene encoding threonine-tRNA ligase
(thrS) is a gene potentially applicable as an identification
and phylogenetic marker in bacteria. It is widely distributed
in bacterial genomes and is subject to evolutionary selection
pressure due to its important function in protein synthesis.
In this study, specific primers were used to amplify a thrS
gene fragment (~740 bp) in 36 type and 30 wild strains classified
under family Bifidobacteriaceae. The full-length gene
has not yet been considered as a possible identification, classification,
and phylogenetic marker in bifidobacteria. The
thrS sequences revealed higher sequence variability (82.7%
of pairwise identities) among members of the family than
that shown by 16S rRNA gene sequences (96.0%). Although
discrepancies were found between the thrS-derived and previously
reported whole genome phylogenetic analyses, the
main phylogenetic groups of bifidobacteria were properly
assigned. Most wild strains of bifidobacteria were better differentiated
based on their thrS sequences than on their 16S
rRNA gene identities. Phylogenetic confidence of the evaluated
gene with respect to other alternative genetic markers
widely used in taxonomy of bifidobacteria (fusA, GroELhsp60,
pyrG, and rplB genes) was confirmed using the localized
incongruence difference - Templeton analysis.
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Corynespora cassiicola is a species of fungus that is a plant
pathogen of many agricultural crop plants, including severe
target spot disease on cucumber. Cassiicolin is an important
effector of pathogenicity of this fungus. In this study, we
collected 141 Corynespora isolates from eighteen hosts, and
the casscolin gene was detected in 82 C. cassiicola strains.
The deduced protein sequences revealed that 72 isolates
contained the Cas2 gene, two strains from Gynura bicolor
harboured the Cas2.2 gene, and 59 isolates without a cassiicolin
gene were classified as Cas0. Phylogenetic analyses was
performed for the 141 isolates using four loci (ITS, ga4, caa5,
and act1) and revealed two genetic clusters. Cluster A is composed
of four subclades: subcluster A1 includes all Cas2
isolates plus 18 Cas0 strains, subcluster A2 includes the eight
Cas5 isolates and one Cas0 isolate, and subclusters A3 and
A4 contain Cas0 strains. Cluster B consists of 21 Cas0 isolates.
Twenty-two C. cassiicola strains from different toxin
classes showed varying degrees of virulence against cucumber.
Cas0 or Cas2 strains induced diverse responses on cucumber,
from no symptoms to symptoms of moderate or severe
infection, but all Cas5 isolates exhibited avirulence on cucumber.
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Varicella-zoster virus (VZV) is a causative agent of chickenpox
in primary infection and shingles after its reactivation
from latency. Complete or almost-complete genomic DNA
sequences for various VZV strains have been reported. Recently,
clinical VZV strains were isolated from Korean patients
whose genome was sequenced using high-throughput
sequencing technology. In this study, we analyzed single nucleotide
polymorphism (SNP) of VZV strains to genetically
characterize Korean clinical isolates. Phylogenetic analyses
revealed that three Korean strains, YC01, YC02, and YC03,
were linked to clade 2. Comprehensive SNP analysis identified
86 sites specific for the 5 VZV clades. VZV strains isolated
from Korea did not form a phylogenetic cluster. Rather,
YC02 and YC03 clustered strongly with Chinese strain 84-7
within clade 2, more specifically cluster 2a. Signature sequences
for the cluster 2a were identified and found to play an
important role in the separation of cluster 2a strains from
other clade 2 strains, as shown in substitution studies. Further
genetic analysis with additional strains isolated from Japan,
China, and other Asian countries would provide a novel insight
into the significance of two distinct subclades within
clade 2.
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A Gram-negative, facultative anaerobic, rod-shaped, motile
by means of a polar flagellum, greenish-yellow-pigmented
bacterial strain (designated strain JJ3220T) was isolated from
an artificial lake in South Korea and characterized using a
polyphasic approach. The 16S rRNA gene sequence of strain
JJ3220T indicated that the isolate belongs to the family Rhodocyclaceae,
and that it exhibits 96.4% similarity to Uliginosibacterium
paludis KBP-13T. The major cellular fatty acids
of the novel strain were C14:0, C16:0, and summed feature 3
(C16:1 ω6c and/or C16:1 ω7c). Strain JJ3220T had flexirubin-type
pigments. The DNA G+C content of the strain was 62.8%.
The major respiratory quinone and major polar lipid of strain
JJ3220T were ubiquinone-8 and phosphatidylethanolamine,
respectively. Based on the morphological and physiological
properties and biochemical evidence presented, it can be concluded
that strain JJ3220T represents a novel species of the
genus Uliginosibacterium. The type strain Uliginosibacterium
flavum is JJ3220T (=KACC 17644T =JCM 19465T).
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There are marked differences between wet and freeze-dried
cells with regard to the identification of polar lipid components.
The determination of the polar lipid composition of
freeze-dried cells is well established. However, several approaches
to identifying polar lipid components in wet cells have
met with limited success owing to the presence of non-polar
compounds in the extracts, resulting in a lipid composition
with a narrow scope. In this study, we surveyed the lipid profiles
of the wet biomasses of three Gram-positive (Microbacterium
lacticum, Rhodococcus koreensis, and Streptomyces
longwoodensis) and two Gram-negative (Pseudomonas aeruginosa
and Novosphingobium capsulatum) bacteria; the results
were comparable in quality to those obtained using a standard
freeze-dried approach. Moreover, our improved method
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A Gram stain-negative, yellowish green-pigmented, facultatively
anaerobic, motile, curved rod-shaped bacterium designated
as strain JJ016T was isolated from an artificial lake in
South Korea, and characterized using a polyphasic approach.
The 16S rRNA gene sequence of strain JJ016T indicated that
the isolate belonged to the family Rhodocyclaceae and exhibited
95.0% identity to Uliginosibacterium gangwonense
5YN10-9T. The major cellular fatty acids of the novel strain
were summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), C16:0, C14:0,
and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The DNA
G+C content of strain JJ016T was 61.9 mol%. The major respiratory
quinone and major polar lipid of strain JJ016T were
ubiquinone-8 and phosphatidylethanolamine, respectively.
Based on the morphological and physiological properties and
the biochemical evidence presented, we concluded that strain
JJ016T represents a novel species of a new genus in the family
Rhodocyclaceae, for which the name Viridibacterium curvum
gen. nov., sp. nov. is proposed. The type strain is JJ016T
(=KACC 16899T =JCM 18715T).
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To understand the isolation and classification state of actinobacterial
species with valid names for Korean indigenous
isolates, isolation source, regional origin, and taxonomic
affiliation of the isolates were studied. At the time of this writing,
the phylum Actinobacteria consisted of only one class,
Actinobacteria, including five subclasses, 10 orders, 56 families,
and 330 genera. Moreover, new taxa of this phylum
continue to be discovered. Korean actinobacterial species with
a valid name has been reported from 1995 as Tsukamurella
inchonensis isolated from a clinical specimen. In 1997, Streptomyces
seoulensis was validated with the isolate from the
natural Korean environment. Until Feb. 2016, 256 actinobacterial
species with valid names originated from Korean
territory were listed on LPSN. The species were affiliated with
three subclasses (Acidimicrobidae, Actinobacteridae, and
Rubrobacteridae), four orders (Acidimicrobiales, Actinomycetales,
Bifidobacteriales, and Solirubrobacterales), 12 suborders,
36 families, and 93 genera. Most of the species belonged
to the subclass Actinobacteridae, and almost of the
members of this subclass were affiliated with the order Actinomycetales.
A number of novel isolates belonged to the families
Nocardioidaceae, Microbacteriaceae, Intrasporangiaceae,
and Streptomycetaceae as well as the genera Nocardioides,
Streptomyces, and Microbacterium. Twenty-six novel
genera and one novel family, Motilibacteraceae, were created
first with Korean indigenous isolates. Most of the Korean
indigenous actionobacterial species were isolated from natural
environments such as soil, seawater, tidal flat sediment,
and fresh-water. A considerable number of species were isolated
from artificial resources such as fermented foods, wastewater,
compost, biofilm, and water-cooling systems or clinical specimens. Korean indigenous actinobacterial species
were isolated from whole territory of Korea, and especially
a large number of species were from Jeju, Gyeonggi, Jeonnam,
Daejeon, and Chungnam. A large number of novel actinobacterial
species continue to be discovered since the Korean
government is encouraging the search for new bacterial species
and researchers are endeavoring to find out novel strains
from extreme or untapped environments.
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