RNA modifications are a common occurrence across all domains
of life. Several chemical modifications, including N6-
methyladenosine, have also been found in viral transcripts
and viral RNA genomes. Some of the modifications increase
the viral replication efficiency while also helping the virus to
evade the host immune system. Nonetheless, there are numerous
examples in which the host's RNA modification enzymes
function as antiviral factors. Although established methods
like MeRIP-seq and miCLIP can provide a transcriptome-
wide overview of how viral RNA is modified, it is difficult
to distinguish between the complex overlapping viral
transcript isoforms using the short read-based techniques.
Nanopore direct RNA sequencing (DRS) provides both long
reads and direct signal readings, which may carry information
about the modifications. Here, we describe a refined protocol
for analyzing the RNA modifications in viral transcriptomes
using nanopore technology.
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Coagulase-negative Staphylococcus (CoNS) species may possess
antibiotic resistance genes and have been associated with
nosocomial infections. In this study, 91 CoNS with decreased
susceptibility to oxacillin were isolated from fresh produce
using oxacillin containing agar plates. Their antibiotic resistances
were determined phenotypically and all isolates were
identified by rep-PCR, 16S rRNA and rpoB gene sequencing.
Furthermore, the genomes of representative strains were sequenced
in order to confirm species identification by phylogenomics.
The majority (64 of 91) of the CoNS strains could
be identified as Mammaliicoccus (M.) fleurettii, while 13 were
identified as M. sciuri, 8 as M. vitulinus, 2 as Staphylococcus
(S.) epidermidis and single strains each as S. warneri, S. xylosus,
Staphylococcus spp. and S. casei. Most of the strains were generally
susceptible to clinically-relevant antibiotics, but only
few (< 7%) strains possessed multiple resistances. Both oxacillin
and cefoxitin resistant isolates were considered to be
presumptive methicillin-resistant CoNS. From whole genome
sequencing data of 6 representative strains, the mecA gene,
accessory genes and the SCC loci were compared, which revealed
high variability between some of the strains. The major
fatty acids of K22-5MT strain included anteiso-C15:0,
iso-C15:0, iso-C17:0, anteiso-C17:0, C18:0, and C20:0. Average nucleotide
identity and digital DNA-DNA hybridization values
indicated that Staphylococcus strain K22-5MT was below the
species delineation cutoff values for ANI (less than 91%) and
DDH (less than 44.4%), with the most closely related species
being the S. pseudoxylosus S04009T type strain. Thus, strain
K22- 5MT (=DSM 112532T, =LMG 32324T) represents a novel
species, for which the name Staphylococcus shinii sp. nov. is
proposed.
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Two novel bacterial strains, KSM-R2A25T and KSM-R2A30T,
were isolated from intestines of Cyclina sinensis (corb shell)
and Channa argus (northern snakehead), respectively. Both
specimens were collected in Korea. The strains were Gramstain-
negative, non-motile, and strictly aerobic. According
to phylogenetic analyses based on 16S rRNA gene sequences,
strains belonged to the genus Flavobacterium within the family
Flavobacteriaceae. 16S rRNA gene sequences of strains KSMR2A25T
and KSM-R2A30T were closely related to Flavobacterium
cucumis DSM 18830T and Flavobacterium aquaticum
JC164T with sequence similarities of 97.77% and 98.54%, respectively.
Further genomic analyses including reconstruction
of the UBCG tree and overall genome-related indices suggested
them as novel species of the genus Flavobacterium.
Both strains contained menaquinone with six isoprene units
(MK-6) as a major isoprenoid quinone and iso-C15:1 G, iso-
C15:0, and iso-C16:0 as major cellular fatty acids. The major polar
lipid in both strains was phosphatidylethanolamine. The
genomic G + C contents of strains KSM-R2A25T and KSMR2A30T
were 31.7 and 31.9%, respectively. Based on the polyphasic
taxonomic study presented here, strains KSM-R2A25T
and KSM-R2A30T represent novel species of the genus Flavobacterium,
for which the names Flavobacterium cyclinae sp.
nov and Flavobacterium channae sp. nov are proposed. The
type strains of F. cyclinae sp. nov and F. channae sp. nov
are KSM-R2A25T (= KCTC 82978T = JCM 34997T) and KSMR2A30T
(= KCTC 82979T = JCM 34998T), respectively.
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International Journal of Systematic and Evolutionary Microbiology
.2023;[Epub] CrossRef
Two novel halophilic archaeal strains, CBA1133T and CBA-
1134, were isolated from solar salt in South Korea. The 16S
rRNA gene sequences of the isolates were identical to each
other and were closely related to the genera Natronomonas
(92.3–93.5%), Salinirubellus (92.2%), Halomarina (91.3–
92.0%), and Haloglomus (91.4%). The isolated strains were
coccoid, Gram-stain-negative, aerobic, oxidase-positive, and
catalase-negative. Growth occurred under temperatures of
25–50°C (optimum, 45°C), NaCl levels of 10–30% (optimum,
15%), pH levels of 6.0–8.5 (optimum, 7.0), and MgCl2 concentrations
of 0–500 mM (optimum, 100 mM). Digital DNADNA
hybridization values between the strains and related
genera ranged from 18.3% to 22.7%. The major polar lipids
of the strains were phosphatidyl glycerol, phosphatidyl glycerol
phosphate methyl ester, and phosphatidyl glycerol sulfate.
Genomic, phenotypic, physiological, and biochemical
analyses of the isolates revealed that they represent a novel
genus and species in the family Halobacteriaceae. The type
strain is CBA1133T (= KACC 22148T = JCM 34265T), for which
the name Sala cibi gen. nov., sp. nov. is proposed.
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Validation List no. 209. Valid publication of new names and new combinations effectively published outside the IJSEM Aharon Oren, Markus Göker
International Journal of Systematic and Evolutionary Microbiology
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A novel bacterium, designated YYF0007T, was isolated from
an agar-degrading co-culture. The strain was found harboring
four CRISPR-Cas systems of two classes in the chromosome
and subsequently subjected to a study on polyphasic
taxonomy. Pairwise analyses of the 16S rRNA gene sequences
indicated that strain YYF0007T had highest 16S rRNA gene
sequence similarity (92.2%) to Jiulongibacter sediminis JN-
14-9T. The phylogenomic trees based on the 16S rRNA gene
and 269 single-copy orthologous gene clusters (OCs) indicated
that strain YYF0007T should be recognized as a novel
genus of the family Spirosomaceae. The cells were Gramstain-
negative, nonmotile, strictly aerobic, and straight long
rods with no flagellum. Optimum growth occurred at 28°C
and pH 7.0 with the presence of NaCl concentration 1.0–3.0%
(w/v). The strain showed oxidase and catalase activities.
The major fatty acids were C16:1ω5c, iso-C15:0 and summed
feature 3 (C16:1 ω7c and/or C16:1 ω6c). The predominant isoprenoid
quinone was MK-7. The complete genome size was
4.64 Mb with a DNA G + C content of 44.4%. Further typing
of CRISPR-Cas systems in the family Spirosomaceae and the
phylum Bacteroidota indicated that it was remarkable for
strain YYF0007T featured by such a set of CRISPR-Cas systems.
This trait highlights the applications of strain YYF-
0007T in studies on the evolutionary dynamics and bacterial
autoimmunity of CRISPR-Cas system as a potential model.
The name Marinilongibacter aquaticus gen. nov., sp. nov. is
proposed, and the type strain is YYF0007T (= MCCC 1K06017T
= GDMCC 1.2428T = JCM 34683T).
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Siboglinid tubeworms thrive in hydrothermal vent and seep
habitats via a symbiotic relationship with chemosynthetic bacteria.
Difficulties in culturing tubeworms and their symbionts
in a laboratory setting have hindered the study of host-microbe
interactions. Therefore, released symbiont genomes are
fragmented, thereby limiting the data available on the genome
that affect subsequent analyses. Here, we present a complete
genome of gammaproteobacterial endosymbiont from the
tubeworm Lamellibrachia satsuma collected from a seep in
Kagoshima Bay, assembled using a hybrid approach that combines
sequences generated from the Illumina and Oxford Nanopore
platforms. The genome consists of a single circular chromosome
with an assembly size of 4,323,754 bp and a GC content
of 53.9% with 3,624 protein-coding genes. The genome
is of high quality and contains no assembly gaps, while the
completeness and contamination are 99.33% and 2.73%,
respectively. Comparative genome analysis revealed a total
of 1,724 gene clusters shared in the vent and seep tubeworm
symbionts, while 294 genes were found exclusively in L. satsuma
symbionts such as transposons, genes for defense mechanisms,
and inorganic ion transportations. The addition of
this complete endosymbiont genome assembly would be valuable
for comparative studies particularly with tubeworm symbiont
genomes as well as with other chemosynthetic microbial
communities.
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Genomic and transcriptomic analyses illuminate the molecular basis of the unique lifestyle of a tubeworm, Lamellibrachia satsuma
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The type VI secretion system (T6SS) is a novel secretion system
found in many Gram-negative bacteria that plays a role
in bacterial competition, virulence, and host immune evasion.
The enterohemorrhagic Escherichia coli (EHEC) O157:H7
strain EDL933 has a single functional T6SS gene cluster. In
this study, we attempted to characterize the transcriptional
pattern of the T6SS effector gene Z0264 in EDL933. Transcriptional
analyses showed that Z0264 and other T6SS genes
were transcribed in vitro in a growth-phase-dependent manner,
but Z0264 was not secreted in the rich medium. Using
adapter- and radioactivity-free transcription start site analysis,
we identified an overlapping divergent promoter between
Z0264 and Z0265. A β-galactosidase assay with truncated promoter
regions showed that the divergent promoter is functional.
In addition, we demonstrated the role of H-NS as a
repressor in the transcription of Z0264. Notably, the cDNA
PCR assay showed that the mRNA transcript from the Z0264
promoter did not include the entire main T6SS cluster, suggesting
segmented gene expression by multiple promoters in
the T6SS cluster. In conclusion, we identified a divergent promoter
for Z0264 located in the T6SS cluster of EDL933 and
characterized its in vitro transcriptional activity during growth.
Our findings provide insights and a preliminary understanding
of the regulatory mechanisms underlying T6SS transcription.
Using a mutant of Mycobacterium smegmatis lacking the major
aa3 cytochrome c oxidase of the electron transport chain
(Δaa3), we demonstrated that inhibition of the respiratory
electron transport chain led to an increase in antibiotic resistance
of M. smegmatis to isoniazid, rifampicin, ethambutol,
and tetracycline. The alternative sigma factors SigB and SigE
were shown to be involved in an increase in rifampicin resistance
of M. smegmatis induced under respiration-inhibitory
conditions. As in Mycobacterium tuberculosis, SigE and SigB
form a hierarchical regulatory pathway in M. smegmatis through
SigE-dependent transcription of sigB. Expression of sigB and
sigE was demonstrated to increase in the Δaa3 mutant, leading
to upregulation of the SigB-dependent genes in the mutant.
The phoU2 (MSMEG_1605) gene implicated in a phosphatesignaling
pathway and the MSMEG_1097 gene encoding a putative
glycosyltransferase were identified to be involved in
the SigB-dependent enhancement of rifampicin resistance observed
for the Δaa3 mutant of M. smegmatis. The significance
of this study is that the direct link between the functionality
of the respiratory electron transport chain and antibiotic resistance
in mycobacteria was demonstrated for the first time
using an electron transport chain mutant rather than inhibitors
of electron transport chain.
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MoaB2, a newly identified transcription factor, binds to σ
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Streptococcus suis type 2 (S. suis type 2, SS2), an infectious
pathogen which is zoonotic and can induce severely public
health concern. Our previous research identified a newly differential
secreted effector of tagatose-bisphosphate aldolase
(LacD) mediated by VirD4 factor within the putative type IV
secretion system of SS2, whereas the functional basis and roles
in virulence of LacD remain elusive. Here in this study, the
LacD was found enzymatic and can be activated to express
under oxidative stress. Gene mutant and its complemental
strain (ΔlacD and cΔlacD) were constructed to analyze the
phenotypes, virulence and transcriptomic profiles as compared
with the parental strain. The lacD gene deletion showed
no effect on growth capability and cells morphology of SS2.
However, reduced tolerance to oxidative and heat stress conditions,
increased antimicrobial susceptibility to ciprofloxacin
and kanamycin were found in ΔlacD strain. Further, the LacD
deficiency led to weakened invasion and attenuated virulence
since an easier phagocytosed and more prone to be cleared of
SS2 in macrophages were shown in ΔlacD mutant. Distinctive
transcriptional profiling in ΔlacD strain and typical downregulated
genes with significant mRNA changes including
alcohol dehydrogenase, GTPase, integrative and conjugative
elements, and iron ABC transporters which were mainly involved
in cell division, stress response, antimicrobial susceptibility
and virulence regulation, were examined and confirmed
by RNA sequencing and real time qPCR. In summary, the results demonstrated for the first time that LacD was a pluripotent
protein mediated the metabolic, stress and virulent
effect of SS2.
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In protein biotechnology, large soluble fusion partners are
widely utilized for increased yield and solubility of recombinant
proteins. However, the production of additional large
fusion partners poses an additional burden to the host, leading
to a decreased protein yield. In this study, we identified
two highly disordered short peptides that were able to increase
the solubility of an artificially engineered aggregationprone
protein, GFP-GFIL4, from 0.6% to 61% (D3-DP00592)
and 46% (D4-DP01038) selected from DisProt database. For
further confirmation, the peptides were applied to two insoluble
E. coli proteins (YagA and YdiU). The peptides also
enhanced solubility from 52% to 90% (YagA) and from 27%
to 93% (YdiU). Their ability to solubilize recombinant proteins
was comparable with strong solubilizing tags, maltosebinding
protein (40 kDa) and TrxA (12 kDa), but much smaller
(< 7 kDa) in size. For practical application, the two peptides
were fused with a restriction enzyme, I-SceI, and they increased
I-SceI solubility from 24% up to 75%. The highly disordered
peptides did not affect the activity of I-SceI while I-SceI fused
with MBP or TrxA displayed no restriction activity. Despite
the small size, the highly disordered peptides were able to
solubilize recombinant proteins as efficiently as conventional
fusion tags and did not interfere with the function of recombinant
proteins. Consequently, the identified two highly disordered
peptides would have practical utility in protein biotechnology
and industry.
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