Transposon mutant libraries are an important resource to
study bacterial metabolism and pathogenesis. The fitness
analysis of mutants in the libraries under various growth conditions
provides important clues to study the physiology and
biogenesis of structural components of a bacterial cell. A transposon
library in conjunction with next-generation sequencing
techniques, collectively named transposon sequencing (Tnseq),
enables high-throughput genome profiling and synthetic
lethality analysis. Tn-seq has also been used to identify essential
genes and to study the mode of action of antibacterials.
To construct a high-density transposon mutant library, an efficient
delivery system for transposition in a model bacterium
is essential. Here, I describe a detailed protocol for generating
a high-density phage-based transposon mutant library in a
Staphylococcus aureus strain, and this protocol is readily applicable
to other S. aureus strains including USA300 and MW2.
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Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus Sally W. Yousief, Nader Abdelmalek, Bianca Paglietti Scientific Reports.2024;[Epub] CrossRef
Recent rapid air temperature increases across the northernlatitude
tundra have prolonged permafrost thawing and snow
melting periods, resulting in increased soil temperature (Ts)
and volumetric soil water content (SWC). Under prolonged
soil warming at 8°C, Alaskan tundra soils were incubated in
a microcosm system and examined for the SWC differential
influence on the microbial decomposition activity of large
molecular weight (MW) humic substances (HS). When one
microcosm soil (AKC1-1) was incubated at a constant SWC
of 41% for 90 days (T = 90) and then SWC was gradually
decreased from 41% to 29% for another T = 90, the initial
HS was partly depolymerized. In contrast, in AKC1-2 incubated
at a gradually decreasing SWC from the initial 32% to
10% for T = 90 and then increasing to 27% for another T =
90, HS depolymerization was undetected. Overall, the microbial
communities in AKC1-1 could maintain metabolic
activity at sufficient and constant SWC during the initial T =
90 incubation. In contrast, AKC1-2 microbes may have been
damaged by drought stress during the drying SWC regimen,
possibly resulting in the loss of HS decomposition activity,
which did not recover even after re-wetting to an optimal
SWC range (20–40%). After T = 90, the CO2 production in
both treatments was attributed to the increased decomposition
of small-MW organic compounds (including aerobic
HS-degradative products) within an optimal SWC range. We
expect this study to provide new insights into the early effects
of warming- and topography-induced SWC variations on
the microbial contribution to CO2 emissions via HS decomposition
in northern-latitude tundra soil.
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Unidirectional freeze–thaw redistributes water and amplifies soil microbial heterogeneity in a mecrocosm experiment Huimin Liu, Yaxian Hu, Yuan Song, Xianwen Li, Xiaorong Wei Geoderma.2025; 453: 117126. CrossRef
Analysis of CO2 Emission from Urban Soils of the Kola Peninsula (European Arctic) M. V. Korneykova, V. I. Vasenev, N. V. Saltan, M. V. Slukovskaya, A. S. Soshina, M. S. Zavodskikh, Yu. L. Sotnikova, A. V. Dolgikh Eurasian Soil Science.2023; 56(11): 1653. CrossRef
Analysis of CO2 Emission by Urban Soils under the Conditions of the Kola North M. V. Korneykova, V. I. Vasenev, N. V. Saltan, M. V. Slukovskaya, A. S. Soshina, M. S. Zavodskikh, Y. L. Sotnikova, A. V. Dolgikh Почвоведение.2023; (11): 1385. CrossRef
Acetyl-CoA synthetase (ACS) is the enzyme that irreversibly
catalyzes the synthesis of acetyl-CoA from acetate, CoA-SH,
and ATP via acetyl-AMP as an intermediate. In this study,
we demonstrated that AcsA1 (MSMEG_6179) is the predominantly
expressed ACS among four ACSs (MSMEG_6179,
MSMEG_0718, MSMEG_3986, and MSMEG_5650) found
in Mycobacterium smegmatis and that a deletion mutation
of acsA1 in M. smegmatis led to its compromised growth on
acetate as the sole carbon source. Expression of acsA1 was
demonstrated to be induced during growth on acetate as the
sole carbon source. The acsA1 gene was shown to be negatively
regulated by Crp1 (MSMEG_6189) that is the major
cAMP receptor protein (CRP) in M. smegmatis. Using DNase
I footprinting analysis and site-directed mutagenesis, a CRPbinding
site (GGTGA-N6-TCACA) was identified in the upstream
regulatory region of acsA1, which is important for repression
of acsA1 expression. We also demonstrated that inhibition
of the respiratory electron transport chain by inactivation
of the major terminal oxidase, aa3 cytochrome c oxidase,
led to a decrease in acsA1 expression probably through
the activation of CRP. In conclusion, AcsA1 is the major ACS
in M. smegmatis and its gene is under the negative regulation
of Crp1, which contributes to some extent to the induction
of acsA1 expression under acetate conditions. The growth of
M. smegmatis is severely impaired on acetate as the sole carbon
source under respiration-inhibitory conditions.
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J. Microbiol. 2022;60(12):1153-1161. Published online November 10, 2022
Aeromonas veronii is a pathogen which can induce diseases in
humans, animals and aquatic organisms, but its pathogenic
mechanism and virulence factors are still elusive. In this study,
we successfully constructed a mutant strain (ΔascP) by homologous
recombination. The results showed that the deletion
of the ascP gene significantly down-regulated the expression
of associated effector proteins in A. veronii compared
to its wild type. The adhesive and invasive abilities of ΔascP to
EPC cells were 0.82-fold lower in contrast to the wild strain.
The toxicity of ΔascP to cells was decreased by about 2.91-fold
(1 h) and 1.74-fold (2 h). Furthermore, the LD50 of the mutant
strain of crucian carp was reduced by 19.94-fold, and
the virulence was considerably attenuated. In contrast to the
wild strain, the ΔascP content in the liver and spleen was considerably
lower. The titers of serum cytokines (IL-8, TNF-α,
and IL-1β) in crucian carp after the infection of the ΔascP strain
were considerably lower in contrast to the wild strain. Hence,
the ascP gene is essential for the etiopathogenesis of A. veronii
TH0426.
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Ribosomes composed of genome-encoded heterogeneous
rRNAs are implicated in the rapid adaptation of bacterial
cells to environmental changes. A previous study showed that
ribosomes bearing the most heterogeneous rRNAs expressed
from the rrnI operon (I-ribosomes) are implicated in the preferential
translation of a subset of mRNAs, including hspA
and tpiA, in Vibrio vulnificus CMCP6. In this study, we show
that HspA nascent peptides were predominantly bound to
I-ribosomes. Specifically, I-ribosomes were enriched more
than two-fold in ribosomes that were pulled down by immunoprecipitation
of HspA peptides compared with the proportion
of I-ribosomes in crude ribosomes and ribosomes pulled
down by immunoprecipitation of RNA polymerase subunit
ß peptides in the wild-type (WT) and rrnI-completed strains.
Other methods that utilized the incorporation of an affinity
tag in 23S rRNA or chimeric rRNA tethering 16S and 23S
rRNAs, which generated specialized functional ribosomes
in Escherichia coli, did not result in functional I-ribosomes
in V. vulnificus CMCP6. This study provides direct evidence
of the preferential translation of hspA mRNA by I-ribosomes.
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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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J. Microbiol. 2022;60(12):1178-1190. Published online November 24, 2022
Microbiome research has been on the rise recently for a more
in-depth understanding of gout. Meanwhile, there is a need to
understand the gut microbiome related to uric acid-lowering
drug resistance. In this study, 16S rRNA gene-based microbiota
analysis was performed for a total of 65 stool samples
from 17 healthy controls and 48 febuxostat-treated gout patients
(including 28 controlled subjects with decreased uric
acid levels and 20 uncontrolled subjects with non-reduced
uric acid levels). Alpha diversity of bacterial community decreased
in the healthy control, controlled, and uncontrolled
groups. In the case of beta diversity, the bacterial community
was significantly different among groups (healthy control, controlled,
and uncontrolled groups). Taxonomic biomarker analysis
revealed the increased population of g-Bifidobacterium
in healthy controls and g-Prevotella in uncontrolled patients.
PCR further confirmed this result at the species level. Additionally,
functional metagenomics predictions led to the exploration
of various functional biomarkers, including purine
metabolism. The results of this study can serve as a basis
for developing potential new strategies for diagnosing and
treating gout from microbiome prospects.
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Terpenes have many applications and are widely found in
nature, but recent progress in synthetic biology has enabled
the use of microorganisms as chassis cells for the synthesis
of these compounds. Candida glycerinogenes (C. glycerinogenes)
is an industrial strain that may be developed as a chassis
for the synthesis of terpenes since it has a tolerance to hyperosmolality
and high sugar, and has a complete mevalonate
(MVA) pathway. However, monoterpenes such as pinene are
highly toxic, and the tolerance of C. glycerinogenes to pinene
was investigated. We also measured the content of mevalonate
and squalene to evaluate the strength of the MVA pathway.
To determine terpene synthesis capacity, a pathway for the synthesis
of pinene was constructed in C. glycerinogenes. Pinene
production was improved by overexpression, gene knockdown
and antisense RNA inhibition. Pinene production was mainly
enhanced by strengthening the upstream MVA pathway and
inhibiting the production of by-products from the downstream
pathway. With these strategies, yield could be increased
by almost 16 times, to 6.0 mg/L. Overall, we successfully constructed
a pinene synthesis pathway in C. glycerinogenes and
enhanced pinene production through metabolic modification.
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Candida species cause the most prevalent fungal illness, candidiasis.
Candida albicans is known to cause bloodstream infections.
This species is a commensal bacterium, but it can
cause hospital–acquired diseases, particularly in COVID-19
patients with impaired immune systems. Candida infections
have increased in patients with acute respiratory distress syndrome.
Coumarins are both naturally occurring and synthetically
produced. In this study, the biological activity of 40 coumarin
derivatives was used to create a three-dimensional quantitative
structure activity relationship (3D-QSAR) model. The
training and test minimum inhibitory concentration values
of C. albicans active compounds were split, and a regression
model based on statistical data was established. This model
served as a foundation for the creation of coumarin derivative
QSARs. This is a unique way to create new therapeutic compounds
for various ailments. We constructed novel structural
coumarin derivatives using the derived QSAR model, and the
models were confirmed using molecular docking and molecular
dynamics simulation.
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