Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation.
Overall, this strain can be further developed to convert acetate and formate into valuable products.
Silver nanoparticles (AgNPs) exhibit strong antibacterial activity and do not easily induce drug resistance; however, the
poor stability and biocompatibility in solution limit their widespread application. In this study, AgNPs were modified with
Polygonatum sibiricum Polysaccharide (PSP) to synthesize PSP@AgNPs with good stability, biocompatibility, and antibacterial
activity. When PSP@AgNP synthesis was performed under a reaction time of 70 min, a reaction temperature of 35 °C,
and an AgNO3-
to-PSP volume ratio of 1:1, the synthesized PSP@AgNPs were more regular and uniform than AgNPs, and
their particle size was around 10 nm. PSP@AgNPs exhibited lower cytotoxicity and hemolysis, and stronger bacteriostatic
activity. PSP@AgNPs damage the integrity and internal structure of cells, resulting in the leakage of intracellular nucleic
acids and proteins. The rate of cell membrane damage in Escherichia coli and Staphylococcus aureus treated with PSP@
AgNPs increased by 38.52% and 43.75%, respectively, compared with that of AgNPs. PSP@AgNPs inhibit the activities
of key enzymes related to antioxidant, energy and substance metabolism in cells. The inhibitory effects on the activities of
superoxide dismutase (SOD), catalase (CAT), adenosine triphosphate enzyme (ATPase), malate dehydrogenase (MDH),
and succinate dehydrogenase (SDH) in E. coli and S. aureus cells were significantly higher than those of AgNPs. In addition,
compared with AgNPs, PSP@AgNPs promote faster healing of infected wounds. Therefore, PSP@AgNPs represent
potential antibacterial agents against wound infections.
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Antibiotic treatment failure threatens our ability to control bacterial infections that can cause chronic diseases. Persister bacteria
are a subpopulation of physiological variants that becomes highly tolerant to antibiotics. Membrane proteins play crucial
roles in all living organisms to regulate cellular physiology. Although a diverse membrane component involved in persistence
can result in antibiotic treatment failure, the regulations of antibiotic persistence by membrane proteins has not been fully
understood. In this review, we summarize the recent advances in our understanding with regards to membrane proteins in
Gram-negative bacteria as a regulator for antibiotic persistence, highlighting various physiological mechanisms in bacteria.
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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.
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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
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Three major proteases, elastase B (LasB), protease IV (PIV),
and elastase A (LasA) expressed in Pseudomonas aeruginosa
play important roles in infections and pathogeneses. These
are activated by a proteolytic cascade initiated by the activation
of LasB. In this study, we investigated whether LasB
could be inhibited using its propeptide (LasBpp). Although
LasA and PIV were inhibited by their propeptides, LasB was
not inhibited by purified LasBpp because LasB degraded LasBpp.
To address this problem, mutant LasBpp variants were constructed
to obtain a mutant LasBpp resistant to LasB degradation.
A C-terminal deletion series of LasBpp was tested in
vivo, and two positive candidates, T2 and T2-1, were selected.
However, both caused growth retardation and were unstably
expressed in vivo. Since deleting the C-terminal end of LasBpp
significantly affected its stable expression, substitution mutations
were introduced at the two amino acids near the
truncation site of T2-1. The resulting mutants, LasBppE172D,
LasBppG173A, and LasBppE172DG173A, significantly diminished LasB
activity when overexpressed in vivo and were stably expressed
in MW1, a quorum sensing mutant that does not produce
LasB. In vitro analysis showed that purified LasBppE172DG173A
inhibited LasB activity to a small extent. Summarizing, Cterminal
modification of LasBpp profoundly affected the stable
expression of LasBpp, and little enhanced the ability of
LasBpp to resist degradation by LasB.
A critical obstacle to the successful treatment of colorectal
cancer (CRC) is chemoresistance. Chemoresistant CRC cells
contribute to treatment failure by providing a mechanism
of drug lethargy and modifying chemoresistance-associated
molecules. The gut microbiota provide prophylactic and therapeutic
effects by targeting CRC through anticancer mechanisms.
Among them, Lactobacillus plantarum contributes
to the health of the host and is clinically effective in treating
CRC. This study confirmed that 5-fluorouracil (5-FU)-resistant
CRC HCT116 (HCT116/5FUR) cells acquired butyrateinsensitive
properties. To date, the relationship between 5-
FU-resistant CRC and butyrate resistance has not been elucidated.
Here, we demonstrated that the acquisition of butyrate
resistance in HCT116/5FUR cells was strongly correlated
with the inhibition of the expression and function of
SMCT1, a major transporter of butyrate in colonocytes. L.
plantarum-cultured cell-free supernatant (LP) restored the
functional expression of SMCT1 in HCT116/5FUR cells, leading
to butyrate-induced antiproliferative effect and apoptosis.
These results suggest that LP has a synergistic effect on the
SMCT1/butyrate-mediated tumor suppressor function and
is a potential chemosensitizer to overcome dual 5-FU and butyrate
resistance in HCT116 cells.
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is an opportunistic pathogen closely related to initiation
and progression of various oral diseases, such as periodontitis
and dental caries. Its biofilm formation is linked
with the development of such diseases by enhanced resistance
against antimicrobial treatment or host immunity. In the
present study, we investigated the effect of short-chain fatty
acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs,
including sodium acetate (NaA), sodium propionate (NaP),
and sodium butyrate (NaB), showed an effective inhibitory
activity on the biofilm formation of S. gordonii without reduction
in bacterial growth. SCFAs suppressed S. gordonii
biofilm formation at early time points whereas SCFAs did
not affect its preformed biofilm. A quorum-sensing system
mediated by competence-stimulating peptide (CSP) is known
to regulate biofilm formation of streptococci. Interestingly,
SCFAs substantially decreased mRNA expression of comD
and comE, which are CSP-sensor and its response regulator
responsible for CSP pathway, respectively. Although S. gordonii
biofilm formation was enhanced by exogenous synthetic
CSP treatment, such effect was not observed in the
presence of SCFAs. Collectively, these results suggest that
SCFAs have an anti-biofilm activity on S. gordonii through
inhibiting comD and comE expression which results in negative
regulation of CSP quorum-sensing system. SCFAs could
be an effective anti-biofilm agent against S. gordonii for the
prevention of oral diseases.
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Flow cytometry is a promising tool used to identify the phenotypic
features of bacterial communities in aquatic ecosystems
by measuring the physical and chemical properties of
cells based on their light scattering behavior and fluorescence.
Compared to molecular or culture-based approaches, flow
cytometry is suitable for the online monitoring of microbial
water quality because of its relatively simple sample preparation
process, rapid analysis time, and high-resolution phenotypic
data. Advanced statistical techniques (e.g., denoising
and binning) can be utilized to successfully calculate phenotypic
diversity by processing the scatter data obtained from
flow cytometry. These phenotypic diversities were well correlated
with taxonomic-based diversity computed using nextgeneration
16S RNA gene sequencing. The protocol provided
in this paper should be a useful guide for a fast and reliable
flow cytometric monitoring of bacterial phenotypic diversity
in aquatic ecosystems.
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such as chronic inflammatory disease, intestinal infection
and colorectal cancer. To identify such dysregulations,
several strategies are being used to create a repertoire of the
microorganisms composing the human gut microbiome. In
this study, we used the “microscomics” approach, which consists
of creating an ultrastructural repertoire of all the cell-like
objects composing stool samples from healthy donors using
transmission electron microscopy (TEM). We used TEM to
screen ultrathin sections of 8 resin-embedded stool samples.
After exploring hundreds of micrographs, we managed to
elaborate ultrastructural categories based on morphological
criteria or features. This approach explained many inconsistencies
observed with other techniques, such as metagenomics
and culturomics. We highlighted the value of our cultureindependent
approach by comparing our microscopic images
to those of cultured bacteria and those reported in the
literature. This study helped to detect “minimicrobes” Candidate
Phyla Radiation (CPR) for the first time in human
stool samples. This “microscomics” approach is non-exhaustive
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PA14, whose transcription is governed by its dual
promoters (katAp1 and katAp2). Here, we observed that KatA
was not required for acute virulence in another wild type P.
aeruginosa strain, PAO1, but that PAO1 exhibited higher
KatA expression than PA14 did. This was in a good agreement
with the observation that PAO1 was more resistant
than PA14 to H2O2 as well as to the antibiotic peptide, polymyxin
B (PMB), supposed to involve reactive oxygen species
(ROS) for its antibacterial activity. The higher KatA expression
in PAO1 than in PA14 was attributed to both katAp1
and katAp2 transcripts, as assessed by S1 nuclease mapping.
In addition, it was confirmed that the PMB resistance is attributed
to both katAp1 and katAp2 in a complementary manner
in PA14 and PAO1, by exploiting the promoter mutants
for each -10 box (p1m, p2m, and p1p2m). These results provide
an evidence that the two widely used P. aeruginosa strains
display different virulence mechanisms associated with OxyR
and Anr, which need to be further characterized for better
understanding of the critical virulence pathways that may
differ in various P. aeruginosa strains.
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Low-density lipoprotein (LDL) was recently reported to be an
opsonin, enhancing the phagocytosis of group A Streptococcus
(GAS) by human monocytic leukemia U937 cells due to the
binding of LDL to some GAS strains. We postulated that LDL
might also promote the opsonophagocytosis of Pseudomonas
aeruginosa by U937 cells since this bacterium interacts with
LDL. In this study, P. aeruginosa (CMCC10104), U937 cells,
and human LDL were used in phagocytosis assays to test our
hypothesis. Escherichia coli strain BL21, which does not interact
with LDL, was used as a negative control. Colony counting
and fluorescence microscopy were used to determine the
bacterial quantity in the opsonophagocytosis assays. After
incubation of U937 cells and P. aeruginosa with LDL (100
μg/ml) for 15 and 30 min, phagocytosis was observed to be
increased by 22.71% and 32.90%, respectively, compared to
that seen in the LDL-free group. However, LDL did not increase
the phagocytosis of E. coli by U937 cells. In addition,
we identified CD36 as a major opsonin receptor on U937 cells,
since an anti-CD36 monoclonal antibody, but not an anti-
CD4 monoclonal antibody, almost completely abolished the
opsonophagocytosis of P. aeruginosa by U937 cells.
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