Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by
recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way
to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward
genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa.
This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating
the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of
P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under
an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a
temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35
gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into
the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA.
The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules.
This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.
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Pilin regions that select for the small RNA phages in
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Two novel Gram-stain-negative, strictly-aerobic, rod-shaped (1.2 ± 3.4 μm × 0.3 ± 0.7 μm), and non-motile marine bacterial
species, designated MEBiC05379T
and MEBiC07777T,
were isolated from a marine sponge Pseudaxinella sp. in Gangneung
City and deep-sea sediments of the Ulleung basin in the East Sea of Korea, respectively. The 16S rRNA gene sequence
analysis revealed high levels of similarities between these strains and members of the genus Flavivirga (97.0–98.4% sequence
identities). Both novel strains revealed as mesophilic, neutrophilic in pH and slightly halophilic. Similar to those of other Flavivirga
members, the primary cellular fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C15:03-OH, and iso-C17:0 3-OH,
with MEBiC05379T
and MEBiC07777T
containing relatively higher proportions of C12:
0 and summed feature 3 (
C16:1ω7c
and/or C16:
1ω6c). In both taxa, the major isoprenoid quinone was MK-6. The DNA G + C contents of MEBiC05379T
and
MEBiC07777T
genomes were 32.62 and 32.46 mol%, respectively. Compared to other members of Flavivirga, both strains
exhibited similar DNA G + C ratio and fatty acids pattern, yet enzyme expression and carbon sources utilization pattern were
different. Genomes of the genus Flavivirga showed enzyme preferences to fucoidan and sulfated galactans. Considering the
monophyly rule, AAI values delineate the genus Flavivirga from adjacent genera calculated to be 76.0–78.7%. Based on
the phenotypic, genomic and biochemical data, strains for MEBiC05379T
and MEBiC07777T
thus represent two novel species
in the genus Flavivirga, for which the names Flavivirga spongiicola sp. nov. (
MEBiC05379T [= KCTC 92527
T = JCM
16662
T]), and Flavivirga abyssicola sp. nov. (
MEBiC07777T [= KCTC 92563
T = JCM 36477
T]) are proposed.
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cell-surface proteins in the Δlkh1 strain. While disruption of the gas2+ gene was not lethal and reduced the flocculation
activity of the Δlkh1 strain, the expression of a secreted form of Gas2, in which the GPI anchor addition sequences had been
removed, conferred the ability to flocculate upon the WT strain. The Gas2-mediated flocculation was strongly inhibited by
galactose but not by glucose. Immunostaining analysis showed that the cell surface localization of Gas2 was crucial for the
flocculation of fission yeast. In addition, we identified the regulation of mbx2+ expression by Lkh1 using RT-qPCR. Taken
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global public health problem. Mycobacterium tuberculosis expresses a unique family of PE_PGRS proteins that have been
implicated in pathogenesis. Despite numerous studies, the functions of most PE_PGRS proteins in the pathogenesis of
mycobacterium infections remain unclear. PE_PGRS45 (Rv2615c) is only found in pathogenic mycobacteria. In this study,
we successfully constructed a recombinant Mycobacterium smegmatis (M. smegmatis) strain which heterologously expresses
the PE_PGRS45 protein. We found that overexpression of this cell wall-associated protein enhanced bacterial viability under
stress in vitro and cell survival in macrophages. MS_PE_PGRS45 decreased the secretion of pro-inflammatory cytokines such
as IL-1β, IL-6, IL-12p40, and TNF-α. We also found that MS_PE_PGRS45 increased the expression of the anti-inflammatory
cytokine IL-10 and altered macrophage-mediated immune responses. Furthermore, PE_PGRS45 enhanced the survival rate
of M. smegmatis in macrophages by inhibiting cell apoptosis. Collectively, our findings show that PE_PGRS45 is a virulent
factor actively involved in the interaction with the host macrophage.
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antimicrobial peptides (AMPs) are potential alternatives as antifungal agents with excellent antifungal activity. We previously
reported that Css54, found in the venom of Centrurodies suffusus suffusus (C. s. suffusus) showed antibacterial activity
against zoonotic bacteria. However, the antifungal activity of Css54 has not yet been elucidated. The obj!ective of this study
was to identify the antifungal activity of Css54 against C. albicans and analyze its mechanism. Css54 showed high antifungal
activity against C. albicans. Css54 also inhibited biofilm formation in fluconazole-resistant fungi. The antifungal mechanism
of action of Css54 was investigated using membrane-related assays, including the membrane depolarization assay and
analysis of the membrane integrity of C. albicans after treatment with Css54. Css54 induced reactive oxygen species (ROS)
production in C. albicans, which affected its antifungal activity. Our results indicate that Css54 causes membrane damage
in C. albicans, highlighting its value as a potential therapeutic agent against C. albicans infection.
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pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of
a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation,
primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming
and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability
of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the
MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication
was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10
7.5 ± 0.15 Ct value
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value for ASFV DNA, HAD50/
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ml assay, and cytopathic effects and hemadsoption were observed similar
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adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell
line for ASFV isolation, replication, and development of live attenuated vaccines.
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We isolated and analyzed a novel, Gram-stain-positive, aerobic, rod-shaped, non-motile actinobacterium, designated as strain ZFBP1038(T), from rock sampled on the north slope of Mount Everest. The growth requirements of this strain were 10-37 °C, pH 4-10, and 0-6% (w/v) NaCl. The sole respiratory quinone was MK-9, and the major fatty acids were anteiso-C(15:0) and iso-C(17:0). Peptidoglycan containing meso-diaminopimelic acid, ribose, and glucose were the major cell wall sugars, while polar lipids included diphosphatidyl glycerol, phosphatidyl glycerol, an unidentified phospholipid, and an unidentified glycolipid. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFBP1038(T) has the highest similarity with Spelaeicoccus albus DSM 26341( T) (96.02%). ZFBP1038(T) formed a distinct monophyletic clade within the family Brevibacteriaceae and was distantly related to the genus Spelaeicoccus. The G + C content of strain ZFBP1038(T) was 63.65 mol% and the genome size was 4.05 Mb.
Digital DNA-DNA hybridization, average nucleotide identity, and average amino acid identity values between the genomes of strain ZFBP1038(T) and representative reference strains were 19.3-25.2, 68.0-71.0, and 52.8-60.1%, respectively.
Phylogenetic, phenotypic, and chemotaxonomic characteristics as well as comparative genome analyses suggested that strain ZFBP1038(T) represents a novel species of a new genus, for which the name Saxibacter gen. nov., sp. nov. was assigned with the type strain Saxibacter everestensis ZFBP1038(T) (= EE 014( T) = GDMCC 1.3024( T) = JCM 35335( T)).
Three novel, Gram-stain-positive, obligate aerobic, catalase- and oxidase-positive bacterial strains, designated B2O-1(T), T2O-4(T), and 0.2-SM1T-5(T), were isolated from jeotgal, a traditional Korean fermented seafood. Strains B2O-1(T), T2O-4(T), and 0.2-SM1T-5(T) exhibited distinct colony colors, characterized by pink, yellow, and red opaque circular colonies, respectively. Phylogenetic analysis revealed that three strains formed a paraphyletic clade within the genus Sporosarcina and shared < 99.0% similarity with Sporosarcina aquimarina KCTC 3840(T) and Sporosarcina saromensis KCTC 13119(T) in their 16S rRNA gene sequences. The three strains exhibiting Orthologous Average Nucleotide Identity values < 79.3% and digital DNA-DNA hybridization values < 23.1% within the genus Sporosarcina affirmed their distinctiveness. Strains B2O-1(T), T2O-4(T), and 0.2-SM1T-5(T) contained MK-7 as a sole respiratory menaquinone and A4α type peptidoglycan based on lysine with alanine, glutamic acid, and aspartic acid. The common polar lipids include diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine.
Strain T2O-4(T) contained one unidentified phospholipid, whereas strain 0.2-SM1T-5(T) contained two unidentified phospholipids. Cellular fatty acid profiles, with C(15:0) anteiso as the major fatty acid, supported the affiliation of the three strains to the genus Sporosarcina. Based on the polyphasic characteristics, strains B2O-1(T) (= KCTC 43506(T) = JCM 36032(T)), T2O-4(T) (= KCTC 43489(T) = JCM 36031(T)), and 0.2-SM1T-5(T) (= KCTC 43519(T) = JCM 36034(T)) represent three novel species within the genus Sporosarcina, named Sporosarcina jeotgali sp. nov., Sporosarcina oncorhynchi sp. nov., and Sporosarcina trichiuri sp. nov., respectively.
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