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Archives of Microbiology Jun 2024A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic...
A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic analysis based on 16S rRNA gene sequences of strain HL-JVS1 revealed its affiliation with the genus Pleionea, with close relatives including Pleionea mediterranea MOLA115 (97.5%) and Pleionea sediminis S1-5-21 (96.2%). The complete genome of strain HL-JVS1 consisted of a circular 4.4 Mb chromosome and two circular plasmids (6.6 and 35.0 kb) with a G + C content of 43.1%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-JVS1 and the type strains of described Pleionea species were 69.7-70.4% and 18.3-18.6%, respectively. Strain HL-JVS1 grew at 10-40 °C (optimum, 30 °C) in the presence of 0.5 - 9.0% (w/v) sea salts (optimum, 2.0 - 2.5%), and at pH range of 5.5 - 10.0 (optimum, pH 6.5). The major fatty acids (> 10%) were summed feature 9 (iso-C ω9c and/or C 10-methyl) (23.3%), iso-C (14.5%), iso-C 3-OH (13.8%) and iso-C (11.0%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, two unidentified aminolipids, and two unidentified lipids. The respiratory quinone was ubiquinone-8. The comprehensive phylogenetic, phylogenomic, phenotypic and chemotaxonomic results showed that strain HL-JVS1 is distinct from other Pleionea species. Hence, we propose strain HL-JVS1 as a novel species belonging to the genus Pleionea, for which the name Pleionea litopenaei sp. nov. is proposed with HL-JVS1 (= KCCM 90514 = JCM 36490) as the type strain.
Topics: Animals; Penaeidae; Phylogeny; Base Composition; RNA, Ribosomal, 16S; Fatty Acids; DNA, Bacterial; Bacterial Typing Techniques; Nucleic Acid Hybridization; Sequence Analysis, DNA; Genome, Bacterial; Planococcaceae; Gastrointestinal Tract; Phospholipids
PubMed: 38951206
DOI: 10.1007/s00203-024-04064-7 -
Applied Microbiology and Biotechnology Jun 2024Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and...
Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.
Topics: Escherichia coli; Cloning, Molecular; Plasmids; Recombination, Genetic; Polymerase Chain Reaction; Genetic Vectors; CRISPR-Cas Systems
PubMed: 38951186
DOI: 10.1007/s00253-024-13239-7 -
Archives of Microbiology Jun 2024A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth...
A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29 was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29 showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166 (96.8%) and Thioclava marina 11.10-0-13 (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29 formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29 and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29, the major fatty acids identified were summed feature 8 (Cω7c) and C and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29 included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29 comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29 and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29 represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29 (= KCTC 82,841 = MCCC 1K08523).
Topics: Seawater; Phylogeny; RNA, Ribosomal, 16S; Fatty Acids; DNA, Bacterial; Base Composition; China; Bacterial Typing Techniques; Phospholipids; Alphaproteobacteria; Sequence Analysis, DNA; Ubiquinone; Nucleic Acid Hybridization
PubMed: 38951168
DOI: 10.1007/s00203-024-04057-6 -
Methods in Molecular Biology (Clifton,... 2024Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which...
Mathematical models have been used to study the spread of infectious diseases from person to person. More recently studies are developing within-host modeling which provides an understanding of how pathogens-bacteria, fungi, parasites, or viruses-develop, spread, and evolve inside a single individual and their interaction with the host's immune system.Such models have the potential to provide a more detailed and complete description of the pathogenesis of diseases within-host and identify other influencing factors that may not be detected otherwise. Mathematical models can be used to aid understanding of the global antibiotic resistance (ABR) crisis and identify new ways of combating this threat.ABR occurs when bacteria respond to random or selective pressures and adapt to new environments through the acquisition of new genetic traits. This is usually through the acquisition of a piece of DNA from other bacteria, a process called horizontal gene transfer (HGT), the modification of a piece of DNA within a bacterium, or through. Bacteria have evolved mechanisms that enable them to respond to environmental threats by mutation, and horizontal gene transfer (HGT): conjugation; transduction; and transformation. A frequent mechanism of HGT responsible for spreading antibiotic resistance on the global scale is conjugation, as it allows the direct transfer of mobile genetic elements (MGEs). Although there are several MGEs, the most important MGEs which promote the development and rapid spread of antimicrobial resistance genes in bacterial populations are plasmids and transposons. Each of the resistance-spread-mechanisms mentioned above can be modeled allowing us to understand the process better and to define strategies to reduce resistance.
Topics: Bacteria; Humans; Gene Transfer, Horizontal; Drug Resistance, Microbial; Models, Theoretical; Drug Resistance, Bacterial; Anti-Bacterial Agents; Host-Pathogen Interactions
PubMed: 38949703
DOI: 10.1007/978-1-0716-3981-8_9 -
Mikrochimica Acta Jun 2024A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for...
A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/μL in a model plasmid containing the malB gene and 3 CFU/μL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.
Topics: Nucleic Acid Amplification Techniques; Escherichia coli; Limit of Detection; Milk; Animals; Molecular Diagnostic Techniques; Microfluidic Analytical Techniques; DNA, Bacterial
PubMed: 38949666
DOI: 10.1007/s00604-024-06509-8 -
Molecular Plant-microbe Interactions :... Jul 2024The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial...
The emergence of plant pathogens is often associated with waves of unique evolutionary and epidemiological events. pv. is one of the major pathogens causing bacterial spot disease of tomatoes. After its first report in the 1950s, there were no formal reports on this pathogen until the 1990s, despite active global research on the pathogens that cause tomato and pepper bacterial spot disease. Given the recently documented global distribution of pv. , our objective was to examine genomic diversification associated with its emergence. We sequenced the genomes of pv. strains collected in eight countries to examine global population structure and pathways of emergence using phylodynamic analysis. We found that strains isolated post-1990 group by region of collection and show minimal impact of recombination on genetic variation. A period of rapid geographic expansion in pv. is associated with acquisition of a large plasmid conferring copper tolerance by horizontal transfer and coincides with the burgeoning hybrid tomato seed industry through the 1980s. The ancestry of pv. is consistent with introduction to hybrid tomato seed production and dissemination during the rapid increase in trade of hybrid seeds.
PubMed: 38949619
DOI: 10.1094/MPMI-04-24-0035-R -
Biomaterials Science Jul 2024Zwitterionic carboxyalkyl poly(1-vinylimidazole) (CA-PVIm) polymers with imidazolium cations and carboxylate anions have been synthesized as a carrier for the delivery...
Zwitterionic carboxyalkyl poly(1-vinylimidazole) (CA-PVIm) polymers with imidazolium cations and carboxylate anions have been synthesized as a carrier for the delivery of plasmid DNA (pDNA) to skeletal muscle. From differential scanning calorimetry measurements, resulting CA-PVIm had intermediate water in hydration water as a biocompatible polymer. Notably, when the pDNA and resulting CA-PVIm were mixed, slight retarded bands of the pDNA were observed in agarose gel electrophoresis, suggesting the polyion complex (PIC) formation between the pDNA and CA-PVIm despite zwitterionic polymers. Resulting PICs maintained the higher-order structure of the pDNA. Using resulting pDNA PICs, the highest pDNA expression by intramuscular injection was achieved in the PIC with 7 mol% carboxymethylated PVIm, that is, CA(7)-PVIm, observed in a widespread area by imaging system. These results suggest that the CA(7)-PVIm/pDNA PIC is effective for the diffusive delivery of the pDNA into skeletal muscle for the treatment of serious muscle diseases.
PubMed: 38949480
DOI: 10.1039/d4bm00510d -
Journal of Visualized Experiments : JoVE Jun 2024Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic...
Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.
Topics: Animals; Neural Stem Cells; Mice; Transfection; Lateral Ventricles; Cytological Techniques
PubMed: 38949388
DOI: 10.3791/66651 -
Microbiology Spectrum Jul 2024The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the...
UNLABELLED
The use of surrogate organisms can enable researchers to safely conduct research on pathogens and in a broader set of conditions. Being able to differentiate between the surrogates used in the experiments and background contamination as well as between different experiments will further improve research efforts. One effective approach is to introduce unique genetic barcodes into the surrogate genome and track their presence using the quantitative polymerase chain reaction (qPCR). In this report, we utilized the CRISPR-Cas9 methodology, which employs a single plasmid and a transformation step to insert five distinct barcodes into , a well-established surrogate for when Risk Group 1 organisms are needed. We subsequently developed qPCR assays for barcode detection and successfully demonstrated the stability of the barcodes within the genome through five cycles of sporulation and germination. Additionally, we conducted whole-genome sequencing on these modified strains and analyzed 187 potential Cas9 off-target sites. We found no correlation between the mutations observed in the engineered strains and the predicted off-target sites, suggesting this genome engineering strategy did not directly result in off-target mutations in the genome. This simple approach has the potential to streamline the creation of barcoded strains for use in future studies on surrogate genomes.
IMPORTANCE
The use of as a biothreat agent poses significant challenges for public health and national security. surrogates, like , are invaluable tools for safely understanding properties without the safety concerns that would arise from using a virulent strain of . We report a simple method for barcode insertion into using the CRISPR-Cas9 methodology and subsequent tracking by quantitative polymerase chain reaction (qPCR). Moreover, whole-genome sequencing data and CRISPR-Cas9 off-target analyses in suggest that this gene-editing method did not directly cause unwanted mutations in the genome. This study should assist in the facile development of barcoded surrogate strains, among other biotechnological applications in species.
PubMed: 38949306
DOI: 10.1128/spectrum.00003-24 -
Molecular Therapy. Nucleic Acids Jun 2024The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality -transcribed mRNA drug substance with specific critical...
The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality -transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.
PubMed: 38948330
DOI: 10.1016/j.omtn.2024.102223