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1.
Enzyme Microb Technol ; 164: 110191, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36608408

RESUMO

Barnase is a ribonuclease used for plasmid purification, targeted gene therapy and studies of protein interactions. To make the use of barnase easier, the barnase gene from Bacillus amyloliquefaciens BH072 was cloned into Lactococcus lactis under the control of the PP5 or PnisA promoters. Four recombinant expression vectors were constructed with one or two signal peptides to control the enzyme secretion. 310 mg/L barnase was obtained in the presence of its inhibitor barstar after 36 h induction. The properties of barnase were investigated, showing that the optimal reaction temperature and pH were 50 °C and 5.0, respectively, and the highest enzyme activity reached 16.5 kU/mL. Barnase stored at 40 °C for 72 h retained 90 % of its initial activity, and maintained more than 80 % of its initial activity after 72 h of storage at pH 5.0-9.0. Furthermore, the optimal conditions for enzymatic reduction of nucleic acids in single-cell proteins (SCP) forages was investigated. 1 % salt solution with an SCP-enzyme ratio of 1000:1, pH 5.0 and incubated at 50 °C for 1 h, allowed 82 % RNA content reduction. Finally, homology modeling of barnase demonstrates its three-dimensional structure, and substrate simulation docking predicts key active residues as well as bonding patterns.


Assuntos
Lactococcus lactis , RNA , RNA/metabolismo , Lactococcus lactis/genética , Ribonucleases/genética , Ribonucleases/química , Ribonucleases/metabolismo , Proteínas de Bactérias/metabolismo , Plasmídeos
2.
Proc Natl Acad Sci U S A ; 120(3): e2210300120, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36634142

RESUMO

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.


Assuntos
Agrobacterium , Proteínas Repressoras , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Plasmídeos , Produtos Agrícolas/genética , Imunidade Vegetal , Raízes de Plantas/metabolismo
3.
Microb Cell Fact ; 22(1): 3, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609377

RESUMO

BACKGROUND: Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum. RESULTS: In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only. CONCLUSIONS: We developed a RecET-assisted CRISPR-Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.


Assuntos
Corynebacterium glutamicum , Edição de Genes , Humanos , Edição de Genes/métodos , Ácido Pantotênico/genética , Ácido Pantotênico/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Plasmídeos/genética , Sistemas CRISPR-Cas
4.
Nucleic Acids Res ; 51(1): 236-252, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36610752

RESUMO

Mobile genetic elements (MGEs) mediate the shuffling of genes among organisms. They contribute to the spread of virulence and antibiotic resistance (AMR) genes in human pathogens, such as the particularly problematic group of ESKAPE pathogens. Here, we performed the first systematic analysis of MGEs, including plasmids, prophages, and integrative and conjugative/mobilizable elements (ICEs/IMEs), across all ESKAPE pathogens. We found that different MGE types are asymmetrically distributed across these pathogens, and that most horizontal gene transfer (HGT) events are restricted by phylum or genus. We show that the MGEs proteome is involved in diverse functional processes and distinguish widespread proteins within the ESKAPE context. Moreover, anti-CRISPRs and AMR genes are overrepresented in the ESKAPE mobilome. Our results also underscore species-specific trends shaping the number of MGEs, AMR, and virulence genes across pairs of conspecific ESKAPE genomes with and without CRISPR-Cas systems. Finally, we observed that CRISPR spacers found on prophages, ICEs/IMEs, and plasmids have different targeting biases: while plasmid and prophage CRISPRs almost exclusively target other plasmids and prophages, respectively, ICEs/IMEs CRISPRs preferentially target prophages. Overall, our study highlights the general importance of the ESKAPE mobilome in contributing to the spread of AMR and mediating conflict among MGEs.


Assuntos
Antibacterianos , Sequências Repetitivas Dispersas , Humanos , Sequências Repetitivas Dispersas/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Plasmídeos/genética , Transferência Genética Horizontal/genética , Prófagos/genética
5.
Int J Mol Sci ; 24(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36614234

RESUMO

The L. lactis IL594 strain contains seven plasmids (pIL1 to pIL7) and is the parental strain of the plasmid-free L. lactis IL1403, one of the most studied lactic acid bacteria (LAB) strain. The genetic sequences of pIL1 to pIL7 plasmids have been recently described, however the knowledge of global changes in host phenotype and transcriptome remains poor. In the present study, global phenotypic analyses were combined with transcriptomic studies to evaluate a potential influence of plasmidic genes on overall gene expression in industrially important L. lactis strains. High-throughput screening of phenotypes differences revealed pronounced phenotypic differences in favor of IL594 during the metabolism of some C-sources, including lactose and ß-glucosides. A plasmids-bearing strain presented increased resistance to unfavorable growth conditions, including the presence of heavy metal ions and antimicrobial compounds. Global comparative transcriptomic study of L. lactis strains revealed variation in the expression of over 370 of chromosomal genes caused by plasmids presence. The general trend presented upregulated energy metabolism and biosynthetic genes, differentially expressed regulators, prophages and cell resistance proteins. Our findings suggest that plasmids maintenance leads to significant perturbation in global gene regulation that provides change in central metabolic pathways and adaptive properties of the IL594 cells.


Assuntos
Lactococcus lactis , Lactococcus lactis/metabolismo , Plasmídeos/genética , Genes Bacterianos , Fenótipo
6.
Mar Genomics ; 67: 100997, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36682852

RESUMO

Ruegeria sp. YS9, an aerobic and chemoheterotrophic bacterium belonging to marine Roseobacter lineage, was a putative new species isolated from red algae Eucheuma okamurai in the South China Sea (Beihai, Guangxi province). The complete genome sequence in strain YS9 comprised one circular chromosome with 3,244,635 bp and five circular plasmids ranging from 38,085 to 748,160 bp, with a total length of 4.30 Mb and average GC content of 58.39%. In total, 4129 CDSs, 52 tRNA genes and 9 rRNA genes were obtained. Genomic analysis of strain YS9 revealed that 85 CAZymes were organized in 147 PUL-associated CAZymes involved in polysaccharides metabolism, which were the highest among its two closely related Ruegeria strains. Numerous PULs related to degradation on the cell wall of algae, especially agar, indicated its major player role in the remineralization of algal-derived carbon. Further, the existence of multiple plasmids provided strain YS9 with distinct advantages to facilitate its rapid environmental adaptation, including polysaccharide metabolism, denitrification, resistance to heavy metal stresses such as copper and cobalt, type IV secretion systems and type IV toxin-antitoxin systems, which were obviously different from the two Ruegeria strains. This study provides evidence for polysaccharide metabolic capacity and functions of five plasmids in strain YS9, broadening our understanding of the ecological roles of bacteria in the environment around red algae and the function patterns of plasmids in marine Roseobacter lineage members for environmental adaptation.


Assuntos
Rhodobacteraceae , Rodófitas , Roseobacter , Roseobacter/genética , DNA Bacteriano/genética , China , Rhodobacteraceae/genética , Plasmídeos/genética , Polissacarídeos , Rodófitas/genética , Filogenia , Análise de Sequência de DNA , RNA Ribossômico 16S
7.
Nat Commun ; 14(1): 294, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653393

RESUMO

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.


Assuntos
Conjugação Genética , Escherichia coli , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/genética , DNA , DNA de Cadeia Simples/genética , Transferência Genética Horizontal
8.
Ann Clin Microbiol Antimicrob ; 22(1): 7, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658572

RESUMO

BACKGROUND: Pathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea. RESULTS: All 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for ß-lactam/ß-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate). CONCLUSION: Our findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Humanos , Animais , Suínos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Escherichia coli/tratamento farmacológico , beta-Lactamases/genética , Diarreia/veterinária , República da Coreia , Plasmídeos
9.
Genome Med ; 15(1): 3, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658655

RESUMO

BACKGROUND: Klebsiella pneumoniae (Kp) Gram-negative bacteria cause nosocomial infections and rapidly acquire antimicrobial resistance (AMR), which makes it a global threat to human health. It also has a comparatively rare hypervirulent phenotype that can lead to severe disease in otherwise healthy individuals. Unlike classic Kp, canonical hypervirulent strains usually have limited AMR. However, after initial case reports in 2015, carbapenem-resistant hypervirulent Kp has increased in prevalence, including in China, but there is limited understanding of its burden  in other geographical regions. METHODS: Here, we examined the largest collection of publicly available sequenced Kp isolates (n=13,178), containing 1603 different sequence types (e.g. ST11 15.0%, ST258 9.5%), and 2174 (16.5%) hypervirulent strains. We analysed the plasmid replicons and carbapenemase and siderophore encoding genes to understand the movement of hypervirulence and AMR genes located on plasmids, and their convergence in carbapenem-resistant hypervirulent Kp. RESULTS: We identified and analysed 3034 unique plasmid replicons to inform the epidemiology and transmission dynamics of carbapenem-resistant hypervirulent Kp (n=1028, 7.8%). We found several outbreaks globally, including one involving ST11 strains in China and another of ST231 in Asia centred on India, Thailand, and Pakistan. There was evidence of global flow of Kp, including across multiple continents. In most cases, clusters of Kp isolates are the result of hypervirulence genes entering classic strains, instead of carbapenem resistance genes entering canonical hypervirulent ones. CONCLUSIONS: Our analysis demonstrates the importance of plasmid analysis in the monitoring of carbapenem-resistant and hypervirulent strains of Kp. With the growing adoption of omics-based technologies for clinical and surveillance applications, including in geographical regions with gaps in data and knowledge (e.g. sub-Saharan Africa), the identification of the spread of AMR will inform infection control globally.


Assuntos
Carbapenêmicos , Infecções por Klebsiella , Humanos , Carbapenêmicos/farmacologia , Klebsiella pneumoniae , Virulência/genética , Plasmídeos/genética , beta-Lactamases/genética , Genômica , Antibacterianos/farmacologia , Infecções por Klebsiella/epidemiologia , Infecções por Klebsiella/microbiologia
10.
Food Microbiol ; 111: 104188, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36681389

RESUMO

The emergence of mobile colistin resistant gene (mcr-1) in Enterobacteriaceae has become a global public health concern. Dissemination of the mcr-1 gene through conjugation of bacteria associated with food may occur. This research investigated the transfer frequency of the mcr-1 gene among Escherichia coli in liquid media and during growth of mung bean sprouts. The donor strain E. coli NCTC 13846 (mcr-1 positive) and recipient strains of E. coli O157:H7 and E. coli O104:H4 were used. Mating experiments in vitro were conducted at 4, 25, and 37 °C for up to 36 h. The in vivo mating experiments (growing sprouts) were conducted in a sprout growth chamber with irrigation of 1 min/h over 6 days following inoculation of mung bean seeds with the donor and a recipient. The highest transfer frequencies in TSB media, 2.86E-07 and 3.24E-07, occurred at 37 °C after mating for 24 h for E. coli O104:H4 and E. coli O157:H7, respectively. Transconjugants were not detected in liquid media at 4 °C. Moreover, transfer frequency (5.68E-05 per recipient) of mcr-1 was greater during mung bean sprout growth for E. coli O104:H4 compared to E. coli O157:H7 (1.02E-05 per recipient) Day 3 to Day 6. This study indicates that the transfer of antibiotic resistant gene(s) among bacteria during mung bean sprout production may facilitate the spread of antibiotic resistant bacteria in the environment and to humans.


Assuntos
Escherichia coli O104 , Escherichia coli O157 , Proteínas de Escherichia coli , Fabaceae , Vigna , Antibacterianos , Colistina , Escherichia coli O104/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Fabaceae/microbiologia , Nutrientes , Plasmídeos , Farmacorresistência Bacteriana/genética
11.
Lett Appl Microbiol ; 76(1)2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36688778

RESUMO

Extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases (AmpCs)-producing Enterobacteriaceae have been increasingly reported and imposing significant threat to public. Livestock production industry might be the important source for clinically important ESBL-producing Enterobacteriaceae. This study aims to investigate the resistance profile, phenotypic ESBL production, beta-lactamase genes, virulence factors, and plasmid replicon types among 59 Enterobacteriaceae strains isolated from poultry faecal samples in Malaysia's commercial poultry farm. There were 38.7% and 32.3% of Escherichia coli resistant to cefotaxime and cefoxitin, respectively, while Klebsiellaspp. demonstrated resistance rate of 52.6% to both mentioned antimicrobials. Majority of the E. coli isolates carried blaTEM and blaCMY-2 group. blaSHV was the most prevalent gene detected in Klebsiellaspp., followed by blaDHA and blaTEM. Resistance to extended spectrum cephalosporin in our isolates was primarily mediated by plasmid mediated AmpC beta-lactamase such as CMY-2 group and DHA enzyme. The CTX-M genes were found in two ESBL-producing E. coli. IncF, IncI1, and IncN plasmids were most frequently detected in E. coli and Klebsiellaspp. The virulence factor, including EAST1 and pAA were identified at low frequency. This study highlights the poultry as a reservoir of resistance and virulence determinants and prevalence of plasmids in Enterobacteriaceae might drive their dissemination.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Animais , Escherichia coli/genética , Aves Domésticas , Infecções por Escherichia coli/veterinária , Fazendas , Enterobacteriaceae/genética , Malásia , Proteínas de Bactérias/genética , beta-Lactamases/genética , Plasmídeos , Antibacterianos
12.
Life Sci Alliance ; 6(4)2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36669792

RESUMO

Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreção Tipo IV , Plasmídeos/genética , DNA , Esterases/genética
13.
Int J Mol Sci ; 24(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36674575

RESUMO

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.


Assuntos
Reparo de Erro de Pareamento de DNA , Quadruplex G , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , DNA/química , Plasmídeos/genética , Reparo do DNA
14.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674913

RESUMO

Insufficient vascular growth in the area of artificial-material implantation contributes to ischemia, fibrosis, the development of bacterial infections, and tissue necrosis around the graft. The purpose of this study was to evaluate angiogenesis after implantation of polycaprolactone microfiber scaffolds modified by a pCMV-VEGF165-plasmid in rats. Influence of vascularization on scaffold degradation was also examined. We investigated flat microfibrous scaffolds obtained by electrospinning polycaprolactone with incorporation of the pCMV-VEGF-165 plasmid into the microfibers at concentrations of 0.005 ng of plasmid per 1 mg of polycaprolactone (0.005 ng/mg) (LCGroup) and 0.05 ng/mg (HCGroup). The samples were subcutaneously implanted in the interscapular area of rats. On days 7, 16, 33, 46, and 64, the scaffolds were removed, and a histological study with a morphometric evaluation of the density and diameter of the vessels and microfiber diameter was performed. The number of vessels was increased in all groups, as well as the resorption of the scaffold. On day 33, the vascular density in the HCGroup was 42% higher compared to the control group (p = 0.0344). The dose-dependent effect of the pCMV-VEGF165-plasmid was confirmed by enhanced angiogenesis in the HCGroup compared to the LCGroup on day 33 (p-value = 0.0259). We did not find a statistically significant correlation between scaffold degradation rate and vessel growth (the Pearson correlation coefficient was ρ = 0.20, p-value = 0.6134). Functionalization of polycaprolactone by incorporation of the pCMV-VEGF165 plasmid provided improved vascularization within 33 days after implantation, however, vessel growth did not seem to correlate with scaffold degradation rate.


Assuntos
Tecidos Suporte , Fator A de Crescimento do Endotélio Vascular , Ratos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neovascularização Fisiológica/genética , Plasmídeos/genética , Engenharia Tecidual
15.
Methods Mol Biol ; 2606: 123-133, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36592312

RESUMO

CRISPR-cas9-guided adenine base editors (ABEs) site-specifically convert the A-T base pair to G-C base pair in genomic DNA. The intracellular delivery of ABE proteins preassembled with guide RNAs (gRNAs) has shown greatly reduced off-target effects compared with that of plasmids or viral vectors containing ABE and gRNA-encoding sequences. For efficient gene editing by the ribonucleoprotein delivery method, the ABE-gRNA complexes need to be prepared in high purity and quantity. Here we describe the expression and purification procedure of ABEmax, one of high-efficiency ABE versions.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Adenina/metabolismo , Edição de Genes/métodos , Plasmídeos/genética , /genética
16.
Nat Commun ; 14(1): 462, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36709320

RESUMO

Shigella sonnei, the main cause of bacillary dysentery in high-income countries, has become increasingly resistant to antibiotics. We monitored the antimicrobial susceptibility of 7121 S. sonnei isolates collected in France between 2005 and 2021. We detected a dramatic increase in the proportion of isolates simultaneously resistant to ciprofloxacin (CIP), third-generation cephalosporins (3GCs) and azithromycin (AZM) from 2015. Our genomic analysis of 164 such extensively drug-resistant (XDR) isolates identified 13 different clusters within CIP-resistant sublineage 3.6.1, which was selected in South Asia ∼15 years ago. AZM resistance was subsequently acquired, principally through IncFII (pKSR100-like) plasmids. The last step in the development of the XDR phenotype involved various extended-spectrum beta-lactamase genes (blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-134) carried by different plasmids (IncFII, IncI1, IncB/O/K/Z) or even integrated into the chromosome, and encoding resistance to 3GCs. This rapid emergence of XDR S. sonnei, including an international epidemic strain, is alarming, and good laboratory-based surveillance of shigellosis will be crucial for informed decision-making and appropriate public health action.


Assuntos
Farmacorresistência Bacteriana Múltipla , Disenteria Bacilar , Shigella sonnei , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Azitromicina/farmacologia , Azitromicina/uso terapêutico , beta-Lactamases/genética , Ciprofloxacina/farmacologia , Disenteria Bacilar/tratamento farmacológico , Disenteria Bacilar/epidemiologia , França/epidemiologia , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Shigella sonnei/efeitos dos fármacos , Shigella sonnei/genética
17.
Virus Res ; 325: 199046, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36657615

RESUMO

Norovirus is the leading cause of viral gastroenteritis globally, and poses substantial threats to public health. Despite substantial progress made in preventing norovirus diseases, the lack of a robust virus culture system has hampered biological research and effective strategies to combat this pathogen. Reverse genetic system is the technique to generate infectious viruses from cloned genetic constructs, which is a powerful tool for the investigation of viral pathogenesis and for the development of novel drugs and vaccines. The strategies of reverse genetics include bacterial artificial chromosomes, vaccinia virus vectors, and entirely plasmid-based systems. Since each strategy has its pros and cons, choosing appropriate approaches will greatly improve the efficiency of virus rescue. Reverse genetic systems that have been employed for norovirus greatly extend its life cycle and facilitate the development of medical countermeasures. In this review, we summarize the current knowledge on the structure, transmission, genetic evolution and clinical manifestations of norovirus, and describe recent advances in the studies of norovirus reverse genetics as well as its future prospects for therapeutics and vaccine development.


Assuntos
Infecções por Caliciviridae , Norovirus , Humanos , Norovirus/genética , Genética Reversa/métodos , Plasmídeos
18.
PeerJ ; 11: e14709, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36718445

RESUMO

Acinetobacter baumannii is one of the most successful pathogens that can cause difficult-to-treat nosocomial infections. Outbreaks and infections caused by multi-drug resistant A. baumannii are prevalent worldwide, with only a few antibiotics are currently available for treatments. Plasmids represent an ideal vehicle for acquiring and transferring resistance genes in A. baumannii. Five extensively drug-resistant A. baumannii clinical isolates from three major Jordanian hospitals were fully sequenced. Whole-Genome Sequences (WGS) were used to study the antimicrobial resistance and virulence genes, sequence types, and phylogenetic relationship of the isolates. Plasmids were characterized In-silico, followed by conjugation, and plasmid curing experiments. Eight plasmids were recovered; resistance plasmids carrying either aminoglycosides or sulfonamide genes were detected. Chromosomal resistance genes included blaOXA-66, blaOXA-91, and blaOXA-23, and the detected virulence factors were involved in biofilm formation, adhesion, and many other mechanisms. Conjugation and plasmid curing experiments resulted in the transfer or loss of several resistance phenotypes. Plasmid profiling along with phylogenetic analyses revealed high similarities between two A. baumannii isolates recovered from two different intensive care units (ICU). The high similarities between the isolates of the study, especially the two ICU isolates, suggest that there is a common A. baumannii strain prevailing in different ICU wards in Jordanian hospitals. Three resistance genes were plasmid-borne, and the transfer of the resistance phenotype emphasizes the role and importance of conjugative plasmids in spreading resistance among A. baumannii clinical strains.


Assuntos
Acinetobacter baumannii , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/genética , Farmacorresistência Bacteriana Múltipla/genética
19.
Antimicrob Agents Chemother ; 67(1): e0135422, 2023 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-36602346

RESUMO

The carbapenem-resistant Klebsiella pneumoniae (CRKP) strain GX34 was recovered from the respiratory tract of an elderly male with severe pneumonia, and only susceptible to amikacin, tigecycline, and colistin. Complete genome suggested that it belonged to K51-ST16 and harbored plasmid-encoded NDM-4 and OXA-181, located on IncFIB plasmid GX34p1_NDM-4 and ColKP3/IncX3 plasmid GX34p4_OXA-181, respectively. A series of transconjugants generated in the plasmid conjugation assays, including Escherichia coli J53-N1 (harboring a self-transmissible and blaNDM-1-producing plasmid Eco-N-1-p), J53-N2 (harboring a blaNDM-4-producing plasmid and a helper plasmid GX34p5), and J53-O (harboring a blaOXA-181-producing plasmid), could be stably inherited after 10 days of serial passage and no significant biological fitness costs were detected. Furthermore, we first reported the blaNDM-1 gene, derived from blaNDM-4 mutation (460C>A) under meropenem pressure, could be in vitro transferred into a self-conjugative, recombined plasmid Eco-N-1-p of J53-N1. Eco-N-1-p was mainly recombined by GX34p4_OXA-181 (40,449 bp, 75.16%) and GX34p1_NDM-4 (8,553 bp, 15.89%), in which IS26 and IS5-like probably played a major role. Eco-N-1-p could be transferred into the conjugation recipient K. pneumoniae KP54 and make the latter sacrifice fitness. The retention rates of blaNDM-1 remained high stability (>80% after 200 generations). The comparative genomic analysis of GX34 and those carrying blaNDM-4 or blaOXA-181 genes retrieved from the NCBI RefSeq database showed all blaNDM-4 (26/26, 100.00%) and blaOXA-181 (13/13, 100.00%) were surrounded by IS26. The immediate environment of blaNDM-4 and blaOXA-181 in GX34 and some retrieved strains shared identical features, hinting at their possible dissemination. Effective measures should be taken to monitor the spread of this clone.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Humanos , Masculino , Idoso , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Elementos de DNA Transponíveis , Antibacterianos/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Escherichia coli/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/genética
20.
PLoS Pathog ; 19(1): e1010961, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36626407

RESUMO

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.


Assuntos
Sistemas CRISPR-Cas , Oryza , Xanthomonas , Proteínas de Bactérias/metabolismo , Edição de Genes/métodos , Genoma Bacteriano , Oryza/microbiologia , Plasmídeos , Xanthomonas/genética
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