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1.
Nat Commun ; 11(1): 5080, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033258

RESUMO

Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.


Assuntos
Sistemas de Secreção Bacterianos/ultraestrutura , Microscopia Crioeletrônica , Fímbrias Bacterianas/ultraestrutura , Secretina/química , Vibrio cholerae/metabolismo , Vibrio cholerae/ultraestrutura , Cisteína/genética , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Mutação/genética , Filogenia , Domínios Proteicos , Transformação Bacteriana
2.
PLoS One ; 15(6): e0234440, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530971

RESUMO

Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous recombination. This is the first example for natural transformation filamentous non-heterocystous cyanobacterium. We found that Phormidium lacuna is polyploid, each cell has about 20-90 chromosomes. Transformed filaments were resistant against up to 14 mg/ml of kanamycin. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.


Assuntos
Cianobactérias/genética , Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Recombinação Homóloga , Transformação Bacteriana , Cromossomos Bacterianos/genética , Biologia Computacional , Canamicina/farmacologia , Poliploidia
3.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32444468

RESUMO

Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are, however, barely tractable outside their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates the reproduction of its host by killing its male progeny at the embryonic stage. S. poulsonii, although a very fastidious bacterium, is closely related to pathogenic Spiroplasma species that are cultivable and genetically modifiable. In this work, we present the transformation of S. poulsonii with a plasmid bearing a fluorescence cassette, leveraging techniques adapted from those used to modify the pathogenic species Spiroplasma citri We demonstrate the feasibility of S. poulsonii transformation and discuss approaches for mutant selection and fly colonization, which are persisting hurdles that must be overcome to allow functional bacterial genetics studies of this endosymbiont in vivo IMPORTANCE Dozens of bacterial endosymbiont species have been described and estimated to infect about half of all insect species. However, only a few them are tractable in vitro, which hampers our understanding of the bacterial determinants of the host-symbiont interaction. Developing a transformation method for S. poulsonii is a major step toward genomic engineering of this symbiont, which will foster basic research on endosymbiosis. This could also open the way to practical uses of endosymbiont engineering through paratransgenesis of vector or pest insects.


Assuntos
Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Spiroplasma/genética , Simbiose , Transformação Bacteriana , Animais , Feminino , Masculino , Reprodução
4.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32385085

RESUMO

Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P cas ) and suppresses the type I-E CRISPR-Cas system in Escherichia coli Although the H-NS paralogue StpA also binds P cas , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P cas Third, by comparing transcriptional activities of the intact P cas and the P cas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P cas Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P cas Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.


Assuntos
Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Chaperonas Moleculares/genética , Transformação Bacteriana , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo
5.
Nat Commun ; 11(1): 1529, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251274

RESUMO

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.


Assuntos
Biotecnologia/métodos , Biblioteca Gênica , Inteínas/genética , Engenharia de Proteínas/métodos , Processamento de Proteína , Clonagem Molecular , Estudos de Viabilidade , Fluorescência , Genes Reporter/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Peptídeos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Transformação Bacteriana
6.
Nucleic Acids Res ; 48(8): 4585-4600, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32232367

RESUMO

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.


Assuntos
Acinetobacter/genética , Engenharia Genética/métodos , Genoma Bacteriano , Acinetobacter/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Deleção de Genes , Genes Bacterianos , Transformação Bacteriana
7.
Nat Commun ; 11(1): 1688, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245943

RESUMO

The cyanobacterium Synechococcus elongatus is a model organism for the study of circadian rhythms. It is naturally competent for transformation-that is, it takes up DNA from the environment, but the underlying mechanisms are unclear. Here, we use a genome-wide screen to identify genes required for natural transformation in S. elongatus, including genes encoding a conserved Type IV pilus, genes known to be associated with competence in other bacteria, and others. Pilus biogenesis occurs daily in the morning, while natural transformation is maximal when the onset of darkness coincides with the dusk circadian peak. Thus, the competence state in cyanobacteria is regulated by the circadian clock and can adapt to seasonal changes of day length.


Assuntos
Relógios Circadianos/fisiologia , Fímbrias Bacterianas/metabolismo , Synechococcus/fisiologia , Transformação Bacteriana/fisiologia , Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Elementos de DNA Transponíveis/genética , Escuridão , Regulação Bacteriana da Expressão Gênica/fisiologia , Transferência Genética Horizontal , Modelos Biológicos , Mutação , Estações do Ano , Fatores de Transcrição/metabolismo
8.
Int J Mol Sci ; 21(5)2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32131382

RESUMO

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5'- and 3'-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.


Assuntos
Engenharia Genética/métodos , Recombinação Homóloga , Plasmídeos/genética , Clonagem Molecular/métodos , Escherichia coli , Transformação Bacteriana
9.
Ecotoxicol Environ Saf ; 195: 110461, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32182530

RESUMO

Antibiotic residues in the environment pose a great risk to global public health. They increase antibiotic resistance by enhancing plasmid conjugation among bacteria or mutations within bacterial genomes. However, little is known about whether the putative environmental levels of antibiotics are sufficient to influence plasmid-mediated transformability. In this study, we explored the effect of eight kinds of representative antibiotics and several other compounds on the plasmid transformability of competent Escherichia coli. Only levofloxacin (LEV) at the putative environmental levels was found to facilitate the frequency of PBR322-or RP4-plasmid-mediated transformation by up to 5.3-fold. Additionally, PBR322 transformation frequency could be further enhanced by copper ion or ammonia nitrogen but inhibited by humic acid. However, when competent E. coli was exposed to the minimal inhibitory concentrations (MIC) of the antibiotics, an enhanced plasmid-assimilation ability was observed and plasmid transformation frequency was increased by up to 98.6-fold for all the tested antibiotics. Furthermore, E. coli exhibited a preference for the uptake of plasmids harbouring the resistance genes to the antibiotics it had been exposed to. Among these antibiotics, cephalexin, tetracycline, and kanamycin induced the highest uptake of RP4. The putative environmental levels of LEV enhanced plasmid transformability regardless of the presence of corresponding antibiotic resistance gene (ARG) on the genetic elements, suggesting environmental LEV residues may facilitate dissemination of antibiotic resistance by any plasmid-mediated transformability, thereby posing a great risk to health.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Levofloxacino/farmacologia , Transformação Bacteriana/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos
10.
Mol Biotechnol ; 62(4): 240-251, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32108286

RESUMO

In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.


Assuntos
Imunoglobulina G/biossíntese , Agricultura Molecular/métodos , Mucuna/genética , Agrobacterium tumefaciens/genética , Expressão Gênica , Técnicas de Transferência de Genes/instrumentação , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Mucuna/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Toxoplasma/imunologia , Transformação Bacteriana
11.
PLoS One ; 15(1): e0217255, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31931516

RESUMO

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ('toxTA'), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy- and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.


Assuntos
DNA/genética , Haemophilus influenzae/genética , Streptococcus/genética , Sistemas Toxina-Antitoxina/genética , Fatores de Transcrição/genética , Antitoxinas/genética , DNA/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Transferência Genética Horizontal/genética , Óperon/genética , Regiões Promotoras Genéticas , Biossíntese de Proteínas/genética , RNA-Seq , Transativadores/genética , Transformação Bacteriana/genética
12.
Curr Issues Mol Biol ; 37: 57-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31950915

RESUMO

Transformation is the process of import and inheritable integration of DNA from the environment. As such, it is believed to be a major driving force for evolution. Competence for transformation is widespread among bacterial species. Recent findings draw a picture of a conserved molecular machine that binds DNA at the cell surface and subsequently transports it through the cell envelope. Within the cytoplasm the DNA is coated by proteins that mediate recombination or self-annealing. The regulatory mechanisms and environmental signals affecting competence are very diverse between different bacterial species. Competence in Bacillus subtilis has become a paradigm for stochastic determination of cell-fate. Quantitative analysis at the single cell level in conjunction with mathematical modelling allowed understanding of induction and decline of competence at the systems level. Currently, the picture is emerging of stochastic differentiation as a fitness trade-off in fluctuating environments.


Assuntos
Bacillus subtilis/fisiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Fenômenos Fisiológicos Bacterianos , Competência de Transformação por DNA , Humanos , Fenótipo , Percepção de Quorum , Transformação Bacteriana/genética
13.
Environ Sci Technol ; 54(3): 1808-1815, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31965791

RESUMO

The practice of urine source-separation for fertilizer production necessitates an understanding of the presence and impact of extracellular DNA in the urine. This study examines the fate of plasmid DNA carrying ampicillin and tetracycline resistance genes in aged urine, including its ability to be taken up and expressed by competent bacteria. Plasmid DNA incubated in aged urine resulted in a >2 log loss of bacterial transformation efficiency in Acinetobacter baylyi within 24 h. The concentration of ampicillin and tetracycline resistance genes, as measured with quantitative polymerase chain reaction, did not correspond with the observed transformation loss. When the plasmid DNA was incubated in aged urine that had been filtered (0.22 µm) or heated (75 °C), the transformation efficiencies were more stable than when the plasmids were incubated in unfiltered and unheated aged urine. Gel electrophoresis results indicated that plasmid linearization by materials larger than 100 kDa in the aged urine caused the observed transformation efficiency decreases. The results of this study suggest that extracellular DNA released into aged urine poses a low potential for the spread of antibiotic resistance genes to bacteria once it is released to the environment.


Assuntos
Fertilizantes , Transformação Bacteriana , Antibacterianos , DNA , DNA Bacteriano , Plasmídeos
14.
J Biomed Sci ; 27(1): 8, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900177

RESUMO

BACKGROUND: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. METHODS: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. RESULTS: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. CONCLUSIONS: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.


Assuntos
Infecções Bacterianas/genética , Proteínas de Bactérias/genética , Plasmídeos/genética , beta-Lactamases/genética , Infecções Bacterianas/microbiologia , Biofilmes/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/patogenicidade , Aptidão Genética/genética , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Transformação Bacteriana/genética , Virulência/genética
15.
J Helminthol ; 94: e118, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31959266

RESUMO

Glutathione S-transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus, but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum, the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum (Ace-GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace-GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace-GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace-GST was 468 bp, which had the highest homology with Ac-GST-1 (60.1%) and clustered into one branch (v-class) with Ac-GST-1 and Na-GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace-GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace-GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace-GST.


Assuntos
Ancylostoma , Glutationa Transferase/metabolismo , Ancylostoma/genética , Ancylostoma/imunologia , Ancylostoma/metabolismo , Ancilostomíase , Animais , Anticorpos Anti-Helmínticos , Antígenos de Helmintos/genética , Antígenos de Helmintos/metabolismo , Clonagem Molecular , DNA de Helmintos/genética , DNA de Helmintos/isolamento & purificação , Escherichia coli/genética , Glutationa Transferase/genética , Imuno-Histoquímica , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Bacteriana
16.
Res Vet Sci ; 128: 308-314, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901569

RESUMO

As most pathogens invade the bodies through the mucosa, it is crucial to develop vaccines that induce mucosal immunity. To this end, we generated a safe and effective vaccine candidate that displayed fimbrial protein 987P of enterotoxigenic Escherichia coli (ETEC) on the surface of Lactobacillus casei (L.casei) CICC 6105 by using poly-γ-glutamate synthetase A (PgsA) as an anchoring matrix. After gavage inoculation of the recombinant strain pLA-987P/L.casei into specific-pathogen-free (SPF) BALB/c mice, high levels of mucosal immunoglobulin A (IgA) were induced in fecal samples, intestine and lung lavage fluids and systemic immunoglobulin G of IgG subclasses (IgG1, IgG2b, and IgG2a) was produced in serum. T-cell proliferation assays showed the stimulation index (SI) of the groups immunized with pLA-987P/L.casei to be significantly higher than that of the control group. The recombinant L.casei promoted T cells to produce both Th1 and Th2 cytokines, while the number of splenic IL-4 Spot forming cells (SFC) exceeded the number of IFN-γ SFC by 2.26-fold (P < .01). >83.3% of the vaccinated mice were protected from challenge with a lethal dose of virulent strain C83916. These results indicate that the recombinant L.casei expressing ETEC 987P fimbrial protein could elicit a protective immune response against ETEC 987P infection effectively.


Assuntos
Adesinas de Escherichia coli/imunologia , Escherichia coli Enterotoxigênica/imunologia , Vacinas contra Escherichia coli/biossíntese , Proteínas de Fímbrias/imunologia , Lactobacillus casei/imunologia , Microrganismos Geneticamente Modificados/imunologia , Adesinas de Escherichia coli/genética , Administração Oral , Animais , Antígenos Heterófilos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/imunologia , Proteínas de Fímbrias/genética , Imunidade Humoral , Imunidade nas Mucosas , Imunogenicidade da Vacina , Lactobacillus casei/genética , Camundongos , Camundongos Endogâmicos BALB C , Transformação Bacteriana/genética , Transformação Bacteriana/imunologia , Vacinação/métodos
17.
PLoS Biol ; 18(1): e3000589, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31922526

RESUMO

Electroporation is a basic yet powerful method for delivering small molecules (RNA, DNA, drugs) across cell membranes by application of an electrical field. It is used for many diverse applications, from genetically engineering cells to drug- and DNA-based vaccine delivery. Despite this broad utility, the high cost of electroporators can keep this approach out of reach for many budget-conscious laboratories. To address this need, we develop a simple, inexpensive, and handheld electroporator inspired by and derived from a common household piezoelectric stove lighter. The proposed "ElectroPen" device can cost as little as 23 cents (US dollars) to manufacture, is portable (weighs 13 g and requires no electricity), can be easily fabricated using 3D printing, and delivers repeatable exponentially decaying pulses of about 2,000 V in 5 ms. We provide a proof-of-concept demonstration by genetically transforming plasmids into Escherichia coli cells, showing transformation efficiency comparable to commercial devices, but at a fraction of the cost. We also demonstrate the potential for rapid dissemination of this approach, with multiple research groups across the globe validating the ease of construction and functionality of our device, supporting the potential for democratization of science through frugal tools. Thus, the simplicity, accessibility, and affordability of our device holds potential for making modern synthetic biology accessible in high school, community, and resource-poor laboratories.


Assuntos
Eletroporação/instrumentação , Técnicas de Transferência de Genes/instrumentação , Análise Custo-Benefício , Eletricidade , Eletroporação/economia , Desenho de Equipamento/economia , Escherichia coli , Técnicas de Transferência de Genes/economia , Humanos , Laboratórios/economia , Manufaturas/economia , Áreas de Pobreza , Impressão Tridimensional , Transformação Bacteriana , Transportes
18.
Biotechnol Lett ; 42(4): 633-640, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31965395

RESUMO

OBJECTIVES: Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. RESULTS: In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. CONCLUSIONS: These results indicate that overexpression of pckA or tpiA can enhance the pyruvate "pool" and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Floroglucinol/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Ácido Pirúvico/metabolismo , Triose-Fosfato Isomerase/metabolismo , Técnicas de Cultura Celular por Lotes/instrumentação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Plasmídeos/genética , Transformação Bacteriana , Triose-Fosfato Isomerase/genética
19.
Anaerobe ; 61: 102085, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31401257

RESUMO

Major advances in Clostridium perfringens genetics have been achieved through the development of electroporation-induced transformation; however, highly transformable strains are still limited. SM101 is the only useful strain for genetic manipulation via transformation in C. perfringens causing foodborne illness (FBI). We focused on the FBI strain NCTC8239, which is transformed at a low frequency, because it has a gene cassette that is predicted to encode enzymes involved in DNA phosphorothioation (PT). The oxidant-dependent degradation of NCTC8239 genomic DNA suggested that the genome is PT-modified. When foreign DNA was PT-modified using a plasmid expressing Salmonella enterica PT modification enzymes, the transformation efficiency of NCTC8239 was significantly higher than that using an unmodified plasmid. We then attempted to establish a highly transformable derivative of NCTC8239, and focused on DptFGH, which are predicted to be PT restriction enzymes. A dptG-null mutant exhibited significantly higher transformation efficiency with unmodified foreign DNA than did the wild-type strain. Furthermore, the mutant was transformed with the unmodified plasmid as efficiently as with a PT-modified plasmid, implying that DptG is involved in PT-dependent restriction. Thus, the present results revealed that PT modifications of foreign DNA influence the transformation frequency of NCTC8239 and suggest that PT is a factor contributing to transformation efficiency in NCTC8239.


Assuntos
Clostridium perfringens/genética , Plasmídeos/genética , Transformação Bacteriana , Clostridium perfringens/metabolismo , Mutação , Oxidantes , Oligonucleotídeos Fosforotioatos , Plasmídeos/metabolismo , Tionucleosídeos/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-31740556

RESUMO

Quinolone resistance is increasing in Neisseria meningitidis, with its prevalence in China being high (>70%), but its origin remains unknown. The aim of this study was to investigate the donors of mutation-harboring gyrA alleles in N. meningitidis A total of 198 N. meningitidis isolates and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China. The MICs of ciprofloxacin were determined using the agar dilution method. The resistance-associated genes gyrA and parC were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the N. meningitidis and commensal Neisseria isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant N. meningitidis isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the gyrA sequence identified six clusters. Among the 71 mutation-harboring gyrA alleles found in 221 N. meningitidis isolates and genomes (n = 221), 12 alleles (n = 103, 46.6%) were included in the N. meningitidis cluster, while 20 alleles (n = 56) were included in the N. lactamica cluster, 27 alleles (n = 49) were included in the N. cinerea cluster, and 9 alleles (n = 10) were included in the N. subflava cluster. Genomic analyses identified the exact N. lactamica donors of seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) and the N. subflava donor isolate of gyrA171, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of gyrA fragments from these donor strains into a meningococcal isolate increased its ciprofloxacin MIC from 0.004 µg/ml to 0.125 or 0.19 µg/ml and to 0.5 µg/ml with further transformation of an additional ParC mutation. Over half of the quinolone-resistant N. meningitidis isolates acquired resistance by horizontal gene transfer from three commensal Neisseria species. Quinolone resistance in N. meningitidis increases in a stepwise manner.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/genética , Neisseria/efeitos dos fármacos , Quinolonas/farmacologia , China/epidemiologia , Ciprofloxacino/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Mutação/genética , Neisseria/genética , Filogenia , Prevalência , Transformação Bacteriana/genética
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