Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.015
Filtrar
1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
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
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 Microbiol ; 58(2): 131-141, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31872373

RESUMO

Response regulator (RR) is known a protein that mediates cell's response to environmental changes. The effect of RR from extremophiles was still under investigation. In this study, response regulator homologs were mined from NGS data of Antarctic bacteria and overexpressed in Escherichia coli. Sixteen amino acid sequences were annotated corresponding to response regulators related to the two-component regulatory systems; of these, 3 amino acid sequences (DRH632, DRH1601 and DRH577) with high homology were selected. These genes were cloned in pRadGro and expressed in E. coli. The transformant strains were subjected to various abiotic stresses including oxidative, osmotic, thermal stress, and acidic stress. There was found that the robustness of E. coli to abiotic stress was increased in the presence of these response regulator homologs. Especially, recombinant E. coli overexpressing drh632 had the highest survival rate in oxidative, hypothermic, osmotic, and acidic conditions. Recombinant E. coli overexpressing drh1601 showed the highest tolerance level to osmotic stress. These results will be applicable for development of recombinant strains with high tolerance to abiotic stress.


Assuntos
Extremófilos/genética , Regulação Bacteriana da Expressão Gênica , Estresse Oxidativo/genética , Estresse Fisiológico/genética , Regiões Antárticas , Bacillus/genética , Bacillus/metabolismo , Bacillus pumilus/genética , Bacillus pumilus/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Biologia Computacional , Escherichia coli/genética , Escherichia coli/fisiologia , Extremófilos/metabolismo , Genoma Bacteriano , Estresse Oxidativo/fisiologia , Estresse Fisiológico/fisiologia , Transformação Bacteriana
15.
Molecules ; 25(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31878031

RESUMO

Microbial transformation of licochalcones B (1), C (2), D (3), and H (4) using the filamentous fungi Aspergillus niger and Mucor hiemalis was investigated. Fungal transformation of the licochalcones followed by chromatographic separations led to the isolation of ten new compounds 5-14, including one hydrogenated, three dihydroxylated, three expoxidized, and three glucosylated metabolites. Their structures were elucidated by combined analyses of UV, IR, MS, NMR, and CD spectroscopic data. Absolute configurations of the 2″,3″-diols in the three dihydroxylated metabolites were determined by ECD experiments according to the Snatzke's method. The trans-cis isomerization was observed for the metabolites 7, 11, 13, and 14 as evidenced by the analysis of their 1H-NMR spectra and HPLC chromatograms. This could be useful in better understanding of the trans-cis isomerization mechanism of retrochalcones. The fungal transformation described herein also provides an effective method to expand the structural diversity of retrochalcones for further biological studies.


Assuntos
Aspergillus niger/metabolismo , Biodegradação Ambiental , Fungos/metabolismo , Estrutura Molecular , Aspergillus niger/química , Chalconas/química , Cromatografia Líquida de Alta Pressão , Fungos/química , Espectroscopia de Ressonância Magnética , Transformação Bacteriana
16.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848285

RESUMO

In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae, expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus.IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus.


Assuntos
Regulação Bacteriana da Expressão Gênica , Transformação Bacteriana , Vibrio/fisiologia , Proteínas de Bactérias/genética , Competência de Transformação por DNA/genética , DNA Bacteriano , Expressão Gênica , Humanos , Modelos Biológicos , Fenótipo , Filogenia , Percepção de Quorum , Transativadores/genética , Vibrio/classificação
17.
Nat Commun ; 10(1): 5726, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844051

RESUMO

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.


Assuntos
Variações do Número de Cópias de DNA/genética , Farmacorresistência Bacteriana/genética , Tecnologia de Impulso Genético/métodos , Genes Bacterianos/genética , Loci Gênicos/genética , Antibacterianos/farmacologia , Tecnologia Biomédica/métodos , Biotecnologia/métodos , Sistemas CRISPR-Cas/genética , Contagem de Colônia Microbiana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Vetores Genéticos/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , RNA Guia/genética , Transformação Bacteriana
18.
Microb Cell Fact ; 18(1): 202, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31739786

RESUMO

BACKGROUND: Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO). RESULTS: PvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield. CONCLUSIONS: In summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.


Assuntos
Evolução Molecular Direcionada , Epóxido Hidrolases/genética , Phaseolus/enzimologia , Escherichia coli , Genes de Plantas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Phaseolus/genética , Transformação Bacteriana
19.
Nat Commun ; 10(1): 5357, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31767852

RESUMO

Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Transferência Genética Horizontal , Helicobacter pylori/metabolismo , Periplasma/metabolismo , Receptores de Superfície Celular/metabolismo , Transformação Bacteriana , Proteínas de Bactérias/genética , Transporte Biológico , DNA/genética , DNA/metabolismo , DNA Bacteriano/genética , Helicobacter pylori/genética , Periplasma/genética , Receptores de Superfície Celular/genética
20.
Appl Microbiol Biotechnol ; 103(23-24): 9205-9215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31650193

RESUMO

The uptake of exogenous DNA materials through the cell membrane by bacteria, known as transformation, is essential for the genetic manipulation of bacteria and, thus, plays key roles in biotechnological and biological research. The efficiency of natural transformation is very low; therefore, various artificial transformation methods have been developed for simple and efficient bacterial transformation. The basic bacterial transformation method is based on chemical, physical, and electrical processes and other means to permeabilize the bacterial cell membrane to allow plasmid DNA uptake. With the introduction of novel chemicals, materials, and devices and the optimization of protocols, new transformation methods have become simpler, cheaper, and more reproducible for use in diverse bacterial species compared with conventional methods. In this review, artificial transformation methods have been classified according to the membrane-permeabilizing mechanisms employed by them. Their influential factors, transformation efficiency, advantages, disadvantages, and practical applications are briefly illustrated. Finally, physicochemical transformation as a new bacterial transformation technique has also been described.


Assuntos
Bactérias/genética , Plasmídeos/genética , Transformação Bacteriana , Transformação Genética , DNA Bacteriano/genética , Microrganismos Geneticamente Modificados
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA