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1.
Arch Microbiol ; 206(3): 116, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38388903

RESUMO

Bacillus subtilis can potentially serve as an efficient expression host for biotechnology due to its ability to secrete extracellular proteins and enzymes directly into the culture medium. One of the important challenges in the biotechnology industry is to optimize the transformation conditions of B. subtilis bacteria. This study aims to provide a new method to optimize the transformation conditions and improve the transformation efficiency of B. subtilis WB600. To increase the transformation efficiency in B. subtilis, two methods of adding CM11 antibacterial peptides to the bacterial medium along with electroporation and optimizing the variables including the growth medium composition, time to adding CM11 peptide, electroporation voltage, recovery medium, and cell recovery time are used. The results of this study showed that the addition of antimicrobial peptides (AMPs) with a concentration of 2 µg/ml increases the transformation efficiency by 4 times compared to the absence of AMP in the bacterial medium. Additionally, the findings from our study indicated that the most optimal rate of transformation for B. subtilis was observed at a voltage of 7.5 kV/cm, with a recovery period of 12 h. With the optimized method, the transformation efficiency came up to 1.69 × 104 CFU/µg DNA. This improvement in transformation efficiency will be attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for transformation of wild-type B. subtilis strains.


Assuntos
Bacillus subtilis , Transformação Bacteriana , Bacillus subtilis/genética , Plasmídeos/genética , Antibacterianos/farmacologia , Peptídeos
2.
Nano Lett ; 24(4): 1145-1152, 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38194429

RESUMO

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m-2 for 180 s at temperatures ranging from 30 to 40 °C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.


Assuntos
Escherichia coli , Transformação Bacteriana , Plasmídeos/genética , DNA/metabolismo , Bactérias/genética , Radiação Eletromagnética
3.
Microbiology (Reading) ; 169(8)2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37526972

RESUMO

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.


Assuntos
Bactérias , DNA , DNA Bacteriano/genética , Bactérias/genética , Transformação Bacteriana/genética , Adaptação Fisiológica
4.
Small ; 19(37): e2301177, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37144438

RESUMO

Graphene quantum dots (GQDs) coexist with antibiotic resistance genes (ARGs) in the environment. Whether GQDs influence ARG spread needs investigation, since the resulting development of multidrug-resistant pathogens would threaten human health. This study investigates the effect of GQDs on the horizontal transfer of extracellular ARGs (i.e., transformation, a pivotal way that ARGs spread) mediated by plasmids into competent Escherichia coli cells. GQDs enhance ARG transfer at lower concentrations, which are close to their environmental residual concentrations. However, with further increases in concentration (closer to working concentrations needed for wastewater remediation), the effects of enhancement weaken or even become inhibitory. At lower concentrations, GQDs promote the gene expression related to pore-forming outer membrane proteins and the generation of intracellular reactive oxygen species, thus inducing pore formation and enhancing membrane permeability. GQDs may also act as carriers to transport ARGs into cells. These factors result in enhanced ARG transfer. At higher concentrations, GQD aggregation occurs, and aggregates attach to the cell surface, reducing the effective contact area of recipients for external plasmids. GQDs also form large agglomerates with plasmids and thus hindering ARG entrance. This study could promote the understanding of the GQD-caused ecological risks and benefit their safe application.


Assuntos
Grafite , Pontos Quânticos , Humanos , Antibacterianos/farmacologia , Grafite/farmacologia , Transformação Bacteriana , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética
5.
Cold Spring Harb Protoc ; 2023(8): 107947, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37117021

RESUMO

This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.


Assuntos
Staphylococcus aureus , Transformação Bacteriana , Staphylococcus aureus/genética , DNA Bacteriano/genética , Plasmídeos/genética , Eletroporação/métodos
6.
Nucleic Acids Res ; 51(8): 3820-3835, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36912090

RESUMO

Natural transformation is one of the major mechanisms of horizontal gene transfer. Although it is usually studied using purified DNA in the laboratory, recent studies showed that many naturally competent bacteria acquired exogenous DNA from neighboring donor cells. Our previous work indicates that cell-to-cell natural transformation (CTCNT) using two different Bacillus subtilis strains is a highly efficient process; however, the mechanism is unclear. In this study, we further characterized CTCNT and mapped the transferred DNA in the recombinants using whole genome sequencing. We found that a recombinant strain generated by CTCNT received up to 66 transferred DNA segments; the average length of acquired continuous DNA stretches was approximately 27 kb with a maximum length of 347 kb. Moreover, up to 1.54 Mb genomic DNA (37% of the chromosome) was transferred from the donors into one recipient cell. These results suggest that B. subtilis CTCNT facilitates horizontal gene transfer by increasing the transfer of DNA segments and fostering the exchange of large continuous genomic regions. This indicates that the potency of bacterial natural transformation is underestimated using traditional approaches and reveals that DNA donor cells may play an important role in the transformation process in natural environments.


Assuntos
Bacillus subtilis , Transformação Bacteriana , Bacillus subtilis/genética , DNA/genética , DNA Bacteriano/genética , Genoma , Genômica
7.
Biomolecules ; 13(3)2023 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-36979449

RESUMO

C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens.


Assuntos
Campylobacter jejuni , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Transformação Bacteriana , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
8.
Sci Total Environ ; 874: 162422, 2023 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-36863585

RESUMO

Excessive presence of antibiotics and their residues can be dangerous to the natural environment. To reduce this negative effect, efficient strategies to remove them from the ecosystem are required. This study aimed to explore the potential of bacterial strains to degrade nitrofurantoin (NFT). Single strains isolated from contaminated areas, namely Stenotrophomonas acidaminiphila N0B, Pseudomonas indoloxydans WB, and Serratia marcescens ODW152 were employed in this study. Degradation efficiency and dynamic changes within the cells during NFT biodegradation were investigated. For this purpose, atomic force microscopy, flow cytometry, zeta potential, and particle size distribution measurements were applied. Serratia marcescens ODW152 showed the highest performance in removal of NFT (96 % in 28 days). The AFM images revealed modifications of cell shape and surface structure induced by NFT. Zeta potential showed significant variations during biodegradation. Cultures exposed to NFT had a broader size distribution than the control cultures due to increased cells agglomeration or aggregation. 1-Aminohydantoin and semicarbazide were detected as nitrofurantoin biotransformation products. They showed increased cytotoxicity toward bacteria as determined by spectroscopy and flow cytometry. Results of this study suggest that nitrofurantoin biodegradation leads to formation of stable transformation products that significantly affect the physiology and structure of bacterial cells.


Assuntos
Nitrofurantoína , Transformação Bacteriana , Nitrofurantoína/toxicidade , Ecossistema , Antibacterianos , Biodegradação Ambiental , Bactérias/metabolismo
9.
J Bacteriol ; 205(2): e0046522, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36695594

RESUMO

Natural transformation enables bacteria to acquire DNA from the environment and contributes to genetic diversity, DNA repair, and nutritional requirements. DNA processing protein A (DprA) receives incoming single-stranded DNA and assists RecA loading for homology-directed natural chromosomal transformation and DNA strand annealing during plasmid transformation. The dprA gene occurs in the genomes of all known bacteria, irrespective of their natural transformation status. The DprA protein has been characterized by its molecular, cellular, biochemical, and biophysical properties in several bacteria. This review summarizes different aspects of DprA biology, collectively describing its biochemical properties, molecular interaction with DNA, and function interaction with bacterial RecA during natural transformation. Furthermore, the roles of DprA in natural transformation, bacterial virulence, and pilin variation are discussed.


Assuntos
Proteínas de Fímbrias , Transformação Bacteriana , Proteínas de Fímbrias/genética , Proteínas de Bactérias/genética , Virulência , DNA , DNA de Cadeia Simples , Recombinases Rec A/metabolismo
10.
Nat Commun ; 13(1): 7724, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36513643

RESUMO

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.


Assuntos
Proteínas de Bactérias , Peptidoglicano , Peptidoglicano/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transformação Bacteriana , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bactérias Gram-Positivas/genética , DNA/metabolismo
11.
Microbiol Spectr ; 10(6): e0249722, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36317996

RESUMO

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/µg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/µg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.


Assuntos
Escherichia coli , Transformação Bacteriana , Escherichia coli/genética , Plasmídeos/genética , DNA/genética
12.
Biomolecules ; 12(10)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36291718

RESUMO

We report the discovery of a new abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism by the rhizosphere bacterium Rhodococcus sp. P1Y. Analogue of (+)-ABA, enriched in tritium in the cyclohexene moiety, was fed in bacterial cells, and extracts containing radioactive metabolites were purified and analyzed to determine their structure. We obtained mass spectral fragmentation patterns and nuclear magnetic resonance spectra of a new metabolite of ABA identified as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, which we named rhodococcal acid (RA) and characterized using several other techniques. This metabolite is the second bacterial ABA degradation product in addition to dehydrovomifoliol that we described earlier. Taken together, these data reveal an unknown ABA catabolic pathway that begins with side chain disassembly, as opposed to the conversion of the cyclohexene moiety in plants. The role of ABA-utilizing bacteria in interactions with other microorganisms and plants is also discussed.


Assuntos
Ácido Abscísico , Ácido Acético , Ácido Abscísico/metabolismo , Trítio , Transformação Bacteriana , Extratos Vegetais
13.
PLoS Biol ; 20(9): e3001727, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36067229

RESUMO

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.


Assuntos
Técnicas de Transferência de Genes , Genes Bacterianos , Microfluídica , Bifidobacterium longum/genética , Eletroporação/métodos , Escherichia coli/genética , Técnicas de Transferência de Genes/instrumentação , Microfluídica/métodos , Transformação Bacteriana/genética
14.
Microbiology (Reading) ; 168(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35920810

RESUMO

Neisseria gonorrhoeae, the causative agent of gonorrhoea, is a major burden on global healthcare systems, with an estimated ~80-90 million new global cases annually. This burden is exacerbated by increasing levels of antimicrobial resistance, which has greatly limited viable antimicrobial therapies. Decreasing gonococcal drug susceptibility has been driven largely by accumulation of chromosomal resistance determinants, which can be acquired through natural transformation, whereby DNA in the extracellular milieu is imported into cells and incorporated into the genome by homologous recombination. N. gonorrhoeae possesses a specialized system for DNA uptake, which strongly biases transformation in favour of DNA from closely related bacteria by recognizing a 10-12 bp DNA uptake sequence (DUS) motif, which is highly overrepresented in their chromosomal DNA. This process relies on numerous proteins, including the DUS-specific receptor ComP, which assemble retractile protein filaments termed type IV pili (T4P) extending from the cell surface, and one model for neisserial DNA uptake proposes that these filaments bind DNA in a DUS-dependent manner before retracting to transport DNA into the periplasm. However, conflicting evidence indicates that elongated pilus filaments may not have such a direct role in DNA binding uptake as this model suggests. Here, we quantitatively measured DNA binding to gonococcal T4P fibres by directly visualizing binding complexes with confocal fluorescence microscopy in order to confirm the sequence-specific, comP-dependent DNA binding capacity of elongated T4P fibres. This supports the idea that pilus filaments could be responsible for initially capturing DNA in the first step of sequence-specific DNA uptake.


Assuntos
Gonorreia , Transformação Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Gonorreia/metabolismo , Humanos , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo
15.
Nat Commun ; 13(1): 1961, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35414142

RESUMO

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein's in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.


Assuntos
DNA de Cadeia Simples , Helicobacter pylori , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Proteínas de Membrana/metabolismo , Transformação Bacteriana
16.
Nat Commun ; 13(1): 1065, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246533

RESUMO

Naturally competent bacteria encode sophisticated protein machinery for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. Here, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We use NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT's role in this process. FimT binds to DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in a conformationally flexible C-terminal tail. FimT orthologues from other Gammaproteobacteria share the ability to bind to DNA. Our results suggest that FimT plays an important role in DNA uptake in a wide range of competent species.


Assuntos
Proteínas de Fímbrias , Legionella pneumophila , Proteínas de Bactérias/metabolismo , DNA/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Transformação Bacteriana
17.
J Bacteriol ; 204(3): e0057221, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34928178

RESUMO

In competent Gram-negative and Gram-positive bacteria, double-stranded DNA is taken up through the outer cell membrane and/or the cell wall and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single-stranded DNA and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C terminus of ComEC, thought to act as a nuclease, is important not only for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single-molecule tracking, we show that only 13% of ComEC molecules are statically localized at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as a nuclease. By tracking fluorescently labeled DNA, we show that taken-up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken-up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken-up DNA, before its entry into the cytosol. IMPORTANCE Bacteria can take up DNA from the environment and incorporate it into their chromosome, which is termed "natural competence" that can result in the uptake of novel genetic information. We show that fluorescently labeled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This finding indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of ComEC at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm and involves a high degree of free diffusion along and within the cell membrane.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , DNA Bacteriano , Proteínas de Ligação a DNA , Proteínas de Membrana , Receptores de Superfície Celular , Transformação Bacteriana , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Receptores de Superfície Celular/metabolismo
18.
J Microbiol Methods ; 192: 106375, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34793853

RESUMO

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.


Assuntos
Comamonadaceae/genética , Eletroporação/métodos , Transformação Bacteriana/genética , DNA Bacteriano/genética , Plasmídeos/genética
19.
Sci Rep ; 11(1): 23964, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34907242

RESUMO

Successful delivery of plasmid DNA into the microbial cells is fundamental in recombinant DNA technology. Natural bacterial transformation is limited to only certain species due in part to the repulsive forces between negatively charged DNA and bacterial membranes. Most common method of DNA delivery into bacteria is artificial transformation through heat shock and electroporation. These methods require sophisticated instruments and tedious steps in preparation of competent cells. Transformation by conjugation is also not applicable to all plasmids. Nanoparticles have been used successfully in therapeutics for drug delivery into animal cells. They are starting to gain popularity in plant sciences as novel DNA nano carriers. Despite their promise as tool for DNA delivery, their use in microbial cell transformation has not been reported yet. Here we report the synthesis of carbon dots (CDs) from citric acid and ß-alanine and their use in DNA delivery into E. coli cells. CDs were fabricated using microwave assisted synthesis. Plasmids carrying RFP reporter and ampicillin resistance genes were transferred to bacterial cells and further confirmed using polymerase chain reaction. Our findings indicate that CDs can be used successfully for delivery of foreign DNA of up to 10 kb into E. coli. We have demonstrated the use of ß-alanine/citric acid carbon dots as nanocarriers of DNA into E. coli cells and identified their limitation in terms of the size of plasmid DNA they could carry. Use of these carbon dots is a novel method in foreign DNA delivery into bacterial cells and have a potential for the transformation of resistant organism for which there is still no reliable DNA delivery systems.


Assuntos
Ácido Cítrico/química , DNA , Escherichia coli/genética , Nanoestruturas/química , Plasmídeos , Transformação Bacteriana , beta-Alanina/química , DNA/química , DNA/genética , Plasmídeos/química , Plasmídeos/genética
20.
PLoS One ; 16(12): e0261088, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34914750

RESUMO

Plasmid transformation of chlamydiae has created new opportunities to investigate host-microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.


Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Cromossomos Bacterianos/genética , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Transformação Bacteriana , Chlamydia trachomatis/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética
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