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 fármacosRESUMO
Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 µm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.
Assuntos
Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/genética , Transformação Bacteriana , Antibacterianos/farmacologia , Comunicação Celular/efeitos dos fármacos , Células Cultivadas , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Testes de Sensibilidade Microbiana , Técnicas Analíticas Microfluídicas/instrumentação , Novobiocina/farmacologia , Tamanho da Partícula , Rifampina/farmacologia , Espectinomicina/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Propriedades de Superfície , Transformação Bacteriana/efeitos dos fármacos , Transformação Bacteriana/genéticaRESUMO
Streptococcus pneumoniae are natural competent bacteria which requires the presence of a pheromone-like molecule to do the transformation process. This study verified the influence of mesoporous silica (SBA-15 and SBA-16) on the transformation process in S. pneumoniae using a donor DNA obtained from a mutant strain of this microorganism (Sp360∆luxS). The results showed that mesoporous silica SBA-15 and SBA-16 particles doubled the transformation ratio frequency compared with negative control (without nanoparticles) in using SBA-15 (ratio 1.81 ± 0.04) and SBA-16 (ratio 2.18 ± 0.22). We demonstrated the how mesoporous silica nanoparticles were able to increase the pneumococcus transformations, which could possibly lead to the acquisition of virulence factor genes and resistance of antibiotics.
Assuntos
Poluentes Atmosféricos/farmacologia , Dióxido de Silício/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Transformação Bacteriana/efeitos dos fármacos , Poluentes Atmosféricos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Tamanho da Partícula , Plasmídeos/genética , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Our previous data show that serum albumin can trigger natural transformation in Acinetobacter baumannii. However, extracellular matrix/basal membrane components, norepinephrine, and mucin did not have a significant effect on this process. Therefore, the effect of human products appears to be albumin specific, as both BSA and HSA have been shown to increase of natural transformation.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Competência de Transformação por DNA/efeitos dos fármacos , Albumina Sérica Humana/metabolismo , Transformação Bacteriana/efeitos dos fármacos , HumanosRESUMO
A practical random mutagenesis system of Ralstonia solanacearum by electroporation with Tn5 transposon was established, which may be utilized to provide genetic approach to study virulence genes of R. solanacearum strains and create nonpathogenic mutants for biological control of bacterial wilt in Pogostemon cablin. R. solanacearum strain PRS-84 used in this study was isolated from P. cablin plants infected with bacterial wilt. The bacterial suspension of R. solanacearum strain PRS-84 was mixed with Tn5 transposome complex and the mixture was transformed by electroporation. The electroporated cells were then spread on the 2, 3, 5-triphenyltetrazolium chloride agar plates containing kanamycin to select the kanamycin-resistant colonies. Several factors which determined the bacterial transformation efficiency were optimized. The transformation process was shown to be optimal at the electric field strength of 12.5 kV cm-1. Bacterial cells harvested at mid-exponential phase gave the highest transformation efficiency. 10 µg mL-1 kanamycin was found to be the optimal concentration for transformant selection. Tn5 insertion mutants of R. solanacearum strain PRS-84 were identified by PCR amplification and Southern blot analysis. Mutants subcultured for 100 passages were also detected by PCR amplification and Southern blot analysis. Furthermore, pathogenicity screening test of mutants was performed by inoculating in vitro regenerated patchouli plants. Results revealed that mutants with a single Tn5 insertion in their genomes were obtained from R. solanacearum strain PRS-84, and the Tn5 insertion could be stably inherited in the mutants. Then, mutants with reduced pathogenicity were selected.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional/métodos , Pogostemon/microbiologia , Ralstonia solanacearum/genética , Fatores de Virulência/genética , Eletroporação/métodos , Genes Bacterianos/genética , Canamicina/farmacologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/efeitos dos fármacos , Ralstonia solanacearum/patogenicidade , Ralstonia solanacearum/fisiologia , Transformação Bacteriana/efeitos dos fármacos , Transformação Bacteriana/genética , Transformação Bacteriana/fisiologia , Virulência/genéticaRESUMO
In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano , Albumina Sérica Humana/farmacologia , Resistência beta-Lactâmica/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Cápsulas Bacterianas/efeitos dos fármacos , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Biofilmes , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Perfilação da Expressão Gênica , Genes MDR/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Ferro/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Transformação Bacteriana/efeitos dos fármacos , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Zinco/metabolismo , Resistência beta-Lactâmica/genética , beta-Lactamas/farmacologiaRESUMO
Natural genetic transformation is a widespread mechanism of horizontal gene transfer. It involves the internalization of exogenous DNA as single strands and chromosomal integration via homologous recombination, promoting acquisition of new genetic traits. Transformation occurs during a distinct physiological state called competence. In Streptococcus pneumoniae, competence is controlled by ComDE, a two-component system induced by an exported peptide pheromone. DprA is universal among transformable species, strongly induced during pneumococcal competence, and crucial for pneumococcal transformation. Pneumococcal DprA plays three crucial roles in transformation and competence. Firstly, DprA protects internalized DNA from degradation. Secondly, DprA loads the homologous recombinase RecA onto transforming DNA to promote transformation. Finally, DprA interacts with the response regulator ComE to shut-off competence. Here, we explored the effect of altering the cellular levels of DprA on these three roles. High cellular levels of DprA were not required for the primary role of DprA as a transformation-dedicated recombinase loader or for protection of transforming DNA. In contrast, full expression of dprA was required for optimal competence shut-off and transformant fitness. High cellular levels of DprA thus ensure the fitness of pneumococcal transformants by mediating competence shut-off. This promotes survival and propagation of transformants, maximizing pneumococcal adaptive potential.
Assuntos
Proteínas de Bactérias/metabolismo , Competência de Transformação por DNA/fisiologia , Proteínas de Membrana/metabolismo , Streptococcus pneumoniae/fisiologia , Streptococcus pneumoniae/patogenicidade , Transformação Bacteriana/fisiologia , Adaptação Fisiológica , Proteínas de Bactérias/genética , Primers do DNA/genética , Primers do DNA/metabolismo , Competência de Transformação por DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Humanos , Isopropiltiogalactosídeo/farmacologia , Proteínas de Membrana/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Transformação Bacteriana/efeitos dos fármacosRESUMO
OBJECTIVES: To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae. RESULTS: The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4 × 105 CFU µg-1 DNA, which is 10 times higher than that of the wild strain. 52.2 g 1,3-propanediol L-1, 30.7 g 2,3-butanediol L-1, and 175.9 g 2-ketogluconic acid L-1 were produced by non-capsulated mutants, which were 10-40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8-2.2 cP. CONCLUSIONS: Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.
Assuntos
Técnicas de Cultura Celular por Lotes , Klebsiella pneumoniae/genética , Proteínas Mutantes/genética , Transformação Bacteriana/efeitos dos fármacos , Butileno Glicóis/farmacologia , Fermentação , Gluconatos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Proteínas Mutantes/química , Propilenoglicóis/farmacologia , Transformação Bacteriana/genéticaRESUMO
Natural transformation is the acquisition of new genetic material via the uptake of exogenous DNA by competent bacteria. Acinetobacter baylyi is model for natural transformation. Here we focus on the natural transformation of A. baylyi ATCC 33305 grown in complex media and seek environmental conditions that appreciably affect transformation efficiency. We find that the transformation efficiency for A. baylyi is a resilient characteristic that remains high under most conditions tested. We do find several distinct conditions that alter natural transformation efficiency including addition of succinate, Fe2+ (ferrous) iron chelation, and substitution of sodium ions with potassium ones. These distinct conditions could be useful to fine tune transformation efficiency for researchers using A. baylyi as a model organism to study natural transformation.
Assuntos
Acinetobacter/efeitos dos fármacos , Cátions Monovalentes/farmacologia , Quelantes de Ferro/farmacologia , Ácido Succínico/farmacologia , Transformação Bacteriana/efeitos dos fármacos , Acinetobacter/genética , Acinetobacter/crescimento & desenvolvimento , Meios de CulturaRESUMO
DNA transformation that delivers plasmid DNAs into bacterial cells is fundamental in genetic manipulation to engineer and study bacteria. Developed transformation methods to date are optimized to specific bacterial species for high efficiency. Thus, there is always a demand for simple and species-independent transformation methods. We herein describe the development of a chemico-physical transformation method that combines a rubidium chloride (RbCl)-based chemical method and sepiolite-based physical method, and report its use for the simple and efficient delivery of DNA into various bacterial species. Using this method, the best transformation efficiency for Escherichia coli DH5α was 4.3×106CFU/µg of pUC19 plasmid, which is higher than or comparable to the reported transformation efficiencies to date. This method also allowed the introduction of plasmid DNAs into Bacillus subtilis (5.7×103CFU/µg of pSEVA3b67Rb), Bacillus megaterium (2.5×103CFU/µg of pSPAsp-hp), Lactococcus lactis subsp. lactis (1.0×102CFU/µg of pTRKH3-ermGFP), and Lactococcus lactis subsp. cremoris (2.2×102CFU/µg of pMSP3535VA). Remarkably, even when the conventional chemical and physical methods failed to generate transformed cells in Bacillus sp. and Enterococcus faecalis, E. malodoratus and E. mundtii, our combined method showed a significant transformation efficiency (2.4×104, 4.5×102, 2×101, and 0.5×101CFU/µg of plasmid DNA). Based on our results, we anticipate that our simple and efficient transformation method should prove usefulness for introducing DNA into various bacterial species without complicated optimization of parameters affecting DNA entry into the cell.
Assuntos
Bactérias/genética , Cloretos/farmacologia , DNA Bacteriano/genética , Técnicas de Transferência de Genes , Silicatos de Magnésio/farmacologia , Rubídio/farmacologia , Transformação Bacteriana/efeitos dos fármacos , Bacillus/genética , Bacillus megaterium/genética , Bacillus subtilis/genética , Enterococcus faecalis/genética , Escherichia coli/genética , Lactococcus lactis , Plasmídeos/genética , Transformação Bacteriana/genéticaRESUMO
The discovery that Streptococcus pneumoniae uses a competence-stimulating peptide (CSP) to induce competence for natural transformation, and that other species of the mitis and the anginosus streptococcal groups use a similar system, has expanded the tools to explore gene function and regulatory pathways in streptococci. Two other classes of pheromones have been discovered since then, comprising the bacteriocin-inducing peptide class found in Streptococcus mutans (also named CSP, although different from the former) and the SigX-inducing peptides (XIP), in the mutans, salivarius, bovis, and pyogenes groups of streptococci. The three classes of peptide pheromones can be ordered from peptide synthesis services at affordable prices, and used in transformation assays to obtain competent cultures consistently at levels usually higher than those achieved during spontaneous competence. In this chapter, we present protocols for natural transformation of oral streptococci that are based on the use of synthetic pheromones, with examples of conditions optimized for transformation of S. mutans and Streptococcus mitis.
Assuntos
Boca/microbiologia , Feromônios/farmacologia , Streptococcus/efeitos dos fármacos , Streptococcus/fisiologia , Transformação Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Humanos , Peptídeos/farmacologia , Feromônios/síntese química , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/fisiologiaRESUMO
Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.
Assuntos
Edição de Genes , Marcadores Genéticos , Genoma Bacteriano , Streptococcus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Genes Bacterianos , Mutação , Deleção de Sequência , Fator sigma/metabolismo , Fator sigma/farmacologia , Transformação Bacteriana/efeitos dos fármacosRESUMO
Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with.
Assuntos
Cromossomos Bacterianos/metabolismo , Clostridioides difficile/genética , Conjugação Genética , Recombinação Homóloga , Plasmídeos/metabolismo , Cromossomos Bacterianos/química , Clostridioides difficile/isolamento & purificação , Clostridioides difficile/metabolismo , Infecções por Clostridium/microbiologia , Meios de Cultura/farmacologia , Temperatura Alta , Humanos , Plasmídeos/química , Ribotipagem , Transformação Bacteriana/efeitos dos fármacosRESUMO
Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 10(3) colony-forming units per microgram of DNA was obtained at 5.0 kV cm(-1) field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng µg(-1), and 3.0 µg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.
Assuntos
Clostridium beijerinckii/metabolismo , Escherichia coli/metabolismo , Vetores Genéticos/metabolismo , Transformação Bacteriana , Acetona/metabolismo , Butanóis/metabolismo , Clostridium beijerinckii/efeitos dos fármacos , Eletroporação , Escherichia coli/efeitos dos fármacos , Etanol/metabolismo , Fermentação/efeitos dos fármacos , Genes Bacterianos , Recombinação Genética/genética , Origem de Replicação/genética , Sacarose/farmacologia , Transformação Bacteriana/efeitos dos fármacosRESUMO
Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.
Assuntos
Antibacterianos/farmacologia , Análise Custo-Benefício , Escherichia coli/genética , Dosagem de Genes , Plasmídeos/genética , Triclosan/farmacologia , Sequência de Bases , DNA Recombinante/genética , Escherichia coli/efeitos dos fármacos , Dados de Sequência Molecular , Transformação Bacteriana/efeitos dos fármacosRESUMO
While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.
Assuntos
Chlamydomonas reinhardtii/genética , Dano ao DNA , Reparo do DNA , Mutagênese Insercional , Chlamydomonas reinhardtii/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Ordem dos Genes/genética , Vetores Genéticos/genética , Hidroxiureia/farmacologia , Hidroxiureia/toxicidade , Mutação , Transformação Bacteriana/efeitos dos fármacosRESUMO
Phospholipase A of the bacterial outer membrane (OMPLA) is a ß-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2--palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²âº-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca²âº-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²âº and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.
Assuntos
Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Fosfolipases A2/química , Fosfolipases A/química , Ácidos Aristolóquicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Cetonas/farmacologia , Modelos Moleculares , Conformação Molecular , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2/metabolismo , Conformação Proteica , Transformação Bacteriana/efeitos dos fármacosRESUMO
Natural competence is a process by which bacteria construct a membrane-associated machine for the uptake and integration of exogenous DNA. Many bacteria harbor genes for the DNA uptake machinery and yet are recalcitrant to DNA uptake for unknown reasons. For example, domesticated laboratory strains of Bacillus subtilis are renowned for high-frequency natural transformation, but the ancestral B. subtilis strain NCIB3610 is poorly competent. Here we find that endogenous plasmid pBS32 encodes a small protein, ComI, that inhibits transformation in the 3610 strain. ComI is a single-pass trans-membrane protein that appears to functionally inhibit the competence DNA uptake machinery. Functional inhibition of transformation may be common, and abolishing such inhibitors could be the key to permitting convenient genetic manipulation of a variety of industrially and medically relevant bacteria.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/farmacologia , Competência de Transformação por DNA/efeitos dos fármacos , Proteínas de Membrana/farmacologia , Plasmídeos/genética , Transformação Bacteriana , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase , Transformação Bacteriana/efeitos dos fármacosRESUMO
Artificial plasmid DNA transformation of Escherichia coli induced by calcium chloride is a routine technique in molecular biology and genetic engineering processes, but its mechanism has remained elusive. Because adenosine monophosphate (AMP) has been found to regulate natural transformation in Haemophilus influenza, we aimed to investigate the effects of AMP and its derivatives on E. coli transformation by treating competence with different concentrations of them. Analysis of the transformation efficiencies revealed that AMP inhibited the artificial plasmid DNA transformation of E. coli in a concentration- and time-dependent manner. Furthermore, we found that AMP had no effect on the expression of the transformed gene but that the intracellular AMP level of the competent cells rose after a 6 h treatment. These results suggested that the intracellular AMP level had an important role in E. coli transformation. And these have useful implications for the further investigation of the mechanism of E. coli transformation.