Putative environmental levels of levofloxacin facilitate the dissemination of antibiotic-resistant Escherichia coli via plasmid-mediated transformability.

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.

Fine-tuning cellular levels of DprA ensures transformant fitness in the human pathogen Streptococcus pneumoniae.

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.

Effect of Host Human Products on Natural Transformation in Acinetobacter baumannii.

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.

Non-capsulated mutants of a chemical-producing Klebsiella pneumoniae strain.

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.

A practical random mutagenesis system for Ralstonia solanacearum strains causing bacterial wilt of Pogostemon cablin using Tn5 transposon.

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.

Succinate, iron chelation, and monovalent cations affect the transformation efficiency of Acinetobacter baylyi ATCC 33305 during growth in complex media.

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.

Heat shock increases conjugation efficiency in Clostridium difficile.

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.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A2 and their effect on the Ca²âº-induced permeabilization of the bacterial membrane.

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 Ca²âº-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 Ca²âº-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 Ca²âº 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.

Plasmid-encoded ComI inhibits competence in the ancestral 3610 strain of Bacillus subtilis.

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.

The animal food supplement sepiolite promotes a direct horizontal transfer of antibiotic resistance plasmids between bacterial species.

Animal fodder is routinely complemented with antibiotics together with other food supplements to improve growth. For instance, sepiolite is currently used as a dietary coadjuvant in animal feed, as it increases animal growth parameters and improves meat and derived final product quality. This type of food additive has so far been considered innocuous for the development and spread of antibiotic resistance. In this study, we demonstrate that sepiolite promotes the direct horizontal transfer of antibiotic resistance plasmids between bacterial species. The conditions needed for plasmid transfer (sepiolite and friction forces) occur in the digestive tracts of farm animals, which routinely receive sepiolite as a food additive. Furthermore, this effect may be aggravated by the use of antibiotics supplied as growth promoters.

Adenosine monophosphate affects competence development and plasmid DNA transformation in Escherichia coli.

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.

Use of ß-galactosidase (lacZ) gene α-complementation as a novel approach for assessment of titanium oxide nanoparticles induced mutagenesis.

The mutagenic potential of titanium dioxide nanoparticles (TiO(2)-NPs) of an average size 30.6nm was investigated using ß-galactosidase (lacZ) gene complementation in plasmid pUC19/lacZ(-)Escherichia coli DH5α system. Plasmid pUC19 was treated with varying concentrations of TiO(2)-NPs and allowed to transfect the CaCl(2)-induced competent DH5α cells. The data revealed loss in transformation efficiency of TiO(2)-NPs treated plasmids as compared to untreated plasmid DNA in DH5α host cells. Induction of multiple mutations in α-fragment of lacZ gene caused synthesis of non-functional ß-galactosidase enzyme, which resulted in a significant number of white (mutant) colonies of transformed E. coli cells. Screening of mutant transformants based on blue:white colony assay and DNA sequence analysis of lacZ gene fragment clearly demonstrated TiO(2)-NPs induced mutagenesis. Multiple alignment of selectable marker lacZ gene sequences from randomly selected mutants and control cells provided a gene specific map of TiO(2)-NPs induced mutations. Mutational analysis suggested that all nucleotide changes were point mutations, predominantly transversions (TVs) and transitions (TSs). A total of 32 TVs and 6 TSs mutations were mapped within 296 nucleotides (nt) long partial sequence of lacZ gene. The region between 102 and 147nt within lacZ gene sequence was found to be most susceptible to mutations with nine detectable point mutations (8 TVs and 1 TSs). Guanine base was determined to be more prone to TiO(2)-NPs induced mutations. This study suggested the pUC19/E. coli DH5αlacZ gene α-complementation system, as a novel genetic approach for determining the mutagenic potential, and specificity of manufactured NPs and nanomaterials.

Bacterial transformation using micro-shock waves.

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 µm thick metal foil, 200 mM CaCl(2), 1 ng/µl plasmid DNA concentration, and 1×10(9) cell density. The highest transformation efficiency achieved (1×10(-5) transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1×10(-6) transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella typhimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation.

Episodic selection and the maintenance of competence and natural transformation in Bacillus subtilis.

We present a new hypothesis for the selective pressures responsible for maintaining natural competence and transformation. Our hypothesis is based in part on the observation that in Bacillus subtilis, where transformation is widespread, competence is associated with periods of nongrowth in otherwise growing populations. As postulated for the phenomenon of persistence, the short-term fitness cost associated with the production of transiently nongrowing bacteria can be compensated for and the capacity to produce these competent cells can be favored due to episodes where the population encounters conditions that kill dividing bacteria. With the aid of a mathematical model, we demonstrate that under realistic conditions this "episodic selection" for transiently nongrowing (persisting) bacteria can maintain competence for the uptake and expression of exogenous DNA transformation. We also show that these conditions for maintaining competence are dramatically augmented even by rare episodes where selection favors transformants. Using experimental populations of B. subtilis and antibiotic-mediated episodic selection, we test and provide support for the validity of the assumptions behind this model and the predictions generated from our analysis of its properties. We discuss the potential generality of episodic selection for the maintenance of competence in other naturally transforming species of bacteria and critically evaluate other hypotheses for the maintenance (and evolution) of competence and their relationship to this hypothesis.

Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes.

In this paper, we report a useful protocol for cloning toxic protein genes. Use of the SOC medium, which is a glucose-containing rich medium, significantly improved the transformation efficiency of a recombinant plasmid containing a toxic plant subtilase SaSBT1 cDNA. Both glucose and rich nutrients present in the SOC medium prevented the unintended activation of the lac promoter carried on the cloning vector, and led to significantly improved transformation efficiency of recombinant plasmids containing toxic protein genes and an increased rate of transformant growth.

Use of cyclodextrin and its derivatives for increased transformation efficiency of competent bacterial cells.

Methodologies for introduction of DNA into cells are essential in molecular genetics and vital for applications such as genetic engineering and gene therapy. The use of cyclodextrins (CyDs) for increased efficiency of introducing DNA into eukaryotic cells (transfection) has been reported, but CyDs' effect on the introduction of DNA into bacterial cells (transformation) is unknown. Here, we have investigated the potential of using CyDs in the transformation of chemically competent in-house, commercially available, and, on non-competent bacterial cells, with plasmid DNA of two different sizes. Possible interactions between CyDs and DNA were studied with nuclear magnetic resonance (NMR) spectroscopy. The presence of CyDs resulted in an up to fourfold increment of the transformation rate for in-house cells, with beta-CyD and derivates giving the strongest effect. For commercial cells and transformation with megaplasmids, a more moderate effect around 1.4-fold was obtained. However, CyDs have little or no effect on DNA uptake by noncompetent cells. Results obtained from NMR spectroscopy show no interactions between CyDs and DNA-like molecules, which indicated that the CyDs' effect is related to the bacterial cell wall.

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

The efficiencies of different transformation methods of E. coli DH5Qalpha train, induced by several cations like Mg2+, Mn2+ Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

Analysis of the effects of mesoporous silica particles SBA-15 and SBA-16 in Streptococcus pneumoniae transformation process.

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.

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

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.

Synthesis and DNA cleaving activity of water-soluble non-conjugated thienyl tetraynes.

Water-soluble non-conjugated thienyl tetraynes (3-6) were synthesized and their DNA cleaving activity was evaluated using electrophoresis, atomic force microscopy (AFM) and Escherichia coli (E. coli) transformation techniques. The amino-functionalized compound 4 was shown to possess an activity to cleave plasmid DNA by both electrophoresis and E. coli transformation techniques. AFM also showed a cleavage of the circular DNA into a linear form with a formation of burst-star-shaped architectures, which were envisaged to be cross-linked DNA oligomers.