Plasmid最新文献

pXO16, the 350 kb-conjugative plasmid from Bacillus thuringiensis sv. israelensis promotes its own transfer at high efficiency, triggers the transfer of mobilizable and non-mobilizable plasmids, as well as the transfer of host chromosomal loci. Naturally found in B. thuringiensis sv. israelensis, pXO16 transfers to various strains of Bacillus cereus sensu lato (s.l.) at a wide range of frequencies. Despite this host diversity, a paradox remains between the relatively large host spectrum and the natural occurrence of pXO16, so far restricted to B. thuringiensis sv. israelensis. Proposing first insights exploring this paradox, we investigated the behaviour of pXO16 amongst different members of the B. cereus group. We first looked at the transfer of pXO16 to two new host clusters of B. cereus s.l., Bacillus mycoides and Bacillus anthracis clusters. This examination brought to light the impairment of the characteristic rhizoidal phenotype of B. mycoides in presence of pXO16. We also explored the stability of pXO16 at different temperatures as some B. cereus group members are well-known for their psychro- or thermo-tolerance. This shed light on the thermo-sensitivity of the plasmid. The influence of pXO16 on its host cell growth and on swimming capacity also revealed no or limited impact on its natural host B. thuringiensis sv. israelensis. On the contrary, pXO16 affected more strongly both the growth and swimming capacity of other B. cereus s.l. hosts. This reinforced the running hypothesis of a co-evolution between pXO16 and B. thuringiensis sv. israelensis, enabling the plasmid maintenance without impairing the host strain development.

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.

Resistance plasmids mediate the rapid spread of antimicrobial resistance, which poses a threat to veterinary and human healthcare. This study addresses the question whether resistance plasmids from Escherichia coli isolated from foodstuffs always transfer unchanged to recipient E. coli cells, or that genetic editing can occur. Strains containing between one and five different plasmids were co-incubated with a standard recipient strain. Plasmids isolated from transconjugant strains were sequenced using short and long read technologies and compared to the original plasmids from the donor strains. After one hour of co-incubation only a single plasmid was transferred from donor to recipient strains. If the donor possessed several plasmids, longer co-incubation resulted in multiple plasmids being transferred. Transferred plasmids showed mutations, mostly in mobile genetic elements, in the conjugative transfer gene pilV and in genes involved in plasmid maintenance. In one transconjugant, a resistance cluster encoding tetracycline resistance was acquired by the IncI1 plasmid from the IncX1 plasmid that was also present in the donor strain, but that was not transferred. A single plasmid transferred twelve times back and forth between E. coli strains resulted in a fully conserved plasmid with no mutations, apart from repetitive rearrangements of pilV from and back to its original conformation in the donor strain. The overall outcome suggests that some genetic mutations and rearrangements can occur during plasmid transfer. The possibility of such mutations should be taken into consideration in epidemiological research aimed at attribution of resistance to specific sources.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

In addition to tumor-inducing agrobacteria, non-pathogenic strains are often isolated from crown gall tumors. Such non-pathogenic strains sometimes contain catabolic plasmids that allow them to take advantage of the opines produced in the tumors. Here we characterize for the first time an octopine catabolic plasmid, pAtAg67, which is derived from an Agrobacterium strain isolated from a grapevine tumor in Crete. By sequence analysis, we deduce that pAtAg67 enables its host to catabolize not only octopine, but also cucumopine and agrocinopine-like compounds. We found that a highly similar set of catabolic genes was present in the Ti plasmids of tumorigenic octopine-cucumopine grapevine strains such as pTiAg57. However, the catabolic genes in octopine-cucumopine Ti plasmids were interrupted by a T-DNA segment. As no T-DNA remnants, virulence genes or border repeats were found in pAtAg67, catabolic plasmid pAtAg67 does not appear to be a degenerate octopine-cucumopine Ti plasmid. In line, plasmid pAtAg67 was found to be compatible with incRh1 octopine Ti plasmids, but to be incompatible with the incRh2 agropine Ti plasmid pTiBo542, forming cointegrates by recombination in the homologous trb genes.

Gateway system is one of the most known cloning systems, which makes it compatible with several expression vectors, including those used for Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays. However, this system is laborious and expensive due to its two-step cloning. In this research, we developed a new cloning strategy named Brick into the Gateway (BiG). This approach uses GoldenBraid/Gate assemblies to create a DNA fragment of interest flanked by attL sites, which can be directly recombined into Gateway destination vectors. BiG method showed a high recombination efficiency and ensured the correct reading frame, which was successfully tested in Y2H and BiFC assays. BiG has proven to be a rapid, low-cost, reusable, and directional cloning method which allows the merged use of systems.

Acinetobacter baumannii RepAci1-RepAci10 plasmids pA388 from a global clone 1 (GC1) isolate from Greece, and pACICU1 and variant pACICU1b from an Italian GC2 isolate were found to share a common ancestor. The ancestor formed via recombination between pdif sites in the widely-distributed RepAci1 plasmid pA1–1 and in a RepAci10 plasmid carrying the oxa58 carbapenem-resistance gene in a dif module. Each plasmid includes copies of IS26 and multiple dif modules surrounded by 28 bp pdif sites resembling the chromosomal dif site, including one carrying the oxa58 gene. Plasmid sequences were compared to identify factors driving their evolution and divergence. IS26-mediated events, recombination between pdif sites and homologous recombination have all contributed. A translocatable unit that includes oxa58, generated by an IS26-mediated adjacent deletion, had been re-inserted by IS26 adjacent to an IS26 in pACICU1b to create the oxa58 gene duplication previously found in pACICU1. The oxa58 duplication has been lost from pACICU1b and the Tn6020 variant carrying the aphA1 (kanamycin, neomycin resistance) gene in pA388 has been lost from pACICU1/1b via recombination between directly-oriented IS26 copies. Two dif modules located between directly-oriented pdif sites in pA388 have been lost from pACICU1/1b and the product of this and other deletion events as well as inversion of dif modules located between inversely-oriented pdif sites were detected experimentally in pA388 DNA by PCR. Also, the new junctions were detected in a minority of reads in pA388 long-read sequence data. Inversion and deletion were only detected when the spacers in the pdif sites were identical and equivalent events involving mismatched spacers were not detected.

Plasmids are important vectors for the spread of genes among diverse populations of bacteria. However, there is no standard method to determine the rate at which they spread horizontally via conjugation. Here, we compare commonly used methods on simulated and experimental data, and show that the resulting conjugation rate estimates often depend strongly on the time of measurement, the initial population densities, or the initial ratio of donor to recipient populations. Differences in growth rate, e.g. induced by sub-lethal antibiotic concentrations or temperature, can also significantly bias conjugation rate estimates. We derive a new ‘end-point’ measure to estimate conjugation rates, which extends the well-known Simonsen method to include the effects of differences in population growth and conjugation rates from donors and transconjugants. We further derive analytical expressions for the parameter range in which these approximations remain valid. We present an easy to use R package and web interface which implement both new and previously existing methods to estimate conjugation rates. The result is a set of tools and guidelines for accurate and comparable measurement of plasmid conjugation rates.

Cell penetrating peptides (CPPs) have been used as alternative delivery vectors to translocate therapeutic cargo molecules across cell membranes. One example of CPPs is the dTAT peptide, which has shown great promise in the design of highly efficient and low-cytotoxic gene vectors when condensed via “soft” calcium cross links. Here, we investigated the effect of fluorination on the formulation of dTAT complexes and explored their potential for pDNA delivery to cells. Fluorinated dTAT complexes achieve excellent gene transfection efficacy compared to fluorinated PEI polyplexes in A549, HeLa, and MCF-7 cell lines. Furthermore, the fluorinated dTAT complexes exhibit excellent serum resistance, high gene transfection efficacy even in 10% FBS medium, and no detectable cytotoxicity on transfected cells. The optimum NaF concentration (14 mM) resulted in an over 1000-fold enhancement in dTAT complexes (N/P 33) transfection efficiency. According to these findings, fluorination seems to be a potential strategy for creating gene vectors without requiring complex syntheses.

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.