Plasmid

The pseudo-compound transposon Tn4352B is unusual in that the translocatable unit (TU) consisting of one of the bounding IS26 copies and the central portion containing the aphA1a gene has been found to be readily lost in the Escherichia coli strains used as host. Rapid loss required the presence of an additional 2 G residues adjacent to the internal end of one of the IS26 that flank the central portion and an active Tnp26 transposase. However, Tn4352B was found to be stable in wild-type Klebsiella pneumoniae strains...

Though IncC and IncA plasmids are compatible, they exert high level exclusion on one another. Here, the question of whether the presence of an SGI1 family element in the donor can overcome the exclusion of an IncC or IncA plasmid exerted by an IncC or IncA plasmid in the recipient was investigated. The transfer of the integrative mobilizable element SGI1 and its many variant forms into a new host is dependent on transfer machinery supplied by IncC or IncA plasmids. SGI1 elements include the determinants of a mobilization system and three genes that encode homologues of transfer proteins including TraG...

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Two phylogenetically distantly-related IncF plasmids, F and pED208, serve as important models for mechanistic and structural studies of F-like type IV secretion systems (T4SSF s) and F pili. Here, we present the pED208 sequence and compare it to F and pUMNF18, the closest match to pED208 in the NCBI database. As expected, gene content of the three cargo regions varies extensively, although the maintenance/leading regions (MLRs) and transfer (Tra) regions also carry novel genes or motifs with predicted modulatory effects on plasmid stability, dissemination and host range...

To characterize IncI1 and IncF18:A-:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014-1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014-2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including blaTEM-1b , blaOXA-1 , oqxAB, dfrA17, aac(6')-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA...

Escherichia coli is ubiquitous in the environment, foods, and mammalian hosts. It can cause disease and exhibits high rates of antibiotic resistance. This study examines the possible role of food strains of E. coli as reservoirs of clinical outbreaks using a genomic approach. We gather a database of fully assembled genomic sequences from E. coli isolated from food (n = 77) and clinical samples (n = 649) deposited in the GenBank by December 2020. We classify the host based on genomic markers and annotate genomic indicators of plasmid content using the most up-to-date genomic analysis pipelines...

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e...

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...

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...

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...

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...

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 Golden Braid/Gate assemblies to create a DNA fragment of interest flanked by attL sites, which can be directly recombined into Gateway destination vectors...

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...

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...

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...

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 identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density...

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...

Plasmids exhibit great diversity of gene content and host ranges and are famous for quick adaptation to the genetic background of the bacterial host cell. In addition to observing ever evolving plasmids, some plasmids have conserved backbones: a stable core composition and arrangement of genes in addition to variable regions. There are a few reports of extremely conserved plasmids. Here we report the complete sequence of pRK100 plasmid - a large, well-characterized conjugative F-like plasmid found in an Escherichia coli strain isolated from a urinary tract infection patient in 1990...

The rapid emergence and spread of antibiotic resistance is a growing global burden. Antibiotic resistance is often associated with large single or low copy number plasmids, which rely upon cytoskeletal proteins for their stable maintenance. While the mechanism of plasmid partitioning has been well established for the R plasmids, the molecular details by which the F plasmid is maintained is only beginning to emerge. The partitioning function of the F plasmid depends upon a ParA/ MinD family of proteins known as SopA...

The bioinformatic analysis that we made of 492 Acinetobacter baumannii plasmid sequences identified 418 genes encoding Replication Initiator (Rep) proteins that fell into at least fourteen groups according to the protein domains that they contained. The most abundant group of Rep proteins contained a Rep_3 superfamily domain, followed by Rep proteins containing Replicase/PriCT_1 superfamily domains, and then by Reps possessing only an HTH_MerR-SF superfamily domain. The remaining eleven groups contain only a few members...