Plasmid最新文献

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 μm while the E589Q rodlets (N = 33) averaged 1.157 μm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons.

Pathogenic Yersinia bacteria, including Y. pseudotubuclosis Y. enterocolitica, and Y. pestis, contain the mosaic plasmid pYV that encodes for, among other things, a number of proteinaceous virulence factors. While the evolutionary histories of many of the biovars and strains of pathogenic Yersinia species are well documented, the origins of many of the individual virulence factors have not been comprehensively examined. Here, the evolutionary origins of the genes coding for a set of Yersinia outer protein (Yop) virulence factors were investigated through phylogenetic reconstruction and subsequence analysis. It was found that many of these genes had only a few sequenced homologs and none of the resolved phylogenies recovered the same relationships as was resolved from chromosomal analyses. Many of the evolutionary relationships differ greatly among genes on the plasmid, and variation is also found across different domains of the same gene, which provides evidence of the mosaic nature of the plasmid as well as multiple genes on the plasmid. This mosaic aspect also relates to patterns of selection, which vary among the studied domains.

The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.

Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial genera that cause human infections and persist in hospital environments. It is crucial that these plasmids are typed accurately so that their clinical and global dissemination can be traced in epidemiological studies. The L/M complex has previously been divided into L, M1 and M2 subtypes. However, those types do not encompass all diversity seen in the group. Here, we have examined 148 complete L/M plasmid sequences in order to understand the diversity of the complex and trace the evolution of distinct lineages. The backbone sequence of each plasmid was determined by removing translocatable genetic elements and reversing their effects in silico. The sequence identities of replication regions and complete backbones were then considered for typing. This supported the distinction of L and M plasmids and revealed that there are five L and eight M types, where each type is comprised of further sub-lineages that are distinguished by variation in their backbone and translocatable element content. Regions containing antibiotic resistance genes in L and M sub-lineages have often formed by initial rare insertion events, followed by insertion of other translocatable elements within the inceptive element. As such, islands evolve in situ to contain genes conferring resistance to multiple antibiotics. In some cases, different plasmid sub-lineages have acquired the same or related resistance genes independently. This highlights the importance of these plasmids in acting as vehicles for the dissemination of emerging resistance genes. Materials are provided here for typing plasmids of the L/M complex from complete sequences or draft genomes. This should enable rapid identification of novel types and facilitate tracking the evolution of existing lineages.

Bacteriophages play an essential role in the transferring of genes that contribute to the bacterial virulence and whose products are dangerous to human health. Interestingly, phages carrying virulence genes are mostly temperate and in contrast to lytic phages undergo both lysogenic and lytic cycles. Importantly, expression of the majority of phage genes and subsequent production of phage encoded proteins is suppressed during lysogeny. The expression of the majority of phage genes is tightly linked to lytic development. Among others, small non-coding RNAs (sRNAs) of phage origin are involved in the regulation of phage gene expression and thus play an important role in both phage and host development. In the case of bacteria, sRNAs affect processes such as virulence, colonization ability, motility and cell growth or death. In turn, in the case of phages, they play essential roles during the early stage of infection, maintaining the state of lysogeny and silencing the expression of late structural genes, thereby regulating the transition between phage life cycles. Interestingly, sRNAs have been identified in both lytic and temperate phages and they have been discussed in this work according to this classification. Particular attention was paid to viral sRNAs resembling eukaryotic microRNAs.

Mobile genetic elements (MGE) carrying resistance genes represent a unique challenge to risk assessment and surveillance of antimicrobial resistance (AMR). Yet determining the mobility of resistance genes within animal microbiomes is essential to evaluating the potential dissemination from livestock to potential human pathogens, as well as evaluating co-selection mechanisms that may impact persistence of resistance genes with changing antibiotic use patterns. Current surveillance efforts utilize phenotypic testing and sequencing of individual isolates for tracking of AMR in livestock. In this work, we investigated the utility of using long-read sequencing of the plasmids from mixed Enterobacterales enrichments of swine fecal samples as a surveillance strategy for AMR plasmids. Enrichments were performed in either MacConkey broth without selection or with selection by addition of tetracycline or ceftriaxone, and plasmids were extracted and sequenced in order to evaluate the diversity of plasmids enriched by each method. Intact resistance plasmids were successfully assembled, as well as complex resistance transposons carrying multiple repeated elements that would interfere with assembly by short read sequencing technologies. Comparison of the assembled plasmids with representatives from public databases confirmed the quality of the assemblies and also revealed the occurrence of IncI2 plasmids carrying bla_CMY-2 in Ontario swine samples, which have not been found in previous studies.

Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and β-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.

A large plasmid, pCERC14, found in an antibiotic resistant commensal Escherichia coli isolate recovered from a healthy adult was sequenced. pCERC14 was 162,926 bp and carried FII-18 and FIB-1 replicons and an F-like transfer region as well as several virulence determinants, some of which are involved in the uptake of iron which would be advantageous for the commensal lifestyle. The plasmid backbone is interrupted in 11 places by complete IS (IS1 (4 copies), IS2 (2), IS629 (2) and single IS100, IS186, ISEc33) and in three places by partial IS copies. The antibiotic resistance genes were found in two IS26-bounded pseudo-compound transposons (PCT). One contained a remnant of a class 1 integron that includes a dfrA5 gene cassette and the sul1 gene conferring resistance to trimethoprim and sulphonamides, respectively. The second, named PTntet(C)-var, contained a 4828 bp DNA segment that includes the tet(C) tetracycline resistance determinant. As tet(C) is relatively rare in E. coli and other Gram-negative bacterial isolates, the structure and evolution of tet(C)-containing PCT in available sequences was examined. The largest identified was PTntet(C), a close relative of PTntet(C)-var, and a potential progenitor for these PCT. Most PCT shared one internal boundary with PTntet(C) but the length of the central tet(C)-containing segment was shorter due to IS26-mediated deletions. The most abundant variant form, previously named Tn6309, was widely distributed and, in a derivative of it, most of the tetA(C) gene has been replaced by the tetA(A) gene presumably by homologous recombination.

The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min⁻¹·(mg protein)⁻¹ from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.

In this study we describe the genetic elements and the antimicrobial resistance units (RUs) harboured by the Salmonella Typhimurium monophasic variant 1,4,[5],12:i:- strain ST1030. Of the three identified RUs two were chromosomal, RU1 (IS26-bla_TEM-1-IS26-strAB-sul2- IS26) and RU2 (IS26-tetR(B)-tetA(B)-ΔIS26), and one, RU3 (a sul3-associated class 1 integron with cassette array dfrA12-orfF-aadA2-cmlA1-aadA1), was embedded in a Tn21-derived element harboured by the conjugative I1 plasmid pST1030-1A. IS26 elements mediated the antimicrobial resistance gene (ARG) shuffling and this gave rise to pST1030-1A derivatives with different sets of ARGs. ST1030 also harboured two ColE1-like plasmids of which one, pST1030-2A, was mobilisable and the target of an intracellular translocation of the Tn21-derived element; the second (pST1030–3) was an orphan mob-associated oriT plasmid co-transferred with pST1030-1A and pST1030-2A. pST1030-2A and pST1030-3 also carried a parA gene and a type III restriction modification system, respectively. Overall analysis of our data reinforces the role played by IS26, Tn21-derived elements and non-conjugative plasmids in the spread of ARGs and supplies the first evidence, at least in Salmonella, for the identification of a natural isolate harbouring a three-element mobilisation system in the same cell.