Plasmid最新文献

Conjugation is a central characteristic of plasmid biology and an important mechanism of horizontal gene transfer in bacteria. However, there is little consensus on how to accurately estimate and report plasmid conjugation rates, in part due to the wide range of available methods. Given the similarity between approaches, we propose general reporting guidelines for plasmid conjugation experiments. These constitute best practices based on recent literature about plasmid conjugation and methods to measure conjugation rates. In addition to the general guidelines, we discuss common theoretical assumptions underlying existing methods to estimate conjugation rates and provide recommendations on how to avoid violating these assumptions. We hope this will aid the implementation and evaluation of conjugation rate measurements, and initiate a broader discussion regarding the practice of quantifying plasmid conjugation rates.

The emergence and spread of antimicrobial resistance results in antibiotic inefficiency against multidrug resistant bacterial strains. Alternative treatment to antibiotics must be investigated to fight bacterial infections and limit this global public health problem. We recently developed an innovative strategy based on mobilizable Targeted-Antibacterial-Plasmids (TAPs) that deliver CRISPR/Cas systems with strain-specific antibacterial activity, using the F plasmid conjugation machinery for transfer into the targeted strains. These TAPs were shown to specifically kill a variety of Enterobacteriaceae strains, including E. coli K12 and the pathogen strains EPEC, Enterobacter cloacae and Citrobacter rodentium. Here, we extend the host-range of TAPs using the RP4 plasmid conjugation system for their mobilization, thus allowing the targeting of E. coli but also phylogenetically distant species, including Salmonella enterica Thyphimurium, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. This work demonstrates the versatility of the TAP strategy and represents a significant step toward the development of non-antibiotic strain-specific antimicrobial treatments.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.

Replication control of many plasmids is mediated by the balance between the positive and negative effects of Rep protein binding repeated sequences (iterons) associated with the replication origin, oriV. Negative control is thought to be mediated by dimeric Rep protein linking iterons in a process termed “handcuffing”. The well-studied oriV region of RK2 contains 9 iterons arranged as a singleton (iteron 1), a group of 3 (iterons 2–4) and a group of 5 (iterons 5–9), but only iterons 5 to 9 are essential for replication. An additional iteron (iteron 10), oriented in the opposite direction, is also involved and reduces copy-number nearly two-fold. Since iterons 1 and 10 share an identical upstream hexamer (5’ TTTCAT 3′) it has been hypothesised that they form a TrfA-mediated loop facilitated by their inverted orientation. Here we report that contrary to the hypothesis, flipping one or other so they are in direct orientation results in marginally lower rather than higher copy-number. In addition, following mutagenesis of the hexamer upstream of iteron 10, we report that the Logo for the hexamer “upstream” of the regulatory iterons (1 to 4 and 10) differs from that of the essential iterons, suggesting functional differences in their interaction with TrfA.

Plasmids are universally present in bacteria and play key roles in the dissemination of genes such as antibiotic resistance determinants. Major concepts in Plasmid Biology derive from the efforts to classify plasmids. Here, we review the main plasmid classification systems, starting by phenotype-based methods, such as fertility inhibition and incompatibility, followed by schemes based on a single gene (replicon type and MOB class), and finishing with recently developed approaches that use genetic distances between whole plasmid sequences. A comparison of the latter highlights significant differences between them. We further discuss the need for an operational definition of plasmid species that reveals their biological features, akin to plasmid taxonomic units (PTUs).

While most detailed analyses of antibiotic resistance plasmids focus on those found in clinical isolates, less is known about the vast environmental reservoir of mobile genetic elements and the resistance and virulence factors they encode. We selectively isolated three strains of cefotaxime-resistant Escherichia coli from a wastewater-impacted coastal wetland. The cefotaxime-resistant phenotype was transmissible to a lab strain of E. coli after one hour, with frequencies as high as 10⁻³ transconjugants per recipient. Two of the plasmids also transferred cefotaxime resistance to Pseudomonas putida, but these were unable to back-transfer this resistance from P. putida to E. coli. In addition to the cephalosporins, E. coli transconjugants inherited resistance to at least seven distinct classes of antibiotics. Complete nucleotide sequences revealed large IncF-type plasmids with globally distributed replicon sequence types F31:A4:B1 and F18:B1:C4 carrying diverse antibiotic resistance and virulence genes. The plasmids encoded extended-spectrum β-lactamases bla_CTX-M-15 or bla_CTX-M-55, each associated with the insertion sequence ISEc9, although in different local arrangements. Despite similar resistance profiles, the plasmids shared only one resistance gene in common, the aminoglycoside acetyltransferase aac(3)-IIe. Plasmid accessory cargo also included virulence factors involved in iron acquisition and defense against host immunity. Despite their sequence similarities, several large-scale recombination events were detected, including rearrangements and inversions. In conclusion, selection with a single antibiotic, cefotaxime, yielded conjugative plasmids conferring multiple resistance and virulence factors. Clearly, efforts to limit the spread of antibiotic resistance and virulence among bacteria must include a greater understanding of mobile elements in the natural and human-impacted environments.

The effective utilization of traditional Chinese medicine (TCM) has been challenged by the difficulty to accurately distinguish between similar plant varieties. The stability and conservation of the chloroplast genome can aid in resolving genotypes. Previous studies using nuclear sequences and molecular markers have not effectively differentiated the species from related taxa, such as Machilus leptophylla, Hanceola exserta, Rubus bambusarum, and Rubus henryi. This study aimed to characterize the chloroplast genomes of these four plant species, and analyze their simple sequence repeats (SSRs) and phylogenetic positions. The results demonstrated the four chloroplast genomes consisted of 152.624 kb, 153.296 kb, 156.309 kb, and 158.953 kb in length, involving 124, 130, 129, and 131 genes, respectively. They also contained four specific regions with mononucleotide being the class with the most members. Moreover, these repeating types of SSR were various in individual class. Phylogenetic analysis showed that M. leptophylla was clustered with M. yunnanensis, and H. exserta was confirmed as belonging to the family Ocimeae. Additionally, R. bambusarum and R. henryi were grouped together but differed in their SSR features, indicating that they were not the same species. This research provides evidence for resolving species and contributes new genetic information for further studies.

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 it was concluded that the difference may be due to the species background, the E. coli strains used were recombination-deficient. Here, we have further investigated the requirements for TU loss in E. coli and found that Tn4352B was stable in recombination-proficient strains. Among several recombination-deficient strains examined, rapid loss occurred only in strains that carry the recA1 allele but not in strains carrying different recA alleles, recA13 and a novel recA allele identified here, that also render the strain deficient in homologous recombination. Hence, it appears that a specific property of the RecA1 protein underlies the observed TU loss from Tn4352B.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.