Plasmid最新文献

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre. One of the resulting mutant plasmids, pSH-CreFM allowed us to remove selection marker genes by Cre-mediated recombination at temperatures up to 70 °C. By using the thermostable Cre/lox system with pSH-CreFM, we successfully constructed two valuable pTT27 megaplasmid mutant strains, a plasmid-free strain and β-galactosidase gene deletion strain, which were produced by different methods. The thermostable Cre/lox system improved the time-consuming nature of the original Cre/lox system, but it was not suitable for multiple markerless gene disruption in T. thermophilus because of its highly efficient induction of Cre-mediated recombination even at 70 °C. However, in vivo megaplasmid manipulations performed at 65 °C were faster and easier than with the original Cre/lox system. Collectively, these results indicate that this system is a powerful tool for engineering T. thermophilus megaplasmids.

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model.

We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases.

The predominant type of plasmids found in Acinetobacter species encode a Rep_3 initiation protein and many of these carry their accessory genes in dif modules. Here, available sequences of the 14 members of the group of Rep_3 plasmids typed as R3-T33, using a threshold of 95% identity in the repA gene, were compiled and compared. These plasmids were from various Acinetobacter species. The pdif sites were identified allowing the backbone and dif modules to be defined. As for other Rep_3 plasmids carrying dif modules, orfX encoding a protein of unknown function was found downstream of repA followed by a pdif site in the orientation XerC binding site–spacer–XerD binding site. Most backbones (n = 12) also included mobA and mobC genes but the two plasmids with the most diverged repA and orfX genes had different backbone contents. Although the gene content of the plasmid backbone was largely conserved, extensive recombinational exchange was detected and only two small groups carried identical or nearly identical backbones. Individual plasmids were associated with 1 to 13 dif modules. Many different dif modules were identified, including ones containing antibiotic or chromate resistance genes and several toxin/antitoxin gene pairs. In some cases, modules carrying the same genes were significantly diverged. Generally, the orientation of the pdif sites alternated such that C modules (XerC binding sites internal) alternated with D modules (XerD binding sites internal). However, fusions of two dif modules via mutational inactivation or loss of a pdif site were also detected.

The length of a plasmid is a key property which is linked to many aspects of plasmid biology. When distributions of plasmid lengths are shown in the literature, they are usually plotted with length on a logarithmic scale. However, a quantity and its logarithm have distinct distributions which may differ considerably in shape. Mistaking the distribution of log-lengths for the distribution of lengths can therefore lead to distorted conclusions about the distribution; in particular, the distribution of log-lengths may be bimodal when the distribution of lengths is only unimodal. This particular confusion has arisen in the literature where the length distribution is often claimed to be bimodal based on examination of what is in fact the log-length distribution. While the length distribution is indeed bimodal within many bacterial families, it is not across the ensemble of all plasmids. We suggest that authors should be careful to show the plasmid length distribution, or to distinguish the two distributions, to avoid misleading inferences.

The complete genome of RBH2, a sporadic, carbapenem resistant ST111 Acinetobacter baumannii isolate from Brisbane, Australia was determined and analysed. RBH2 is extensively resistant and the chromosome includes two transposons carrying antibiotic resistance genes, AbaR4 (oxa23 in Tn2006) and Tn7::Tn2006 (dfrA1, sat2, aadA1, oxa23). The chromosome also includes two copies of Tn6175, a transposon carrying putative copper resistance genes, and 1–17 copies of six different insertion sequences. RBH2 has six plasmids ranging in size from 6 kb – 141 kb, four carrying antibiotic resistance genes. Plasmids pRBH2–1 (aadB) and pRBH2–2 (aphA6 in TnaphA6) were found to be essentially identical to known plasmids pRAY*-v1 and pS21–1, respectively. The largest plasmids, pRBH2–5 (oxa23 in AbaR4) and pRBH2–6 (oxa23 in AbaR4::ISAba11 and sul2, tet(B), strA and strB in Tn6172) have known transfer-proficient relatives. pRBH2–5, an RP-T1 (RepAci6) plasmid, also carries a different putative copper resistance transposon related to Tn6177 found in pS21–2. The backbone of pRBH2–5 is related to those of previously described RepAci6 plasmids pAb-G7–2 and pA85–3 but has some distinctive features. Three different RepAci6 backbone types were distinguished, Type 1 (pAb-G7–2), Type 2 (pA85–3) and Type 3 (pRBH2–5 and pS21–2). pRBH2–6 is closely related to pAB3 and their backbones differ by only 5 SNPs. Plasmids pRBH2–3 and pRBH2–4 do not carry antibiotic resistance genes. pRBH2–3 does not include an identifiable rep gene and is a novel plasmid type. pRBH2–4 is of the R3-T3 type and includes segments of the larger pABTJ2 that heads this group. Other ST111 genomes carry different plasmids.

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the −35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from “read-through” transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, P_rnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

Antimicrobial resistance (AR) mechanisms encoded on plasmids can affect other phenotypic traits in bacteria, including biofilm formation. These effects may be important contributors to the spread of AR and the evolutionary success of plasmids, but it is not yet clear how common such effects are for clinical plasmids/bacteria, and how they vary among different plasmids and host strains. Here, we used a combinatorial approach to test the effects of clinical AR plasmids on biofilm formation and population growth in clinical and laboratory Escherichia coli strains. In most of the 25 plasmid-bacterium combinations tested, we observed no significant change in biofilm formation upon plasmid introduction, contrary to the notion that plasmids frequently alter biofilm formation. In a few cases we detected altered biofilm formation, and these effects were specific to particular plasmid-bacterium combinations. By contrast, we found a relatively strong effect of a chromosomal streptomycin-resistance mutation (in rpsL) on biofilm formation. Further supporting weak and host-strain-dependent effects of clinical plasmids on bacterial phenotypes in the combinations we tested, we found growth costs associated with plasmid carriage (measured in the absence of antibiotics) were moderate and varied among bacterial strains. These findings suggest some key clinical resistance plasmids cause only mild phenotypic disruption to their host bacteria, which may contribute to the persistence of plasmids in the absence of antibiotics.

Lactiplantibacillus plantarum is one of the important species of lactic acid bacterium (LAB) found in diverse environments, with many strains exhibiting probiotic properties. In our previous study, 41.6% of protein families (PFs) encoded by 395 plasmids from several L. plantarum strains were found to be hypothetical proteins with no predicted function. This study aimed at predicting the functions of these 647 hypothetical proteins using 21 different bioinformatics methods. As a result, 160 PFs could be newly annotated. A lower proportion of plasmid-specific functions was annotated as compared to the functions shared between plasmids and chromosomes. Also, hypothetical proteins were less conserved than the annotated proteins across L. plantarum plasmids. Based on the subcellular localization, cell envelope proteins represented the biggest category in the newly annotated proteins. Transporters (112 PFs) which was a part of cell envelop proteins represented the largest functional group. Additionally, 40 and 25 other PFs were predicted to contain signal peptides and transmembrane helices, respectively. We speculate that such hypothetical proteins might be involved in the transport of various chemicals and environmental interactions in L. plantarum. In the future, functional characterization of these proteins through wet-lab experimental approach can provide novel insights into their contribution to the physiology, probiotic properties, and industrial utility of these bacteria.

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.