Project description:Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls.
Project description:Transient plasmid transfection is common approach for studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We found that plasmids generate different levels of transcripts virtually everywhere. Spurious transcription may have undesirable effects as some co-transfected plasmids inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to kan/neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression of transiently transfected plasmids highlights the importance of appropriate experimental controls. HEK293 cells (human origin) transiently transfected with 4 various plasmids
Project description:Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 μg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.
Project description:Plasmid-free Lactococcus lactis IL1403 is one of the best-characterized representatives of lactic acid bacteria (LAB), intensively used in broad microbiology worldwide. Its parent strain, L. lactis IL594, contains seven plasmids (pIL1-pIL7) with resolved DNA sequences and an indicated role for overall plasmid load in enhancing host adaptive potential. To determine how individual plasmids manipulate the expression of phenotypes and chromosomal genes, we conducted global comparative phenotypic analyses combined with transcriptomic studies in plasmid-free L. lactis IL1403, multi-plasmid L. lactis IL594 and its single-plasmid derivatives. The presence of pIL2, pIL4 and pIL5 led to the most pronounced phenotypic differences in the metabolism of several carbon sources, including some β-glycosides and organic acids. The pIL5 plasmid also contributed to increased tolerance to some antimicrobial compounds and heavy metal ions, especially those in the toxic cation group. Comparative transcriptomics showed significant variation in the expression levels of up to 189 chromosomal genes due to the presence of single plasmids, and 435 unique chromosomal genes that are resultant of the activity of all plasmids, which may suggest that the observed phenotypic changes are not only the result of direct action of their own genes, but also originate from indirect actions through cross-talk between plasmids and the chromosome. The data obtained here indicate that plasmid maintenance leads to the development of important mechanisms of global gene regulation that provide changes in the central metabolic pathways and adaptive properties of L. lactis, and suggest the possibility of a similar phenomenon among other groups of bacteria.
Project description:The plasmids introduced into the E. coli cells affect the expression of chromosomal genes. Therefore we aimed at comparing the protein expression profiles of a strain not containing any plasmid with strains that do carry plasmids.