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: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 L. lactis IL594 strain contains seven plasmids (pIL1 to pIL7) and is the parental strain of the plasmid-free L. lactis IL1403, one of the most studied lactic acid bacteria (LAB) strain. The genetic sequences of pIL1 to pIL7 plasmids have been recently described, however the knowledge of global changes in host phenotype and transcriptome remains poor. In presented study, global phenotypic analyses were combined with transcriptomic studies to evaluate a potential influence of plasmidic genes on overall gene expression in industrially important L. lactis strains. High-throughput screening of phenotypes differences revealed pronounced phenotypic differences in favor of IL594 during the metabolism of seven C-sources, including carbohydrates and β-glucosides. A plasmid-bearing strain presented increased resistance to unfavorable growth conditions, including the presence of heavy metal ions, antibiotics and antimicrobial compounds. Global comparative transcriptomic study of L. lactis strains revealed variation in the expression of over 370 of chromosomal genes caused by plasmids presence. The general trend presented upregulated energy metabolism and biosynthetic genes, differentially expressed regulators, prophages and cell resistance proteins. Our findings suggest that plasmids maintenance leads to significant perturbation in global gene regulation that provide changes in central metabolic pathways and adaptive properties of the IL594 cells.
Project description:Gut plasmids show heightened sensitivity to gut microenvironmental changes compared to their bacterial hosts. To explore their significance in colorectal cancer (CRC), we analyzed metagenomic data from 863 participants (312 CRC, 387 high-risk, 164 low-risk). Plasmid and bacterial profiles were characterized, along with trace elements and metabolites. Differential analysis, functional gene assessment (ARG, MGE, MRG, VFGB), random forest modeling, and structural equation modeling (SEM) were applied. In terms of overall abundance, plasmids in both the high-risk and CRC groups exhibited a decreasing trend. Gut plasmids significantly influenced the functional genes (ARG, MGE, MRG, VFGB) of their bacterial hosts. Six key bacterial hosts (Enterobacterales, Burkholderiales, Hyphomicrobiales, Lactobacillales, Bacteroidales, Campylobacterales) and 12 plasmid markers were identified. The plasmid-based model effectively predicted CRC risk. SEM revealed that trace elements (e.g., Ni), metabolites (e.g., 5-Hydroxytryptophol), and host bacteria (e.g., Campylobacterales, Enterobacterales) predominantly exerted negative effects on most plasmids, whereas Ni exhibited a positive influence on plasmids NZ_CP013564.1, NZ_CP024312.1, and NZ_CP48284.1. We characterized the composition of gut plasmids and their bacterial hosts, explored the impacts of gut plasmids on bacterial functionality, and mapped multi-omics interaction networks linking plasmids, hosts, and metabolic features.