Identification of hundreds of novel small plasmids
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REPOSITORIES: ENA
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Project description:Rhizobia are gram-negative bacteria able to establish a symbiotic interaction with leguminous plants. Due to their nitrogen fixing capacity, the study of these microorganisms has acquired great relevance for the agriculture. Rhizobia usually harbor many plasmids in their genome which can be transferred to other organisms by conjugation. Two main mechanisms of regulation of rhizobial plasmid transfer have been described: Quorum sensing (QS) and rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have been recently described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pLPU83a from Rhizobium favelukesii LPU83, a plasmid that showed a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory mechanism, we show evidence of the functions of these genes in different genomic backgrounds, and confirmed that homologous proteins from non-closely related organisms play the same function. These findings set up a cornerstone for a new molecular circuit of conjugative transfer of plasmids.
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Project description:Compared with HKE9, HKE9-M-rpoS (rpoS knockout in hypervirulent Klebsiella pneumoniae strain HKE9) had 624 genes expressed significantly different, among which 148 genes were significantly up-regulated and 476 genes were significantly down-regulated, 586 genes on chromosomes, 10 genes on circular plasmid and 28 genes on linear plasmid; HKE9-C-M-rpoS had 746 genes expressed significantly different, among which 286 genes were significantly up-regulated and 460 genes were significantly down-regulated, 727 genes on chromosomes, 11 genes on circular plasmid and 8 genes on linear plasmid. Compared with HKE9-M-rpoS, HKE9-C-M-rpoS (rpoS complement in strain HKE9-M-rpoS) had 509 genes expressed significantly different, among which 324 genes were significantly up-regulated and 185 genes were significantly down-regulated, 458 genes on chromosomes, 3 genes on circular plasmid and 48 genes on linear plasmid.
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Project description:Plasmid maintenance costs to bacterial hosts is closely linked to the mechanisms that underlie plasmid fitness and how these costs are resolved. Herein, we performed multiple (63) serial passage to explore the compensatory mechanisms of co-evolution of multidrug-resistant IncHI2 plasmid pJXP9 and S. Typhimurium strain ATCC 14028 with or without antibiotic selection. pJXP9 could be maintained at for hundreds of generations even without drug exposure. Decreased lag times and higher competitive advantages were observed in end-point evolved strains bearing pJXP9 compared to ancestral strains. Genomic and transcriptomic analyses revealed that the fitness costs of pJXP9 in ATCC 14028 were derived from not only specific plasmid genes, particularly the multidrug-resistant region and conjugation transfer region I, but also the conflicts resulting from chromosomal gene interactions. Correspondingly, plasmid-borne deletions of these regions could compensate the fitness cost due to the presence of the plasmid. Furthermore, mutations and mRNA alterations in chromosomal genes involved in physiological functions were also adaptative. These functions included decreased flagellar motility, oxidative stress resistance and fumaric acid synthesis, and increased Cu resistance. Our findings suggest that plasmid maintenance through plasmid-bacteria co-evolution is a trade-off between increasing plasmid vertical transmission and impairing its horizontal transmission and bacterial physiological phenotypes.
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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.
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Project description:The siRNA transfection includes JAK3 and EZH2 siRNAs. The plasmid transfection includes EZH2 WT and its mutants. We used gene expression data from JAK3 siRNA to test whether it affected epigenetic regulation, and used EZH2 mutants to study how it affected EZH2 canonical function.
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