Project description:Plasmids are one of the important mobile genetic elements in bacterial evolution. In this study, to evaluate the generality of the impact of plasmid carriage on host cell between different plasmids, we compared the response of Pseudomonas putida KT2440 to harboring three natural plasmids; RP4 (IncP-1, multidrug resistance, 60,099-bp), pCAR1 (IncP-7, carbazole-degradative, 200,231-bp) and NAH7 (IncP-9, naphthalene-degradative, 82,232-bp). We prepared two sets of plasmid-harboring strains from independent conjugation events to elucidate the reproducibility of the impact of the plasmid carriage. As results, the fitness was reduced by the carriage of RP4 and pCAR1 in liquid medium, while it was unaffected or even improved for NAH7-harboring strains. RP4-harboring KT2440 formed smaller colonies than the plasmid-free strain on solid medium (1.6% agar). The host cells were elongated by the carriage of the all plasmids, respectively. Copy number determination by quantitative PCR showed that the amount of each plasmid DNA in the host cell did not differed drastically. Whole genome resequencing showed that 13 SNPs (RP4), 24 SNPs (pCAR1) and 5 SNPs (NAH7) were the total differences between the two substrains for each plasmid-harboring strains. Transcriptome analyses showed that the impact of plasmid carriage was constantly larger in RP4-harboring strain than the other two plasmid-harboring strains. Genes involved in metal acquisition and metabolism were commonly affected by the carriage of the three plasmid. Indeed, plasmid-harboring strains showed greater growth inhibition than plasmid-free strains under iron-limiting condition. This feature could become future target to control plasmid spreading.
Project description:Comparative genome hybridization of transconjugants of E. faecalis OG1RF mated with V583. The total DNA of transconjugants was compared with wildtype strains to ascertain the amount of DNA that was transferred from E. faecalis V583 to E. faecalis OG1RF.
Project description:Plasmids were constructed harboring protospacers that are perfect targets for spacers #4 and #21 of the array, but contain NNN (all possible PAM combinations) immediately upstream of the protospacer. The plasmids were introduced into V. cholerae with or without a functional CRISPR-cas system and cells were plated on selective media. Cells were collected and the protospacer plasmids were sequenced in a high throughput manner. PAMs were counted using a custom python script
Project description:V. cholerae A50 has a functional CRISPR-cas system with a conserved boxA sequence. Plasmids harboring protospacers that are perfect targets for each spacer of the array are introduced into wt and boxA mutant V. cholerae. After a period of growth without selection, cells are collected and the protospacer plasmids are sequenced in a high throughput manner.
Project description:Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.
Project description:Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.
Project description:We compared the binding patterns in embryonic stem cells of KAP1 and KRAB Zinc Finger (KZNF) proteins as well as H3K4me3 DNA under several conditions. Native human stem cells. Mouse stem cells containing a transchromosomic copy of human chromosome 11, with and without the introduction of plasmids containing KRAB Zinc Finger sequences. We show that certain KZNFs are responsible for the repression of certain retrotransposons in embryonic stem cells, preventing their spread across the genome. ChIP-seq of HESCs and mouse TC11 ESCs with ZNF91 plasmids and with empty vector plasmids. ChIP of KAP1, ZNF486, H3K4me3. At least 2 replicates of each condition.