Project description:We report the genome-wide analysis from chromatin immunoprecipitated DNA (ChIP-sequencing) at very high resolution of the DNA binding pattern of ParBF (SopB) either on the full length plasmid F or on E. coli chromosome carrying the parSF centromere sequence. We also varied the intracellular ParBF concentration to discriminate between the several proposed mechanism of partition complexes assembly.
Project description:Purpose: We investigate the evolutionary footprints of a bacteria-plasmid association consisting of Escherichia coli K-12 MG1655 and plasmid RP4 undergoing a long-term sub-MIC antibiotic stress. Methods: Bacterial mRNA profiles of evolved RP4-carrying strains (E:H:p) and ancestral RP4-carrying strains (A:H:p) were generated by deep sequencing on an Illumina Hiseq platform. The sequence reads that passed quality filters were analyzed by Burrows–Wheeler Aligner (BWA), followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads of E:H:p and 12 million sequence reads of A:H:p to the E. coli MG1655 genome (GCF_000801205.1) and differential expressed genes were identified with TopHat workflow. RNA-seq data showed that approximately 15% of the transcripts showed differential expression between the E:H:p and A:H:p strains, with a fold change ≥1 and p value <0.005. Altered expression of 26 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Data analysis with bowtie and TopHat workflows provided complementary insights in transcriptome profiling. Conclusions: Our study showed the coevolved bacteria-plasmid pairs has colonization traits superior to the wild-type parent strain. Antibiotic stress was necessary for bacterial evolution and evolved strains mostly employed transcriptional modifications to reduce plasmid-related cost in evolutionary adaptations. Several genes related to chromosome-encoded efflux pumps were transcriptionally upregulated, while most plasmid-harboring genes were downregulated based on RNA gene sequencing. These transcriptional modifications endowed evolved strains with resistant phenotype modifications, including the enhanced bacterial growth and biofilm formation.
