BACKGROUND: Enterococcus faecium is a Gram-positive commensal bacterium of the mammalian intestinal tract. In the last two decades it has also emerged as a multi-resistant nosocomial pathogen. In order to survive in and colonize the human intestinal tract E. faecium must resist the deleterious actions of bile. The molecular mechanisms exploited by this bacterium to tolerate bile are as yet unexplored. RESULTS: In this study we used a high-throughput quantitative screening approach of transposon ...[more]
Project description:In this study, RNA-seq was used to compare the transcriptomes of L. monocytogenes strain H7858 grown on cold smoked salmon (CSS) and in modified brain heart infusion broth (MBHIB, water-phase salt 4.65%, pH 6.1) at 7oC. RNA-seq was performed on H7858 RNA samples representing four independent biological replicates of growth experiments that involved growth of H7858 on CSS or in MBHIB. Samples for RNA preparation were collected when H7858 was grown to late log phase on these two matrices. Indexed and purified cDNA libraries (8 libraries including 4 replicates for each CSS and MBHIB) were loaded together onto an independent flow cell without any other samples; sequencing was carried out by running Hiseq 2000 (single-end, 100-bp per read). Reads alignment was carried out using the Burrows-Wheeler Aligner (BWA). Differential expression of genes in different strains was statistically assessed using the BaySeq method. Significant differential transcription of 149 genes in H7858 was observed between these two treatments including 88 genes and 61 genes up- and downregulated, respectively, in H7858 grown on CSS compared to in MBHIB. Our results show that genes involved in cobalamin biosynthesis, ethanolamine and 1,2-propanediol utilization, and specific carbohydrate transport systems have significantly higher transcript level in H7858 grown on CSS compared to in MBHIB. PrfA-dependent gene enrichment analysis showed that, genes regulated by PrfA were overrepresented among L. monocytogenes genes with higher transcript level on CSS than in MBHIB. Transcriptome profiles of L. monocytogenes grown on cold smoked salmon and in modified BHI broth at 7oC were generated by deep sequencing, in quadruplicate, using Illumina Hiseq 2000 (100 bp per read, single-end).
Project description:Two independent aliquots containing approximately 10^7 cells from the transposon mutant of E. faecium E1162 were grown at 37 C for 20 hours in 20 ml of BHI broth. Genomic DNA was isolated from the two replicate cultures and used for the generation of cDNA. The cDNA samples were labeled with Cy3 and Cy5 respectively and hybridized to a microarray that was designed using the E. faecium E1162 genome sequence.