Project description:The enterococci comprise a genus of 49 low-GC content Gram-positive commensal species within the Firmicutes phylum that are known to occupy diverse habitats, notably the gastrointestinal core microbiota of nearly every phylum, including human. Of particular clinical relevance are two rogue species of enterococci, Enterococcus faecalis and the distantly related Enterococcus faecium, standing among the nefarious multi-drug resistant and hospital-acquired pathogens. Despite increasing evidence for RNA-based regulation in the enterococci, including regulation of virulence factors, their transcriptome structure and arsenal of regulatory small sRNAs (sRNAs) are not thoroughly understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptomes of E. faecalis V583 and E. faecium AUS0004. We identified 2517 and 2771 transcription start sites (TSS) in E. faecalis and E. faecium, respectively. Based on the identified TSS, we created a global map of s70 promoter motifs. We also revealed features of 5’ and 3’UTRs across the genomes. The transcriptome maps also predicted 150 and 128 sRNA candidates in E. faecalis and E. faecium, respectively, some of which have been identified in previous studies and many of which are new. Finally, we validated several of the predicted sRNAs by Northern Blot in biologically relevant conditions. Comprehensive TSS mapping of two representative strains will provide a valuable resource for the continued development of RNA biology in the Enterococci.
Project description:Young adult N2 Caenorhabditis elegans were infected with Enterococcus faecalis or Enterococcus faecium for 8 h to determine the transcriptional host response to each enterococcal species. Analysis of differential gene expression in C. elegans young adults exposed to four different bacteria: heat-killed Escherichia coli strain OP50 (control), wild-type E. faecalis MMH594, wild-type E. faecium E007, or Bacillus subtilis PY79 (sigF::kan). Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Brain-heart infusion agar plates (10 ug/ml kanamycin) were used.
Project description:Enterococcus faecium is a member of the human gut microbiota that has evolved to be a problematic nosocomial pathogen and a leading cause of bloodstream infections in hospitalized patients. Treatment of E. faecium infections is complicated by antibiotic resistance, making it important to understand resistance mechanisms and their broader consequences in this pathogen. Here we explored the collateral effects of rifampin resistance-associated mutations in the E. faecium RNA polymerase β-subunit (RpoB). Of 14,384 publicly available E. faecium genomes, nearly one-third carried a mutation in the rifampin resistance-determining region (RRDR) of RpoB. In a local population of 710 E. faecium isolates collected from patients at a single medical center, we found significant associations between the presence of RRDR mutations and prior exposure to rifamycin class antibiotics, as well as associations between RRDR mutations and altered daptomycin susceptibility. To investigate the phenotypic impacts of RRDR mutations, we studied four isogenic strains with distinct RRDR mutations (Q473K, G482D, H486Y, S491L) that overlapped with clinical isolate variants. Transcriptomic and phenotypic analyses revealed allele-specific effects on E. faecium gene expression, growth dynamics, antibiotic susceptibility, isopropanol tolerance, and cell wall physiology. One frequently observed mutation, H486Y, caused minimal transcriptional changes and enhanced bacterial fitness. In contrast, the S491L mutation induced extensive transcriptional changes and slowed bacterial growth, but also conferred increased isopropanol tolerance, potentially enhancing bacterial survival in the hospital environment. Overall, our findings highlight the multifaceted impacts of RRDR mutations in shaping E. faecium physiology and antibiotic resistance, two important features of this hospital-associated pathogen.
Project description:Mapping of transposon mutant library in Enterococcus faecium during growth in Brain Heart Infusion (BHI) broth and in a semi-static biofilm model. The goal of this study was to identify factors that play a role in E. faecium biofilm formation by selection of transposon insertion mutants that lost the capacity to form biofilm in vitro.
Project description:The transcriptome of Enterococcus faecium E1162 growing in Brain heart Infusion Broth was compared in the mid-exponential growth phase (A660 = 0.3) at 25 C and 37 C.
Project description:Enterococcus faecium was grown in open circuit (control) and closed circuit (current collecting) to observe the difference in extracellular electron transfer (EET) mechanisms. mRNA was extracted, amplified and used on Combimatrix custom arrays to obtain a
