SRA

Glycocin F (GccF) is a bacteriocin produced by Lactobacillus plantarum which causes susceptible cells to enter bacteriostasis within minutes of exposure. GccF is diglycosylated with two N-acetylglucosamine (GlcNAc) moieties and is active against strains of Lb. plantarum, as well as potential pathogens such as Enterococcus faecalis and E. faecium. Its mechanism of action, however, is unknown. To better understand how GccF inhibits growth, we carried out RNA sequencing (RNA-seq) on E. faecalis strain JH2-2 exposed to different concentrations of GccF for different lengths of time. Here we demonstrate that free GlcNAc both protects E. faecalis from the effects of GccF, as well as pre-sensitises it to GccF. Transcriptional analysis showed that exposure to free GlcNAc resulted in up-regulation of a GlcNAc-specific phosphotransferase system (PTS) transporter, which is a putative GccF receptor. Time course analysis showed over 100 genes were up- or down-regulated within 10 minutes of exposure, a number that increased to over 300 genes after 40 minutes. A large number of these genes were found to be regulated by the alternative sigma factor 54, which was shown to not be required for susceptibility to GccF. Interestingly, genes encoding the V-type ATPases and sodium:proton antiporters were found to be up-regulated at all times and all concentrations of GccF tested. This suggests an indirect response to GccF, possibly due to low-level permeabilization of the cell membrane. Overall design: Enterococcus faecalis cells were exposed to the bacteriocin glycocin F at different concentrations and different times, and transcriptional response profiled usind RNA sequncing on an Illumina HiSeq 2000. Samples were carried out in triplicate, with the necessary untreated controls.