PMC

Time-course accumulation of (p)ppGpp in Ent. faecalis after mupirocin treatment. Mid-exponential phase cultures were labelled with [³²P]orthophosphate and were treated with 50 µg mupirocin ml⁻¹ (Mup) or untreated (Control) for the time indicated. Nucleotide acid extracts were spotted onto polyethyleneimine-cellulose plates for TLC in 1.5 M KH₂PO₄.

Heat map of genes involved in transcription, translation and DNA replication repressed by mupirocin treatment in OG1RF and ΔrelQ. Column titles indicate comparisons between mupirocin-treated cells (15 or 30 min) and control cells for each strain (e.g. OG1RF mupirocin 15 min versus OG1RF control). Colours represent fold repression: pink, 2≤x<5; magenta, 5≤x<10; red, ≥10.

Venn diagrams of the differentially expressed genes (upregulated in white type, downregulated in black type) of OG1RF and Δrel strains treated with mupirocin for 15 and 30 min compared with control (non-stressed) samples of each strain. (a) Comparisons of OG1RF and ΔrelQ strains. (b) Comparisons of OG1RF, ΔrelA and ΔrelAΔrelQ strains. All genes were subjected to a twofold differential expression cut-off at P≤0.001.

The ΔrelAΔrelQ mutant shows reduced survival in macrophages and reduced virulence in G. mellonella. (a) Intracellular survival of OG1RF and its derivatives within murine macrophage cell line J774.A1 (m.o.i. 100 : 1). The curves shown are means±sds of the results from two independent experiments (n = 6). A Student’s t test indicated that the differences observed between ΔrelAΔrelQ and the other three strains at 72 h were statistically significant (P = 0.042). (b) Kaplan–Meier plots of larvae that received injections of Ent. faecalis OG1RF and its Δrel derivatives. The experiments were repeated three times, and the results are representative of a typical experiment. Compared with the wild-type strain, ΔrelAΔrelQ showed attenuated virulence (P = 0.0039).