Fig 5. The stringent response synthetase Rel_Sau interacts with Era and CshA.

A) Top: Bacterial two-hybrid showing an interaction between CshA-T18/T18-CshA and both Rel_Sau-T25 and T25-Rel_Sau. CshA did not interact with the small (p)ppGpp synthetase enzymes RelP or RelQ. Bottom: Bacterial two-hybrid showing an interaction between Rel_Sau-T25 and both Era-T18 and T18-Era. N = 3 with one representative image shown. B) Affinity pulldown assay using GST, GST-tagged CshA and GST-tagged Era coupled to glutathione beads. Beads alone, GST, GST-Era or GST-CshA coupled beads were incubated with His-tagged Rel_Sau. After washing, bound Rel_Sau-His was detected using HRP-conjugated anti-His antibodies. N = 3 or 4 with one representative image shown above. Below, the mean signal intensities and standard deviations from the repeats are plotted. Statistical analysis was performed using a one-way ANOVA, followed by Tukey’s multiple comparisons test (* P < 0.05, *** P < 0.0005). C, D & E) Split luciferase assays demonstrating an interaction between CshA (C), Era (D) or Era 1–180 (E) and Rel_Sau in S. aureus. The negative controls have genes singly fused to either SB or LB. The average values and standard deviations of triplicate experiments (N = 3) are plotted. All statistical analysis was performed using a one-way ANOVA, followed by Tukey’s multiple comparisons test (** P < 0.005, *** P < 0.0005, **** P < 0.0001).