Fig. 4

Involvement of stringent response in induced persistence. a Persistence in (s) WT cells plus SHX and then alkaline phosphatase (CIAP) or in (s) cells expressing xseB_asRNA plus SHX. SHX increased persistence in (s) WT cells and CIAP neutralized the persistence enhancement. SHX failed to increase persistence in (s) cells expressing xseB_asRNA. b Quantitative real-time PCR assays for expression of xseB and xseA in (s) WT cells before and after plus 1 mg ml⁻¹ SHX. Copy numbers of genes were set in relation to copy numbers of housekeeping gene rpsL. Significant changes were not detected. c Protein involved in triggering the degradation of XseA. WT, Δ(lon), Δ(ppk1 ppk2A ppk2B ppk2C ppx), Δ(ppk1 ppx), Δ(relA spoT), Δ(exsA), and Δ(exsA lon) cells harboring pBBR1MCS3-xseA (His-tagged XseA) were grown in LB at 37 °C for 18 h ((s) phase). After addition of 1 mg ml⁻¹ SHX for 30 min, samples were removed at the indicated times for western blot analysis. (s) Δ(relA spoT) mutant harboring pBBR1MCS3-xseA was incubated with 5 μg ml⁻¹ pp(G)pp at 37 °C for 30 min. Samples were then removed at the indicated times for western blot analysis. GAPDH was used as an internal reference. d Persistence in washed (s) mutants or SHX-treated (s) mutants. Both washing and SHX induction increased persistence in the (s) Δ(lon), (s) Δ(ppk1 ppk2A ppk2B ppk2C ppx), (s) Δ(ppk1 ppx), and (s) Δ(exsA) mutants, respectively. e SDS-PAGE analysis of in vitro degradation of XseA by Lon. His-tagged Lon failed to degrade His-tagged XseA in absence of polyP or in presence of low polyP (0.5 μM), but did it in presence of high polyP (1.5 μM). f Persistence in (s) Δ(relA spoT) mutant treated by washing, SHX induction, or ppGpp induction. Both washing and ppGpp induction increased persistence in the (s) Δ(relA spoT) mutant, but SHX induction did not. CIAP neutralized the ppGpp-triggered persistence enhancement. All experiments were performed in biological triplicates. Error bars represent standard deviations. Dot plots overlaid on bar graphs represent individual data points. Black spot, treatment step or object; arrow, treatment order; hash sign, inactivated protein