Fig. 2
hok induction disturbs essential membrane functions of E. amylovora. Effect of hok induction on the proton motive force (PMF) and membrane integrity without (panel labelled as “None”) or with the addition of 10 mM mannitol (panel labelled as “Mannitol”) immediately before IPTG supplementation (a), and effect of mannitol in reversing the toxicity of Hok (b). E. amylovora cultures grown overnight for 20 h in LB broth were washed twice and diluted to OD600 = 0.2 in fresh LB broth. The concentrations of IPTG supplemented are indicated in parentheses. After incubation at 28 °C with 200 rpm shaking for 1 h, the PMF of cultures was examined using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4 [3]), and membrane integrity was determined via propidium iodide (PI). Fluorescence was measured using a BD LSR II flow cytometer. Ten thousand events were examined with a 488 nm laser and a 530/30 emission filter (DiBAC4 [3]) staining and a 561 nm laser and a 620/15 emission filter (PI). Subsequent analyses were conducted on Flowing Software 2.5.1 and R v3.4.0. Increased fluorescence after treatment with DiBAC4 [3] or PI indicates greater collapse of the PMF or compromised membrane integrity, respectively. To test the effect of bacterial metabolites on the toxicity of Hok, 10 mM mannitol or 10 mM arabinose was added to the cultures (OD600 = 0.2) immediately before IPTG was added, and the OD600 was measured 4 h after the incubation using a Tecan spectrophotometer. Bacterial growth was monitored by measuring OD600 of the cultures using a Tecan spectrophotometer. Results represent the means of three biological replicates and error bars indicate the standard deviation. Different letters indicate significant differences (P < 0.05) using Student’s t-test. The assays were done three times with similar results