HPLC measurement of total polyamines

Strains were cultured as described above for the determination of DNA supercoiling. Five OD₆₀₀ units were pelleted and washed three times with PBS. Cells were then resuspended in 200 μL lysis buffer (100 mM MOPS, 50 mM NaCl, 20 mM MgCl₂, pH 8.0) and plated to obtain Colony Forming Units (CFUs).

Cells were frozen in liquid nitrogen, then thawed at 37°C three times. 60 μL trichloroacetic acid 40% was added and the mixture was incubated on ice for 5 min. The supernatant was submitted for derivatization.

Derivatization was performed by adding 250 μL NaOH 2M, 10 μL benzoyl chloride and incubating at room temperature for 20 min. The reaction was stopped by adding 400 μL NaCl, then polyamines were extracted using 300 μL diethyl ether. The ether was evaporated at 37°C and polyamines were dissolved in methanol/water 45%/55% (v/v).

For the standard curve, a mixture of 20 mM putrescine, 10 mM spermidine and 10 mM spermine was made. This mixture was serially diluted in 4-fold increments. 50 μL of the polyamine mix was added to 200 μL lysis buffer, then derivatized as before.

HPLC was performed on a Waters Acquity UPLC H Class with PDA detector (Waters, Milford, MA). The column was a Waters Acquity UPLC BEH C18, 1.7 μm (Waters 186005604), kept at 60°C throughout the experiment. Flow rate was 0.3 mL/min. Solvent was 45% methanol/55% water (v/v). Detection was performed in UV at 254 nm.

Cell volume was measured in a separate experiment by phase microscopy, and estimated to be 4 μm³ for the tested strains and conditions.

Intracellular polyamine concentrations were then calculated from the measured concentrations by HPLC, the CFUs and the cell volume.