a, Overview of ripcord mutagenesis. b, Co-expression of the WT pyocin and mutant 626TEV with the TEV protease. Pyocin killing activity in the lysates was assessed with the help of a spot assay with the P. aeruginosa 13 s strain as prey. Both pyocin and protease expression levels are arabinose dependent, with the rate of protease production being proportional to arabinose concentration and pyocin expression reaching the maximum at the lowest concentrations of arabinose tested (0.01%). Each experiment was repeated biologically three times (also for c–g). c, Representative negative staining EM images of the crude lysates shown in panel b induced with 0.01% arabinose. Despite showing killing activity in the lysates, no extended particles were found in the mutant 626TEV on EM grids. d–f, Temperature-dependent sheath contraction rates of the WT pyocins (d) and mutants (e and f) measured with the help of circular dichroism. g, The rate constants k(T) of WT pyocins, 626ΔWL and 626TEV fitted to the Arrhenius model k(T) = A exp(−E a /RT) where T is the absolute temperature, A is a temperature independent constant, E a is the activation energy and R is the ideal gas constant. The dots on the graph are individual values for three biologically independent measurements of ln k(T), and the error bars show the 95% confidence interval calculated for them.