Fig. 4. Evolved tolerance against benzalkonium chloride is related to membrane surface charge, motility, and biofilm formation.

a Mutants of outer membrane biogenesis and envelope stress, but not flagella, show increased levels of survival against BAC. Survival fractions were scaled to the survival fraction of the corresponding wild-type. Bars represent the geometric mean ± 95% C.I., n = 4 biological replicates. Δ indicates knock-out mutants; P_lac, P_BAD indicate inducible promoter; for strain details, refer to Table S3. Significance of difference to wild-type is indicated by asterisks: ${}^{*}$p < 0.05; ${}^{**}$p < 0.01 (two-tailed t-test of log-transformed survival fraction. b The evolved clones and a knock-out of lpxM absorb significantly less positively charged cytochrome c than the ancestor, indicating a reduction in net-negative surface charge caused by mutations in lpxM. Shown is the mean ± 95% C.I., n = 3 biological replicates. Significance of decreased cytochrome c absorption against ancestor is indicated by asterisks: ${}^{*}$p < 0.05; ${}^{**}$p < 0.01 (one-tailed one-sample t-test for difference from 0). c Quantitative relationship between tolerance against BAC and changes in surface charge in the evolved clones (circles). The wild-type is shown as triangle, the lpxM knock-out is shown as square. The Pearson correlation coefficient r for the evolved clones is indicated in the plot and was calculated with the data from the evolved clones only. Significance of correlation: two-sided test with t-distribution of the test statistic. Source data are provided as a Source Data file. Exact p-values are indicated above the asterisks.