Fig. 4: Bioaccumulation of PFNA affects bacterial physiology.

a, Proteomics analysis shows proteins that are differentially abundant between B. uniformis treated with 20 µM PFNA and those treated with DMSO. The red and green dots mark proteins with a log₂(abundance ratio) > 1 or < −1 (that is, twofold increase or decrease) and a multiple-testing corrected P value of less than 0.05; n = 6 biological replicates (Supplementary Table 24). The circle marks a protein from the RND efflux system (gene ID 962 corresponds to protein R9I2L8), for which nine missense variants within the coding region of the gene were identified in populations evolved under high PFAS concentrations (Supplementary Table 4). P values were calculated using analysis of variance followed by Benjamini–Hochberg correction for multiple testing. b,c, TPP analysis of E. coli BW25113 wt (wild type; low-PFNA bioaccumulating) (b) and E. coli BW25113 ΔtolC (high-PFNA bioaccumulating) (c). Lysate and live cells incubated with PFNA look more similar for E. coli BW25113 ΔtolC mutant compared with the wild type, supporting increased bioaccumulation in ΔtolC mutants. Each data point represents the summed log₂(FC) across all temperatures for a specific protein. Black dashed line, diagonal; blue line, linear regression with 95% confidence interval (Supplementary Table 25). d, Principal component (PC) analysis shows a clear distinction between B. uniformis pellet samples treated with 20 µM PFNA and the control; n = 6 biological replicates (Supplementary Table 26). e, Aspartic acid, glutamic acid and glutamine concentrations in B. uniformis pellet and supernatant samples; n = 6 biological replicates (Supplementary Table 26). P values were calculated using two-sided t-test and corrected for multiple testing using the Benjamini–Hochberg method.