Figure 3

Abnormal proliferation under treatment with pentanol. (A) Representative micrographs of E. coli cultures supplemented with pentanol (C5OH) at the different concentrations specified (shoots at the same microscopy conditions as in Fig. 2): Control, untreated E. coli (top panels); added pentanol at variable doses (lower panels). (B) Abnormal proliferation scenario: in the presence of the membrane softener (pentanol), we observed anomalous bacterial growth as a marginal population of normal specimens coexisting with a prevailing population of bacterial persisters as long filaments that grow and eventually divide under elongation (see micrographs on the right; the arrows mark sites of division in the elongated filaments). (C) Cell counting with cytometric size specification for cultures treated with pentanol (as in A, five different micrographs were analysed at each condition; \(n>100\) specimens): (left) cell length \(L\); (right) cell width \(D\). The normal size distributions for the untreated cells are included for comparison (yellowish Gaussians from Fig. 2B rescaled down dividing fivefold). (D) Cytometric kinetics by phenotype (at variable pentanol concentration as specified in the legend): (left panel) normal cells with length compatible with the canonical dimensions as determined for the untreated cells (\(L={L}_{0}\pm {\sigma }_{L}\) with \({L}_{0}=1.9\,\upmu {\mathrm{m}}\) and \({\sigma }_{L}=1\,\upmu {\mathrm{m}}\)); (central panel) filaments with an elongated cell length (\(L>{L}_{0}+{\sigma }_{L}=2.9\,\upmu {\mathrm{m}}\)). The right panel plots the time evolution of the produced biomass (at the different pentanol concentrations; same colour code). The biomass index is evaluated as the microbe coverage by all the bacterial specimens relative to the whole surface of the microscopy slides. (E) Zoomed fluorescence micrograph (×5) of a flaccid filamentous specimen representing typical bacterial persisters that proliferate longitudinally without division but with several nucleoids replicated along the filament (yellow arrows; DNA appears blue by Hoechst staining). (F) Kinetic turbidity plots (same experimental rationale and legend as in Fig. 2D but corresponding to heterogenous bacterial cultures including normal and filamentous cells). Three kinetic regimes of bacterial proliferation are clearly discernible: (I) induction phase (at \(t<{t}_{0}\)); (II) exponential phase; (III) stationary phase (at \(t>{t}_{\infty }\approx 9h\)) (see inset). The kinetic plots experience progressive delay in the induction phase and acceleration in the exponential phase upon increasing pentanol concentration (see legend). Complete growth inhibition is observed at \({c}_{inh}=90 \,{\mathrm{mM}}\) (magenta). The straight lines correspond to the best fits to the logistic curve in Eq. (S12) (in terms of the lag time \({t}_{0}\), and an effective growth rate \(G\)). The dependencies of the fitted parameters in terms of pentanol concentration appear in: (G) lag times; (H) effective growth rates (the straight line represents the best fit to the Monod’s law; see main text for details).