FIG 5.

Separation of filaments from the new progeny after 3 h of resuspension in fresh medium. (a) Workflow for separation of filaments from the new progeny. A mid-exponential culture of Evo3A was treated with ampicillin (∼15× MIC) for 5 h, washed to remove antibiotic, and resuspended in fresh LB medium. After 3 h of resuspension, there was a mixture of filaments and newly divided cells. The mixture was filtered with a PVDF membrane (5-μm pore size, 25-mm diameter) to separate the filaments (residue) and the normal-sized new progeny (filtrate). (b) Epifluorescence microscopy of the cells before and after filtration. The top row shows filtration using one filter for 3 ml culture, and the bottom row shows filtration using 6 filters for 3 ml culture (one filter per 500 μl). (c and d) Venn diagrams for proteome comparison of the enriched filaments to those before resuspension (c) and the enriched new progeny to the enriched filaments (d). (e and f) Volcano plots of the enriched filaments compared to those before resuspension (e) and the enriched new progeny compared to the enriched filaments (f). Differentially expressed proteins are defined as those with Benjamini-Hochberg-corrected P values below 0.1, and fold change higher or lower than ±1.5, corresponding to the colored dots. Red dots are the downregulated proteins, and blue dots are the upregulated proteins. (g and h) Functional classification of the differentially expressed proteins identified in the enriched filaments compared to those before resuspension (g) and the enriched new progeny compared to the enriched filaments (h) by gene ontology (GO) analysis using DAVID, classified by biological process. Dot size is proportional to the y axis. (i) ATP level of Evo3A after 5 h of ampicillin treatment, Evo3A after 3 h of resuscitation, and WT after 3 h of resuscitation, compared to those before treatment, measured using the BacTiter-Glo assay. To calculate the abundance on a per-cell basis, the measured values were normalized by CFU counts by serially diluting and plating washed aliquots. Data are presented relative to the untreated cells (marked by the horizontal dashed line). Data represent three biological replicates, and each data point is denoted as mean ± SEM.