Figure 1.
Flow cytometry analysis of E. coli populations of varying cell lengths.
(A) Cell length distributions for E. coli DH5α populations either not exposed to cephalexin (0) or exposed to cephalexin for 1 hour (1), 1.5 hours (1.5) or 2 hours (2). (B–E) Flow cytometry analysis of the corresponding populations displayed as dot plots with SSC-H plotted against SSC-W. (B) Not exposed to cephalexin, (C) 1 hour exposure, (C) 1.5 hours exposure, (D) 2 hours exposure. The percentage of events in each gate for each population is displayed at the top of each gate, 100 000 events from each population are displayed.
Figure 2.
Cell length distributions of sorted populations.
Sorted from the gates “short”, “long” and ”longer as defined in Figure 1. Long and longer sorted populations were re-sorted from their respective gates to yield the resort-long and resort-longer populations. Cell lengths were measured via phase contrast microscopy.
Figure 3.
Filamentous cells are effectively sorted from mixed populations by flow cytometry sorting.
(A) Mixed populations of various cell lengths were sorted from “short” and “filamentous” gates, and re-sorted from the filamentous gate. (B) Cell length distributions of populations sorted from “short” (grey bars), and “filamentous” (open bars) gates, and re-sorted from “filamentous” gate (black bars). Data was collated from 2 independent sorts each of fixed and live cells.
Figure 4.
Detection of filamentous clones in a mixed population.
Dot plots of (A) control population (EC764, short cells) and (B) a mixed population of predominantly short cells (EC764) spiked with filamentous cells (induced EC766 (ftsZ*, PBAD). Numbers at the top of each gate represent the percentage of events contained within that gate. An increase in events (filamentous cells) is observed in the “filamentous” gate between the control and mixed populations.
Figure 5.
Representative images of control cells and clones with a filamentous phenotype isolated from the screen.
(A) Control cells of strains EC766 (DH5α, pBAD24). (B–D) examples of phenotypes of E. coli clones isolated from the screen. Different degrees of filamentation were observed, along with a chaining phenotype as shown in (D). Scale bar = 10 µm.
Figure 6.
Cell length distribution of re-sorted filamentous cells from induced control EC764 (DH5α, pBAD24) and DH5α genomic library populations.
Data based on cell length measurements from 96 (control) and 75 (DH5α library) cells.
Figure 7.
Representative images of the phenotype observed for each clone listed in Table 1 with arabinose induction.
(A) Negative control EC764, (B–D) clones containing the aroB-dam geneic locus, (B) D2–3D, (C) D2–8G, (D) D2–4B. (E) D1–2H encoding yejH. (F) D2–3B and (G) D2–7H encoding ycjY. (H) D2–5G and (I) D2–7D encoding ytfA-B. (J) D2–10F encoding rplL-rpoB. (K) D1–8E encoding a region of the e14 prophage element. (L) D2–8D and (M) D1–9C encoding a region of the CP4-6 prophage element. (N) D2–7F and (O) D2–8F encoding a portion of the histidine biosynthesis operon. (P) D1–5C encoding the Kil protein from the Rac prophage element. (Q) D1–5F and (R) D2–11E encoding two distinct peptidylprolyl-cis-transisomerase genes ppiA and ppiC respectively. (S) D1–9G encoding mutT. Images taken using phase contrast, scale bars = 10 µm.
Table 1.
Genetic loci, genes and phenotypes for filamentous clones.
Table 2.
Bacterial strains used in this study.
Table 3.
Primers used in this study.