Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium
Fig 3
Influence of the multicopy reporter pPcib gfp and its derivatives on expression of native S. Tm p2cib-HA at the single cell level.
S. Tm p2 cib-HA transformed with pPcib gfp or the derivatives (pcontrol, pPcib, pgfp and pPrpsm gfp), respectively, were grown in LB (+ampicillin) overnight (12h; stationary phase) or subcultured for 4h in LB (late logarithmic phase) with or without indicated supplements (0.25μg/ml MitC, 100μM DTPA). Bacteria were fixed, permeabilized and intracellular ColIb-HA was detected using HA-specific antiserum and a DyLight 594-conjugated secondary antibody, DNA was stained with DAPI. Bacteria were imaged by confocal microscopy and fluorescence of DAPI, GFP and DyLight 594-conjugate was recorded. Mean fluorescence intensity (MFI) of ColIb-HA-DyLight594 (A,C,E,G,I) and GFP (B,D,F,H,J) as determined by ImageJ is shown. Dots represent MFI of individual objects (bacteria). Bars represent the median and the dotted line the detection limit (background fluorescence of S. Tmwt pcontrol). Values indicate the fraction (%) of the population above detection limit (blue). Statistical analysis was done using Kruskal-Wallis test with Dunn's post test (* p < 0.05).