Journal: PLoS Genetics

Article Title: P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

doi: 10.1371/journal.pgen.1005797

Figure Lengend Snippet: A. Induction of the SOS response in P1 lysogens by 8 ng/mL ciprofloxacin. E . coli strains EAR15, EAR16, and EAR17 (non-lysogen, P1 ref + lysogen, and P1 Δref lysogen, respectively) were grown at 30°C. Each strain contained a plasmid (pEAW903) expressing SuperGlo GFP from the recN promoter, allowing the SOS response to be reported as fluorescence. After reaching log phase, ciprofloxacin (8 ng/mL) or water was added (time = 0) and growth continued under the same conditions. Δfluorescence (as calculated in Methods) average and standard deviation of three biological and four technical replicates is reported. B. SOS induction by strains expressing Ref in the absence of phage P1. E . coli strains EAR86 (EV), EAR87 (pRef) EAR88 (pRefΔC110), EAR 120 (pRefΔN76), EAR121 (pRef nuc- ) and EAR123 (pRbsR) harboring protein expression plasmids and an SOS reporter plasmid were grown to log-phase at 30°C. Cultures were treated with 1% arabinose to induce protein expression (time = 0). Fluorescence and optical density were measured as in panel A. Normalized fluorescence average and standard deviation of at least two biological and two technical replicates is reported for each condition. C. Microscopy of E . coli cells expressing Ref variants. Log phase cultures of EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), EAR98 (pRefΔN76), and EAR105 (pRef nuc- ) at 1x10 8 CFU/mL were treated with 1% arabinose. Cells were outgrown for 4 hours and incubated with DAPI before imaging at 600x magnification using brightfield and fluorescence channels. Scale bar = 10 μm, representative images shown. D. Quantification of cell length data from (C) was obtained using the MicrobeTracker plugin for MatLab . Each counted cell is represented by a single data point with the average and standard deviation for the data shown. At least 100 cells were measured for each condition, except WT Ref in which only 39 cells could be found. **** = p-value <0.0001.

Article Snippet: Cell length measurements were obtained using the MicrobeTracker plugin for Matlab [ ] and were converted to μm using the conversion factor 0.1083 μm/pixel.

Techniques: Plasmid Preparation, Expressing, Fluorescence, Standard Deviation, Microscopy, Incubation, Imaging

Journal: PLoS Genetics

Article Title: P1 Ref Endonuclease: A Molecular Mechanism for Phage-Enhanced Antibiotic Lethality

doi: 10.1371/journal.pgen.1005797

Figure Lengend Snippet: A . Microscopy of cells expressing Ref or RefΔC110. WT E . coli strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), ΔrecA strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110), sulA - strains EAR77 (EV), EAR78 (pRef), EAR79 (pRefΔC110), and sulA - lexA3 strains EAR69 (EV), EAR70 (pRef), and EAR75 (pRefΔC110) were grown to 1x10 8 CFU/mL, treated with 1% arabinose, outgrown for 4 hours, and images were obtained as in . Scale bar = 10 μm, representative images shown. B. Quantification of cell length data from (A) was obtained using the MicrobeTracker plugin for MatLab. Each counted cell is represented by a single data point with the average and standard deviation for the data shown. An average of 90 cells (range: 39–188) were counted for each condition. ** = p-value <0.0001 when compared to EV in same background, * = p-value <0.05 when compared to same vector in WT background. C. Cell survival after expression of Ref. WT E . coli strains EAR61 (EV), EAR62 (pRef), EAR73 (pRefΔC110), ΔrecA strains EAR64 (EV), EAR65 (pRef), EAR74 (pRefΔC110) were treated as in (A). Cells were plated for viability and the average and standard deviation of at least three biological replicates for each condition are reported (error bars small and not visible in some cases). Significant p-values are noted in .

Article Snippet: Cell length measurements were obtained using the MicrobeTracker plugin for Matlab [ ] and were converted to μm using the conversion factor 0.1083 μm/pixel.

Techniques: Microscopy, Expressing, Standard Deviation, Plasmid Preparation