Fig. 4

PIP-treated stationary phase cells contain more PBPs and exhibit more uniform DNA gyrase supercoiling activity upon dilution into fresh media. a Cell cultures were treated with piperacillin (PIP-treated) or water (untreated) at t = 4 h. At t = 24 h, an aliquot was removed for staining with Bocillin-FL (before sample). Further, at t = 24 h, cell cultures were washed to remove piperacillin and diluted in fresh LB. Aliquots were taken at the indicated time points for staining with Bocillin-FL (t = 0, 30, and 60 min). Bocillin-FL stained samples were analyzed by flow cytometry (solid lines and filled histogram) and unstained samples were used to control for autofluorescence (dashed lines and hollow histogram). Data shown correspond to one replicate of at least three biological replicates. b Cultures of MG1655 with pQE-80 L-kan were treated with piperacillin or water at t = 4.5 h (OD₆₀₀ ~ 1). Piperacillin was removed at t = 24 h by washes in fresh LB, in the presence or absence of novobiocin, before dilution and incubation. Plasmid DNA was extracted at t = 24 h and after 5 min incubation in fresh media with or without novobiocin. Plasmid DNA concentration was determined and equal amounts of plasmid DNA were loaded onto an agarose gel containing chloroquine (top gel) and an agarose gel without intercalator as a loading control (bottom gel). Top and bottom gels were run for 21 h and 1 h, respectively. c-e Densitometry scans of untreated and PIP-treated samples that were either processed at t = 24 h (c), washed and incubated in fresh LB for 5 min (d), or washed and incubated for 5 min in fresh LB in the presence of novobiocin (NVB) prior to plasmid extraction (e). Two more replicates are presented in Additional file 8: Figure S8