Quantification of ICE excision. (A) Localization of amplicons used for quantitative PCR. The total ICE copy number is quantified by amplification of ICE internal fragments corresponding to orfJ/orfI and orfM/orfL junctions (J/I and M/L, respectively) whereas the total chromosome number is quantified by amplification of an internal fragment of fda. The two products of excision, i.e circular ICE and chromosome devoid of ICE, are quantified by amplification of the recombination sites resulting from excision, attI and attB respectively. The star represents the putative transfer origin. (B) Effect of growth phase on excision. qPCR amplifications were performed on total DNA extracted from cells harvested during exponential growth in LM17 medium at OD600 nm = 0.2 (expo0.2) or OD600 nm = 0.6 (expo0.6) or after 1.5 hours in stationary phase (stat). (C) Effect of MMC treatment on excision. qPCR amplifications were performed on total DNA extracted from cells grown in LM17 medium treated or not (expo0.6) during 2.5 hours with MMC at MIC/2 and harvested at OD600 nm = 0.6 (MMC). Excision percentage is calculated as (attB/fda)×100. Data are presented as average and standard deviation from three independent biological replicates.
Carraro et al. BMC Microbiology 2011 11:238 doi:10.1186/1471-2180-11-238