Regardless of conditions, no amplification was detected at the junction between the two operons (orfQ/orfP junction), which corroborates the lack of cotranscription of these

genes. For ICESt3, the level of arp1 and orf385A/arp2 transcripts increased after MMC treatment (40-fold) and in stationary phase (about 10-fold) (Figure 3B). Co-transcription of the two operons was quantified by considering the orfQ/orf385B Fedratinib order junction. During exponential growth phase and MMC exposure, co-transcription represented 20 and 38% of transcripts respectively, indicating that the terminator and the promoter PorfQ were active. However, in stationary phase, the amount of this junction was similar to that of the two operons, probably selleck inhibitor reflecting an activity of the Parp2s promoter. After MMC exposure during

stationary phase, transcript quantities were found to be similar to the ones observed in stationary phase without MMC. Therefore, MMC has an impact on DNA metabolism (lower level of DNA) during stationary phase but does not affect levels or organization of transcripts (data not shown). Growth phase and mitomycin C affect ICESt1 and ICESt3 excision Excision is the first step of ICE transfer from host chromosome to a recipient cell, leading to a circular intermediate and an empty chromosomal integration site, attB (Figure 4A). The influence of the growth phase (early, mid exponential growth phase or stationary phase) and MMC treatment on ICE excision was analyzed by quantitative PCR on genomic

DNA. The excision percentage was calculated as the copy number of attB sites per fda copy (adjacent chromosomal locus). As a control, the amount of attB sites was determined in strain CNRZ368ΔICESt1 (X. Bellanger unpublished data) and in CNRZ385ΔICESt3 [21] and was found equal to the amount of fda. Figure 4 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 C1GALT1 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 Selleck Akt inhibitor 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.