The ICESt3 precise start point could not be deduced from 5′RACE experiments because all the obtained products ended in a region located 100 bp downstream from the corresponding start point of ICESt1. For ICESt1, several 5′RACE products also ended in this region. mFold software analysis [19] revealed a conserved putative stem loop structure (ΔG = -6.7 kcal.mol-1

for ICESt1 and ΔG = -6.4 kcal.mol-1 for ICESt3), which could affect RNA stability. Although it could not be experimentally demonstrated, we propose, based on sequence conservation (Akt activity Figure 1B), a GW2580 same location of the Pcr promoter for ICESt3 and ICESt1. Figure 2 Transcriptional analysis of the arp2 / orfM region of ICE St3. (A) Schematic representation of the arp2/orfM intergenic region of ICESt3. Primers used for PCR analysis are represented by triangles see more and promoters are represented by angled arrows. (B) RT-PCR mapping Pcr promoter of ICESt3. Amplicons are generated with primers mentioned

above the gels on genomic DNA (gDNA) or cDNA synthesized from RNA extracted from cells in exponential growth phase (expo0.6). Amplicon size is given on the left. Results were identical for three independent biological replicates. (C) RT-PCR mapping Parp2 promoter of ICESt3. Amplicons are generated with primers mentioned on the left of the gels on genomic DNA (gDNA) or cDNA synthesized from RNA extracted from exponential growth phase (expo0.6) and stationary phase (stat) cells. The transcriptional activity upstream from the Parp2 promoter was detected during stationary phase. Results were identical for three independent biological replicates. For both elements, the functionality of the predicted arp2 promoter Parp2 was established with a (A) start site located seven nucleotides downstream from a -10 box (TACAAT) (Figure 1B). For both ICEs, transcriptional

analyses showed that all the promoters (Pcr, PorfQ and Parp2), which are active during the stationary phase, are also active during exponential the growth phase Endonuclease (data not shown). However, an additional promoter was identified in ICESt3 upstream from the Parp2 promoter during stationary phase. Amplicons were obtained using arp2.f/r3 and arp2.f/r4 primers (Figure 2C). 5′RACE experiments revealed a start site located within a (A)6 stretch in this region (between the r4 and r5 primers, Figure 2C). Therefore, an alternative transcript originating from a distal arp2 promoter in ICESt3 (called “”Parp2s”") is expressed during the stationary phase (Figure 1C). This promoter does not match the classical promoter consensus as its -35 (TTATCA) and -10 (TGTAAT) boxes are separated by only 15 nucleotides (Figure 1C). The functionality of this promoter was highlighted only during stationary phase (Figure 2C) and only in ICESt3 (data not shown), although its sequence is strictly identical in ICESt1 (Figure 1C).