This study of ICESt1 and ICESt3, showed that their respective transcriptional organization and their mobility behaviors differ. As previously proposed from sequence analyses, all genes included in the conjugation and recombination modules of the two elements were found to be transcriptionally linked and controlled by a single promoter. This organization allows a coordinated regulation of genes involved in conjugation and recombination, which are functionally associated during ICE transfer.
For ICESt1 and ICESt3 regulation module, the cI-like encoding gene and one to two genes located downstream are expressed from the convergent promoter Parp2 or from a distal conditional promoter Parp2s. The genes encoding metalloprotease (orfQ) and cI homologs belong to a different operon expressed from another promoter PorfQ. These two operons are separated by a rho-independent transcription terminator. The ICESt1 regulation module includes two independent transcriptional units. By contrast, co-transcription of all the ORFs belonging to the regulation module was observed for ICESt3. This is probably enabled by a weaker transcriptional terminator and perhaps a higher transcription level and the activation of the stationary phase promoter Parp2s. These differences probably induce ICESt3 and ICESt1 differential regulations.
The mechanisms of ICE regulation based on cI or ImmR repressors, previously described for SXT and ICEBs1, are characterized by a decrease of transcript level of the cI or immR gene and an activation of the conjugation-recombination module transcription . By contrast, in ICESt3 from S. thermophilus, a transcriptional derepression was observed for the two operons of the regulation module, whereas in ICESt1, only the transcript level of the operon containing arp1 was affected. Under all tested conditions, ICESt3 is more transcriptionally active than ICESt1. The partial derepression of transcription of the regulation module may explain the lower activation of ICESt1 (conjugation-recombination transcript level, excision, replication) compared to ICESt3. So far, ICESt1 and ICESt3 were the only known elements (ICEs and prophages) encoding homologs of both cI and ImmR repressors. The gene encoding a putative metalloprotease is generally cotranscribed and located immediately downstream from the gene encoding the ImmR repressor [12, 16]. However, in ICESt1 and ICESt3, the metalloprotease gene (orfQ) is adjacent to the cI gene (arp1) but not to the cI-like gene (arp2), suggesting that the regulation involving both cI and cI-like regulators fundamentally differs from those identified in ICEs and related elements encoding only one regulator. Genomic analyses revealed, in various streptococci, ICEs that harbor conjugation module related to the ICESt1/3 ones These elements carry a regulation module related to the ICESt1/3 ones, suggesting that they could share a similar regulation.
After MMC treatment, the transcript levels of the recombination module increases 16-fold for ICESt1 and 84-fold for ICESt3. The 10-fold increase in ICESt3 copy number, after MMC treatment, could contribute to this increase of transcript levels but is not sufficient to explain its range. MMC exposure could induce an overinitiation of DNA replication with an apparent increase in origin-proximal gene expression for a short distance (≈50 kb) , but ICESt1 and ICESt3 are out of this area on the chromosome. MMC thus stimulates ICE transfer [10, 15, 25], but also increases transcription of both ICESt3 and ICESt1.
As copy number of ICESt3 increases after MMC treatment, the quantification of the empty chromosomal integration site underestimates the level of extrachromosomal ICEs. It is worth noticing that the increase of excision after MMC exposure does not lead to an increase of ICESt1 transfer. Additionally, a similar excision level was obtained for ICESt3 in HJGL medium, although this medium does not support ICE transfer. It shows that, besides excision, additional factors affect transfer of these elements. Similarly, although prior excision is required to observe the conjugative transfer of Tn916, which is an ICE that harbors a conjugation module very distantly related to the one of ICESt1/3, the transfer frequency of this ICE is not correlated with excision .
Some preliminary results favor the hypothesis of multiple extrachromosomal copies of ICESt3 (data not shown). ICEs, as their name implies, are able to excise from their host chromosome. Then the circular extrachromosomal ICE transfers to recipient cell per conjugation and simultaneously replicates by rolling-circle mechanism. The site-specific recombination leads to integration in donor and recipient chromosomes. During division, ICE transmission to the daughter cells is thought to depend on the replication and partition of the host chromosome. However, it has been recently reported that at least some ICEs can replicate independently of their conjugative transfer. In particular, the amount of excised forms of ICEBs1 increases two- to five-fold under inducing conditions  ICEBs1 replication is initiated within oriT and is unidirectional . This replication is involved in the stability of ICEBs1 and required the relaxase encoded by the element. In silico analysis of the putative relaxases of ICESt1/3 and of ICEBs1 indicated that they are distantly related (27.4% amino acid identity for relaxase), suggesting that replication could have similar role for the two ICEs.
Furthermore, the ICE RD2 from S. pyogenes related to ICESt1/3  and the putative ICE pKLC102 from Pseudomonas aeruginosa  were reported to be simultaneously integrated and at extrachromosomal multiple copies while pP36 from Legionella pneumophila is present as a multiple extrachromosomal copies in some conditions . Whereas, in firmicutes, none of the known ICEs was found to encode a partitioning system; in proteobacteria, the ICEs belonging to pKLC102-ICEclc family encode a putative partition system [30, 31].
In its host strain CNRZ368, ICESt1 exhibits a stable copy number, even after a stimulation of its excision and core region transcription by MMC exposure. In this strain, ICESt3 excision percentage is reduced 3-fold in stationary phase and nine-fold after MMC treatment and ICESt3 copy number is not increased compared to the one observed in the strain CNRZ385. Additional factor(s) could explain these differences (excision percentage and copy number) of ICESt3 in different S. thermophilus strains. Some host factors are likely involved in key steps of the ICE behavior, like B. subtilis PolC, DnaN and PcrA for ICEBs1 replication  and IHF for SXT excision in V. cholerae . To our knowledge, our work is the first report of partial shutdown of ICE activity by a strain belonging to the primary host species.
Analysis of recently available sequences led us to identify a set of closely related putative ICEs among various streptococcal species. All of them exhibit closely related conjugation modules but highly variable recombination modules. This suggests that these elements can transfer between various streptococcal species and exchange modules between one another. However, these regulation modules all share arp2, orfQ and arp1 genes (Figure 6), suggesting a fundamental function of these 3 genes in governing transfer of this ICE family. Further investigations will be required to characterize these genes and of their functional interactions with host regulators.