BACKGROUND
acquisition of genomic islands  plays a key role in bacterial evolution  <cit> .
in silico analyses revealed that numerous gis probably belong to integrative and conjugative elements  or are ice-deriving elements  <cit> .
ices, including conjugative transposons, were defined as autonomous mobile elements that encode the functions needed for their excision, conjugative transfer and integration  <cit> .
cis-acting sequences and genes involved in a same biological process  are generally grouped in a module, such as orit and genes encoding relaxosome and conjugation pore.
the recombination, conjugation and regulation modules are frequently grouped to form the core region of the ices.
although ices replicate during their conjugative transfer, it was originally assumed that they are incapable of autonomous intracellular replication and that their maintenance during cell growth and division only relies on their integration in the chromosome.
besides one or few core regions, they also harbor highly variable regions that encode functions potentially useful for the bacterial host  <cit> .
comparison of the organization of related ices, such as tn <dig> and its close relatives, revealed that they evolve by deletion, acquisition and/or exchange of modules.
the conjugation, tetracycline resistance and regulation modules of tn <dig> and tn <dig> are closely related whereas their recombination modules are unrelated  <cit> .
likewise, the tn <dig> recombination module is closely related to the one of tn <dig>  but their conjugation and resistance modules are unrelated  <cit> .
the closely related ices of the lactic acid bacterium streptococcus thermophilus, icest <dig> and icest <dig>  are integrated within the 3' end of the fda gene encoding a putative fructose  <dig>  6-diphosphate aldolase  <cit> .
they carry recombination and conjugation modules that are almost identical , related regulation modules  and various modules that could be advantageous for their hosts .
their conjugation modules are very distantly related to modules of a large group of ices found in firmicutes, including tn <dig> and icebs <dig>  <cit> .
as the conjugative transfer of icest <dig> occurs at a frequency one thousand times lower than that of icest <dig>  their divergent regulation modules might be involved in these very different transfer activities  <cit> .
the activity of almost all prophages and at least some ices is controlled by a central repressor that can belong to two unrelated families, either ci or immr .
both types of repressor carry a hth xre domain that allows their binding to promoter sequences upstream from their target genes.
transfer of the element requires the inactivation of the corresponding regulator, as shown during the reca-dependent sos response  <cit>  of many ci-encoding prophages and two ices, sxt from vibrio cholerae  <cit>  and icebs <dig> from bacillus subtilis  <cit> , which encode respectively a ci and an immr repressor.
derepression of the ice is due to the cleavage of the transcriptional regulator catalyzed by either the ci autopeptidase function  <cit>  or a metalloprotease encoded by a gene adjacent to the gene encoding immr  <cit> .
previous studies showed that various stimuli can activate ices, such as antibiotic treatment, cell density, stationary phase, dna damage or presence of chlorocatechol  <cit> .
within the regulation module of icest <dig> and icest <dig>  genes encoding homologs of ci  and immr  and its associated protease  were identified.
icest <dig> and icest <dig> are the only two characterized elements which encode both ci and immr repressors, suggesting a novel and complex regulatory mechanism.
in order to explain the differences of transfer frequency previously observed for icest <dig> and icest <dig> of s. thermophilus, a transcriptional mapping of these elements was undertaken.
furthermore their excision/replication rates were investigated in different conditions .
finally the influence of the host background was also explored.
these experiments revealed that the two ices harbor closely related core regions, differ in their transcriptional organization and regulation.
they provide further evidence of ice replication.
our results also pointed out an impact of host cell on the ice behavior.
RESULTS
transcriptional organization and promoter analyses of the icest <dig> and icest <dig> core region
previous sequences analyses suggested that the thirteen orfs belonging to the conjugation module and the genes encoding the excisionase and integrase  of icest1/ <dig> could be transcribed as a unique polycistronic mrna while the regulation module could have a two-operon organization  <cit> .
gene organization, position of predicted promoters and rho-independent transcription terminators of the icest1/ <dig> core region are schematically presented in the figure  <dig>  as some ice activities were reported to be affected by growth phase and/or cell density  <cit> , cnrz <dig> and cnrz <dig>  strains carrying icest <dig> and icest <dig> respectively, were harvested in exponential growth phase as well as in stationary phase for total rna extraction and subsequent transcriptional organization studies.
to determine which genes were co-transcribed, rt-pcr amplification of core region was performed by grouping orfs two by two or three by three.
for icest <dig>  amplifications of orfr/arp1/orfq and orfp/arp <dig>  respectively, were positive while that of the orfq/orfp junction was negative .
these data comfort the hypothesis of a two-operon organization for icest <dig>  with a functional rho-independent transcription terminator located between the two operons.
by contrast, for icest <dig>  all the rt-pcr amplifications of the regulation module were positive  indicating a co-transcription of all the regulation genes .
the free energy of the transcriptional terminator detected between orf385b and orfq genes in icest <dig>  was calculated with the mfold software  <cit> .
it is different from the one for icest <dig> .
this difference could explain why all genes of the regulation module of icest <dig> can be co-transcribed while two independent transcriptional units were found in icest <dig>
we then examined the activity of the promoter located upstream from the orfq gene by rapid amplification of cdna ends .
for both elements, the start point  was located seven nucleotides downstream from a - <dig> box separated by  <dig> nt from a - <dig> box, which overlapped the rho-independent transcription terminator .
this result is consistent with the s. thermophilus promoter consensus sequence   <cit> .
therefore, both ices possess a functional porfq promoter.
however, it was previously showed that icest <dig> differs from icest <dig> by a - <dig> frameshift in the 5' end of its orfq gene   <cit> .
a second rbs, that could enable the translation from an initiation codon located downstream, was identified in silico .
all together, these data suggest that the orfq <dig> gene of icest <dig> is truncated of  <dig> nucleotides at its 5' end compared to the orfq gene of icest <dig>
all rt-pcr amplifications targeting co-transcription of the sixteen conjugation-recombination genes of icest <dig> and icest <dig> gave amplicons .
therefore, these genes are transcribed as a single polycistronic mrna of about  <dig>  kb .
to map more precisely the 5' end of these transcripts, other sets of primers were designed in the arp2/orfn intergenic region.
for icest <dig>  these results  combined with 5' race experiments confirmed the predicted conjugation-recombination promoter, pcr, with a - <dig> box  located seven nucleotides upstream from the transcription start point  nucleotide .
rt-pcr experiments also localized the icest <dig> pcr promoter in the same region, between the f <dig> and f <dig> primers .
the icest <dig> precise start point could not be deduced from 5'race experiments because all the obtained products ended in a region located  <dig> bp downstream from the corresponding start point of icest <dig>  for icest <dig>  several 5'race products also ended in this region.
mfold software analysis  <cit>  revealed a conserved putative stem loop structure , which could affect rna stability.
although it could not be experimentally demonstrated, we propose, based on sequence conservation , a same location of the pcr promoter for icest <dig> and icest <dig>
for both elements, the functionality of the predicted arp <dig> promoter parp <dig> was established with a  start site located seven nucleotides downstream from a - <dig> box  .
for both ices, transcriptional analyses showed that all the promoters , which are active during the stationary phase, are also active during exponential the growth phase .
however, an additional promoter was identified in icest <dig> upstream from the parp <dig> promoter during stationary phase.
amplicons were obtained using arp <dig> f/r <dig> and arp <dig> f/r <dig> primers .
5'race experiments revealed a start site located within a  <dig> stretch in this region .
therefore, an alternative transcript originating from a distal arp <dig> promoter in icest <dig>  is expressed during the stationary phase .
this promoter does not match the classical promoter consensus as its - <dig>  and - <dig>  boxes are separated by only  <dig> nucleotides .
the functionality of this promoter was highlighted only during stationary phase  and only in icest <dig> , although its sequence is strictly identical in icest <dig> .
sequence analyses failed to detect any orf in the  <dig> nucleotides between the parp2s and parp <dig> promoters.
taken together, these data demonstrate that icest <dig> and icest <dig> do not share the same transcriptional organization of their regulation module: icest <dig> is organized as two operons, while in icest <dig> the whole module can be co-transcribed.
furthermore, icest <dig> possesses an additional distal promoter upstream the module, which is activated during stationary phase.
growth phase and mmc exposure modulate the transcription of the icest <dig> and icest <dig> core genes
previous analyses showed a derepression of conjugative transfer of icest <dig> but not of icest <dig> after exposure to mitomycin c   <cit> .
in order to explain this difference, we quantified by real-time rt-pcr, three regions  of the conjugation-recombination transcript of icest <dig> and icest <dig>
quantification was done from cells harvested in exponential growth phase treated or not with mmc at the half of the minimal inhibitory concentration  as well as in stationary phase .
of note, in preliminary experiments, mmc exposure did not affect the transcriptional organization , cell morphology or chain length but, as expected for a dna damaging agent, it delayed growth, reduced dna quantity and increased reca transcript levels .
transcription of the icest <dig> conjugation-recombination modules was found up-regulated upon dna damage  and in stationary phase  compared to exponential growth phase without mmc treatment .
the same observation was made for icest <dig> with a 84-fold and 11-fold increase of int transcript levels after mmc treatment and stationary phase, respectively , indicating a probable transcriptional regulation of ice excision.
whatever the considered region of the conjugation-recombination transcript, higher amounts were found for icest <dig> than for icest <dig> .
for both elements, quantitative rt-pcr was also performed on three loci of the regulation module .
in icest <dig>  the amount of arp2-orfp transcripts was similar whatever the conditions considered, while the amount of arp <dig> transcripts increased 10-fold after mmc treatment .
regardless of conditions, no amplification was detected at the junction between the two operons , which corroborates the lack of cotranscription of these genes.
for icest <dig>  the level of arp <dig> and orf385a/arp <dig> transcripts increased after mmc treatment  and in stationary phase  .
co-transcription of the two operons was quantified by considering the orfq/orf385b junction.
during exponential growth phase and mmc exposure, co-transcription represented  <dig> 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 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  during stationary phase but does not affect levels or organization of transcripts .
growth phase and mitomycin c affect icest <dig> and icest <dig> 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 .
the influence of the growth 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 .
as a control, the amount of attb sites was determined in strain cnrz368Δicest <dig>  and in cnrz385Δicest <dig>  <cit>  and was found equal to the amount of fda.
the excision percentage of icest <dig> was found seven-fold higher than the one of icest <dig> in exponential growth phase , consistent with the higher level of icest <dig> conjugation-recombination transcript , and its higher transfer frequency  <cit> .
for both ices, excision frequency was higher in stationary phase compared to exponential growth phase .
for these experiments, cells were grown in lm <dig> rich medium, in which transfer has been demonstrated  <cit> .
a similar excision rate of icest <dig> was measured in another rich medium  that do not support the transfer of the two ices .
therefore, the lack of icest <dig> transfer in this medium can not be due to a low excision level.
transcriptional analyses have shown an increase of core transcript level for icest <dig> and icest <dig> after mmc treatment during exponential growth.
this dna damaging agent leads to an increase of excision percentage up to 90% for icest <dig>  but only  <dig> % for icest <dig> .
however, the increase is higher for icest <dig>  compare to icest <dig> .
therefore, under all tested conditions, icest <dig> is more active in excision than icest <dig>
dna damage induces replication of icest3
quantitative pcr was performed to measure the amounts of excised and integrated ices at different growth phases and after mmc treatment.
according to the previously proposed ice model  atti and attb were expected to have the same copy number after ice excision.
this was found for both ices whatever the tested conditions, except for icest <dig> dna extracted from strain cnrz <dig> exposed to mmc .
to confirm this data, the orfm/orfl junction localized in the conjugation module was quantified and normalized to levels of different chromosomal loci: fda, dnaa and xers .
the same result was obtained with an amount of m/l reaching about nine-fold the one of fda .
as fda is adjacent to integrated icest <dig> and replicates prior to the ice during host chromosome replication, icest <dig> could be able to replicate autonomously under this condition.
different loci along ices  were quantified at similar levels  and thus did not allow us to propose a replicative mechanism .
icest <dig> excision and replication depend on the host strain
to test the icest <dig> behavior in different s. thermophilus strain background, its excision percentage × <dig> and copy number  were quantified.
icest <dig> was transferred by conjugation to lmg <dig>  a strain initially devoid of ice and in cnrz368Δicest <dig>  the strain that originally carries icest <dig> but has been deleted of it.
icest <dig> excision percentage was lower in strain lmg <dig> and much lower in cnrz368Δicest <dig> compared to that observed in cnrz <dig> strain but mmc treatment increased its excision percentage in all strains .
in cnrz <dig>  excision rates of icest <dig> were higher than those of icest <dig> .
furthermore, the quantification showed a single copy of icest <dig>  per chromosome even after mmc exposure .
this indicates a preponderant effect of the host strain on the ice replication.
a family of streptococcal ices shares related regulation and conjugation modules
protein and nucleic acid sequences from the regulation, conjugation and recombination modules of icest <dig> and icest <dig> were compared with sequences from firmicutes.
closely related conjugation modules  were found in the putative icespn <dig> from s. pneumoniae  <dig>  <cit>  and in the partially or completely sequenced genomes of s. parasanguinis atcc <dig> and f <dig>  s. infantis atcc  <dig> and s. australis atcc <dig> .
all these conjugation modules are adjacent to putative recombination modules that are unrelated or very distantly related to the ones of icest1/ <dig> .
nevertheless, they could be cotranscribed with the conjugation module from a pcr promoter similar to the one identified above since it is present at the same position as in icest1/ <dig> with high sequence conservation .
therefore, these conjugation-recombination modules probably belong to non identified ices.
all these putative elements harbor closely related regulation modules that would be transcribed divergently from the conjugation and recombination modules.
all these modules possess a similar organization and encode putative ci repressors, immr repressors and metalloproteases related to the ones of icest1/ <dig>  and one to four unrelated proteins .
sequence comparison of the intergenic core regions of the closely related streptococci ices revealed similar regulatory signals at the same positions as in icest1/ <dig> with high sequence conservation , suggesting a similar regulation.
more distantly related conjugation modules  are found not only in previously described elements - rd <dig> from s. pyogenes  <cit>  and four elements integrated in a trnalys gene from four s. agalactiae strains  <cit>  - but also in novel putative ices that we found in various streptococci including s. agalactiae atcc <dig> , s. dysgalactiae atcc <dig> , s. downei f <dig>  streptococcus sp.
2_1_36faa and s. gallolyticus ucn <dig>  only the elements found in s. dysgalactiae encode a putative ci repressor, immr repressor and metalloprotease.
discussion
this study of icest <dig> and icest <dig>  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 icest <dig> and icest <dig> regulation module, the ci-like encoding gene and one to two genes located downstream are expressed from the convergent promoter parp <dig> or from a distal conditional promoter parp2s.
the genes encoding metalloprotease  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 icest <dig> regulation module includes two independent transcriptional units.
by contrast, co-transcription of all the orfs belonging to the regulation module was observed for icest <dig>  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 icest <dig> and icest <dig> differential regulations.
the mechanisms of ice regulation based on ci or immr repressors, previously described for sxt and icebs <dig>  are characterized by a decrease of transcript level of the ci or immr gene and an activation of the conjugation-recombination module transcription  <cit> .
by contrast, in icest <dig> from s. thermophilus, a transcriptional derepression was observed for the two operons of the regulation module, whereas in icest <dig>  only the transcript level of the operon containing arp <dig> was affected.
under all tested conditions, icest <dig> is more transcriptionally active than icest <dig>  the partial derepression of transcription of the regulation module may explain the lower activation of icest <dig>  compared to icest <dig>  so far, icest <dig> and icest <dig> were the only known elements  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  <cit> .
however, in icest <dig> and icest <dig>  the metalloprotease gene  is adjacent to the ci gene  but not to the ci-like gene , 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/ <dig> ones these elements carry a regulation module related to the icest1/ <dig> ones, suggesting that they could share a similar regulation.
after mmc treatment, the transcript levels of the recombination module increases 16-fold for icest <dig> and 84-fold for icest <dig>  the 10-fold increase in icest <dig> 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   <cit> , but icest <dig> and icest <dig> are out of this area on the chromosome.
mmc thus stimulates ice transfer  <cit> , but also increases transcription of both icest <dig> and icest <dig>
as copy number of icest <dig> 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 icest <dig> transfer.
additionally, a similar excision level was obtained for icest <dig> 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 tn <dig>  which is an ice that harbors a conjugation module very distantly related to the one of icest1/ <dig>  the transfer frequency of this ice is not correlated with excision  <cit> .
some preliminary results favor the hypothesis of multiple extrachromosomal copies of icest <dig> .
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 icebs <dig> increases two- to five-fold under inducing conditions  <cit>  icebs <dig> replication is initiated within orit and is unidirectional  <cit> .
this replication is involved in the stability of icebs <dig> and required the relaxase encoded by the element.
in silico analysis of the putative relaxases of icest1/ <dig> and of icebs <dig> indicated that they are distantly related , suggesting that replication could have similar role for the two ices.
furthermore, the ice rd <dig> from s. pyogenes related to icest1/ <dig>  <cit>  and the putative ice pklc <dig> from pseudomonas aeruginosa  <cit>  were reported to be simultaneously integrated and at extrachromosomal multiple copies while pp <dig> from legionella pneumophila is present as a multiple extrachromosomal copies in some conditions  <cit> .
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  <cit> .
in its host strain cnrz <dig>  icest <dig> exhibits a stable copy number, even after a stimulation of its excision and core region transcription by mmc exposure.
in this strain, icest <dig> excision percentage is reduced 3-fold in stationary phase and nine-fold after mmc treatment and icest <dig> copy number is not increased compared to the one observed in the strain cnrz <dig>  additional factor could explain these differences  of icest <dig> 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 icebs <dig> replication  <cit>  and ihf for sxt excision in v. cholerae  <cit> .
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 arp <dig>  orfq and arp <dig> genes , suggesting a fundamental function of these  <dig> 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.
CONCLUSIONS
in conclusion, the transcriptional organization of the conjugation and recombination modules of two closely related ices from s. thermophilus, icest <dig> and icest <dig>  is identical, while that of their regulation module is somewhat different.
transcripts of core region and excision levels are higher for icest <dig>  which is consistent with its higher transfer frequency.
despite these differences, the excision of both ices is stimulated by exposure to a dna damaging agent and stationary phase.
data generated by the transcriptional study suggest a new mechanism of regulation of icest1/ <dig>  this behavior could be due to the atypical regulation module of these elements that encode homologues of both ci and immr repressors.
analyses of sequenced genomes revealed, among streptococci, a family of ices that encode ci and immr homologs and therefore could share similar regulation.
furthermore, our results suggest that dna damage induces not only the excision and transfer of icest <dig> but also its intracellular replication.
this characteristic, which is not considered in the initial ice model, may be shared by other ices.
this study also revealed that icest <dig> has very different behaviors depending on its primary host species, suggesting a major role of host factor in its excision and replication.

 acquisition of genomic islands plays a key role in bacterial evolution. in silico analyses revealed that numerous gis probably belong to integrative and conjugative elements or are ice-deriving elements. ices, including conjugative transposons, were defined as autonomous mobile elements that encode the functions needed for their excision, conjugative transfer and integration. cis-acting sequences and genes involved in a same biological process are generally grouped in a module, such as orit and genes encoding relaxosome and conjugation pore. the recombination, conjugation and regulation modules are frequently grouped to form the core region of the ices. although ices replicate during their conjugative transfer, it was originally assumed that they are incapable of autonomous intracellular replication and that their maintenance during cell growth and division only relies on their integration in the chromosome. besides one or few core regions, they also harbor highly variable regions that encode functions potentially useful for the bacterial host. comparison of the organization of related ices, such as tn and its close relatives, revealed that they evolve by deletion, acquisition and/or exchange of modules. the conjugation, tetracycline resistance and regulation modules of tn and tn are closely related whereas their recombination modules are unrelated. likewise, the tn recombination module is closely related to the one of tn but their conjugation and resistance modules are unrelated. the closely related ices of the lactic acid bacterium streptococcus thermophilus, icest and icest are integrated within the 3' end of the fda gene encoding a putative fructose 6-diphosphate aldolase. they carry recombination and conjugation modules that are almost identical, related regulation modules and various modules that could be advantageous for their hosts. their conjugation modules are very distantly related to modules of a large group of ices found in firmicutes, including tn and icebs. as the conjugative transfer of icest occurs at a frequency one thousand times lower than that of icest their divergent regulation modules might be involved in these very different transfer activities. the activity of almost all prophages and at least some ices is controlled by a central repressor that can belong to two unrelated families, either ci or immr. both types of repressor carry a hth xre domain that allows their binding to promoter sequences upstream from their target genes. transfer of the element requires the inactivation of the corresponding regulator, as shown during the reca-dependent sos response of many ci-encoding prophages and two ices, sxt from vibrio cholerae and icebs from bacillus subtilis, which encode respectively a ci and an immr repressor. derepression of the ice is due to the cleavage of the transcriptional regulator catalyzed by either the ci autopeptidase function or a metalloprotease encoded by a gene adjacent to the gene encoding immr. previous studies showed that various stimuli can activate ices, such as antibiotic treatment, cell density, stationary phase, dna damage or presence of chlorocatechol. within the regulation module of icest and icest genes encoding homologs of ci and immr and its associated protease were identified. icest and icest are the only two characterized elements which encode both ci and immr repressors, suggesting a novel and complex regulatory mechanism. in order to explain the differences of transfer frequency previously observed for icest and icest of s. thermophilus, a transcriptional mapping of these elements was undertaken. furthermore their excision/replication rates were investigated in different conditions. finally the influence of the host background was also explored. these experiments revealed that the two ices harbor closely related core regions, differ in their transcriptional organization and regulation. they provide further evidence of ice replication. our results also pointed out an impact of host cell on the ice behavior.  transcriptional organization and promoter analyses of the icest and icest core region previous sequences analyses suggested that the thirteen orfs belonging to the conjugation module and the genes encoding the excisionase and integrase of icest1/ could be transcribed as a unique polycistronic mrna while the regulation module could have a two-operon organization. gene organization, position of predicted promoters and rho-independent transcription terminators of the icest1/ core region are schematically presented in the figure as some ice activities were reported to be affected by growth phase and/or cell density, cnrz and cnrz strains carrying icest and icest respectively, were harvested in exponential growth phase as well as in stationary phase for total rna extraction and subsequent transcriptional organization studies. to determine which genes were co-transcribed, rt-pcr amplification of core region was performed by grouping orfs two by two or three by three. for icest amplifications of orfr/arp1/orfq and orfp/arp respectively, were positive while that of the orfq/orfp junction was negative. these data comfort the hypothesis of a two-operon organization for icest with a functional rho-independent transcription terminator located between the two operons. by contrast, for icest all the rt-pcr amplifications of the regulation module were positive indicating a co-transcription of all the regulation genes. the free energy of the transcriptional terminator detected between orf385b and orfq genes in icest was calculated with the mfold software. it is different from the one for icest. this difference could explain why all genes of the regulation module of icest can be co-transcribed while two independent transcriptional units were found in icest we then examined the activity of the promoter located upstream from the orfq gene by rapid amplification of cdna ends. for both elements, the start point was located seven nucleotides downstream from a - box separated by nt from a - box, which overlapped the rho-independent transcription terminator. this result is consistent with the s. thermophilus promoter consensus sequence. therefore, both ices possess a functional porfq promoter. however, it was previously showed that icest differs from icest by a - frameshift in the 5' end of its orfq gene. a second rbs, that could enable the translation from an initiation codon located downstream, was identified in silico. all together, these data suggest that the orfq gene of icest is truncated of nucleotides at its 5' end compared to the orfq gene of icest all rt-pcr amplifications targeting co-transcription of the sixteen conjugation-recombination genes of icest and icest gave amplicons. therefore, these genes are transcribed as a single polycistronic mrna of about kb. to map more precisely the 5' end of these transcripts, other sets of primers were designed in the arp2/orfn intergenic region. for icest these results combined with 5' race experiments confirmed the predicted conjugation-recombination promoter, pcr, with a - box located seven nucleotides upstream from the transcription start point nucleotide. rt-pcr experiments also localized the icest pcr promoter in the same region, between the f and f primers. the icest precise start point could not be deduced from 5'race experiments because all the obtained products ended in a region located bp downstream from the corresponding start point of icest for icest several 5'race products also ended in this region. mfold software analysis revealed a conserved putative stem loop structure, which could affect rna stability. although it could not be experimentally demonstrated, we propose, based on sequence conservation, a same location of the pcr promoter for icest and icest for both elements, the functionality of the predicted arp promoter parp was established with a start site located seven nucleotides downstream from a - box. for both ices, transcriptional analyses showed that all the promoters, which are active during the stationary phase, are also active during exponential the growth phase. however, an additional promoter was identified in icest upstream from the parp promoter during stationary phase. amplicons were obtained using arp f/r and arp f/r primers. 5'race experiments revealed a start site located within a stretch in this region. therefore, an alternative transcript originating from a distal arp promoter in icest is expressed during the stationary phase. this promoter does not match the classical promoter consensus as its - and - boxes are separated by only nucleotides. the functionality of this promoter was highlighted only during stationary phase and only in icest, although its sequence is strictly identical in icest. sequence analyses failed to detect any orf in the nucleotides between the parp2s and parp promoters. taken together, these data demonstrate that icest and icest do not share the same transcriptional organization of their regulation module: icest is organized as two operons, while in icest the whole module can be co-transcribed. furthermore, icest possesses an additional distal promoter upstream the module, which is activated during stationary phase. growth phase and mmc exposure modulate the transcription of the icest and icest core genes previous analyses showed a derepression of conjugative transfer of icest but not of icest after exposure to mitomycin c. in order to explain this difference, we quantified by real-time rt-pcr, three regions of the conjugation-recombination transcript of icest and icest quantification was done from cells harvested in exponential growth phase treated or not with mmc at the half of the minimal inhibitory concentration as well as in stationary phase. of note, in preliminary experiments, mmc exposure did not affect the transcriptional organization, cell morphology or chain length but, as expected for a dna damaging agent, it delayed growth, reduced dna quantity and increased reca transcript levels. transcription of the icest conjugation-recombination modules was found up-regulated upon dna damage and in stationary phase compared to exponential growth phase without mmc treatment. the same observation was made for icest with a 84-fold and 11-fold increase of int transcript levels after mmc treatment and stationary phase, respectively, indicating a probable transcriptional regulation of ice excision. whatever the considered region of the conjugation-recombination transcript, higher amounts were found for icest than for icest. for both elements, quantitative rt-pcr was also performed on three loci of the regulation module. in icest the amount of arp2-orfp transcripts was similar whatever the conditions considered, while the amount of arp transcripts increased 10-fold after mmc treatment. regardless of conditions, no amplification was detected at the junction between the two operons, which corroborates the lack of cotranscription of these genes. for icest the level of arp and orf385a/arp transcripts increased after mmc treatment and in stationary phase. co-transcription of the two operons was quantified by considering the orfq/orf385b junction. during exponential growth phase and mmc exposure, co-transcription represented 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 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 during stationary phase but does not affect levels or organization of transcripts. growth phase and mitomycin c affect icest and icest 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. the influence of the growth 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. as a control, the amount of attb sites was determined in strain cnrz368Δicest and in cnrz385Δicest and was found equal to the amount of fda. the excision percentage of icest was found seven-fold higher than the one of icest in exponential growth phase, consistent with the higher level of icest conjugation-recombination transcript, and its higher transfer frequency. for both ices, excision frequency was higher in stationary phase compared to exponential growth phase. for these experiments, cells were grown in lm rich medium, in which transfer has been demonstrated. a similar excision rate of icest was measured in another rich medium that do not support the transfer of the two ices. therefore, the lack of icest transfer in this medium can not be due to a low excision level. transcriptional analyses have shown an increase of core transcript level for icest and icest after mmc treatment during exponential growth. this dna damaging agent leads to an increase of excision percentage up to 90% for icest but only % for icest. however, the increase is higher for icest compare to icest. therefore, under all tested conditions, icest is more active in excision than icest dna damage induces replication of icest3 quantitative pcr was performed to measure the amounts of excised and integrated ices at different growth phases and after mmc treatment. according to the previously proposed ice model atti and attb were expected to have the same copy number after ice excision. this was found for both ices whatever the tested conditions, except for icest dna extracted from strain cnrz exposed to mmc. to confirm this data, the orfm/orfl junction localized in the conjugation module was quantified and normalized to levels of different chromosomal loci: fda, dnaa and xers. the same result was obtained with an amount of m/l reaching about nine-fold the one of fda. as fda is adjacent to integrated icest and replicates prior to the ice during host chromosome replication, icest could be able to replicate autonomously under this condition. different loci along ices were quantified at similar levels and thus did not allow us to propose a replicative mechanism. icest excision and replication depend on the host strain to test the icest behavior in different s. thermophilus strain background, its excision percentage × and copy number were quantified. icest was transferred by conjugation to lmg a strain initially devoid of ice and in cnrz368Δicest the strain that originally carries icest but has been deleted of it. icest excision percentage was lower in strain lmg and much lower in cnrz368Δicest compared to that observed in cnrz strain but mmc treatment increased its excision percentage in all strains. in cnrz excision rates of icest were higher than those of icest. furthermore, the quantification showed a single copy of icest per chromosome even after mmc exposure. this indicates a preponderant effect of the host strain on the ice replication. a family of streptococcal ices shares related regulation and conjugation modules protein and nucleic acid sequences from the regulation, conjugation and recombination modules of icest and icest were compared with sequences from firmicutes. closely related conjugation modules were found in the putative icespn from s. pneumoniae and in the partially or completely sequenced genomes of s. parasanguinis atcc and f s. infantis atcc and s. australis atcc. all these conjugation modules are adjacent to putative recombination modules that are unrelated or very distantly related to the ones of icest1/. nevertheless, they could be cotranscribed with the conjugation module from a pcr promoter similar to the one identified above since it is present at the same position as in icest1/ with high sequence conservation. therefore, these conjugation-recombination modules probably belong to non identified ices. all these putative elements harbor closely related regulation modules that would be transcribed divergently from the conjugation and recombination modules. all these modules possess a similar organization and encode putative ci repressors, immr repressors and metalloproteases related to the ones of icest1/ and one to four unrelated proteins. sequence comparison of the intergenic core regions of the closely related streptococci ices revealed similar regulatory signals at the same positions as in icest1/ with high sequence conservation, suggesting a similar regulation. more distantly related conjugation modules are found not only in previously described elements - rd from s. pyogenes and four elements integrated in a trnalys gene from four s. agalactiae strains - but also in novel putative ices that we found in various streptococci including s. agalactiae atcc, s. dysgalactiae atcc, s. downei f streptococcus sp. 2_1_36faa and s. gallolyticus ucn only the elements found in s. dysgalactiae encode a putative ci repressor, immr repressor and metalloprotease. discussion this study of icest and icest 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 icest and icest regulation module, the ci-like encoding gene and one to two genes located downstream are expressed from the convergent promoter parp or from a distal conditional promoter parp2s. the genes encoding metalloprotease 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 icest regulation module includes two independent transcriptional units. by contrast, co-transcription of all the orfs belonging to the regulation module was observed for icest 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 icest and icest differential regulations. the mechanisms of ice regulation based on ci or immr repressors, previously described for sxt and icebs 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 icest from s. thermophilus, a transcriptional derepression was observed for the two operons of the regulation module, whereas in icest only the transcript level of the operon containing arp was affected. under all tested conditions, icest is more transcriptionally active than icest the partial derepression of transcription of the regulation module may explain the lower activation of icest compared to icest so far, icest and icest were the only known elements 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. however, in icest and icest the metalloprotease gene is adjacent to the ci gene but not to the ci-like gene, 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/ ones these elements carry a regulation module related to the icest1/ ones, suggesting that they could share a similar regulation. after mmc treatment, the transcript levels of the recombination module increases 16-fold for icest and 84-fold for icest the 10-fold increase in icest 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, but icest and icest are out of this area on the chromosome. mmc thus stimulates ice transfer, but also increases transcription of both icest and icest as copy number of icest 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 icest transfer. additionally, a similar excision level was obtained for icest 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 tn which is an ice that harbors a conjugation module very distantly related to the one of icest1/ the transfer frequency of this ice is not correlated with excision. some preliminary results favor the hypothesis of multiple extrachromosomal copies of icest. 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 icebs increases two- to five-fold under inducing conditions icebs replication is initiated within orit and is unidirectional. this replication is involved in the stability of icebs and required the relaxase encoded by the element. in silico analysis of the putative relaxases of icest1/ and of icebs indicated that they are distantly related, suggesting that replication could have similar role for the two ices. furthermore, the ice rd from s. pyogenes related to icest1/ and the putative ice pklc from pseudomonas aeruginosa were reported to be simultaneously integrated and at extrachromosomal multiple copies while pp 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. in its host strain cnrz icest exhibits a stable copy number, even after a stimulation of its excision and core region transcription by mmc exposure. in this strain, icest excision percentage is reduced 3-fold in stationary phase and nine-fold after mmc treatment and icest copy number is not increased compared to the one observed in the strain cnrz additional factor could explain these differences of icest 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 icebs 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 arp orfq and arp genes, suggesting a fundamental function of these 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.  in conclusion, the transcriptional organization of the conjugation and recombination modules of two closely related ices from s. thermophilus, icest and icest is identical, while that of their regulation module is somewhat different. transcripts of core region and excision levels are higher for icest which is consistent with its higher transfer frequency. despite these differences, the excision of both ices is stimulated by exposure to a dna damaging agent and stationary phase. data generated by the transcriptional study suggest a new mechanism of regulation of icest1/ this behavior could be due to the atypical regulation module of these elements that encode homologues of both ci and immr repressors. analyses of sequenced genomes revealed, among streptococci, a family of ices that encode ci and immr homologs and therefore could share similar regulation. furthermore, our results suggest that dna damage induces not only the excision and transfer of icest but also its intracellular replication. this characteristic, which is not considered in the initial ice model, may be shared by other ices. this study also revealed that icest has very different behaviors depending on its primary host species, suggesting a major role of host factor in its excision and replication. 
analysis revealed an identical transcriptional organization of their recombination and conjugation modules whereas the transcriptional organization of their regulation modules were found to be different and to depend on the conditions. this finding suggests an impact of the host cell on ice behavior. the functional regulation of icest and icest transcription, excision and replication were investigated under different conditions. for both elements, stationary phase and dna damage lead to the rise of transcript levels of the conjugation-recombination and regulation modules. although ices are generally not described as replicative elements, the copy number of icest exhibited a sharp increase after mitomycin c exposure of its harboring strain cnrz this result was not observed when icest was introduced in a strain deriving icest host strain cnrz deleted for this element. the sharp increase of icest copy number suggests an induction of replication; such conditional intracellular replication may be common among ices. this is the first report of a partial shutdown of the activity of an ice executed by a strain belonging to its primary host species. all together, these results suggest a novel mechanism of regulation shared by icest icest and closely related ices, which we identified by analysis of recently sequenced genomes of firmicutes. two closely related ices, icest and icest have been identified in the lactic acid bacterium streptococcus thermophilus. while their conjugation and recombination modules are almost identical and their regulation modules related, previous work has demonstrated that transconjugants carrying icest were generated at rate exceeding by a factor that of icest furthermore, for both elements, excision increases in stationary phase and is strongly enhanced by dna damage.
 two closely related ices, icest and icest have been identified in the lactic acid bacterium streptococcus thermophilus. while their conjugation and recombination modules are almost identical and their regulation modules related, previous work has demonstrated that transconjugants carrying icest were generated at rate exceeding by a factor that of icest  the functional regulation of icest and icest transcription, excision and replication were investigated under different conditions. analysis revealed an identical transcriptional organization of their recombination and conjugation modules whereas the transcriptional organization of their regulation modules were found to be different and to depend on the conditions. for both elements, stationary phase and dna damage lead to the rise of transcript levels of the conjugation-recombination and regulation modules. whatever the growth culture conditions, excision of icest was found to be lower than that of icest which is consistent with weaker transfer frequencies. furthermore, for both elements, excision increases in stationary phase and is strongly enhanced by dna damage. although ices are generally not described as replicative elements, the copy number of icest exhibited a sharp increase after mitomycin c exposure of its harboring strain cnrz this result was not observed when icest was introduced in a strain deriving icest host strain cnrz deleted for this element. this finding suggests an impact of the host cell on ice behavior.  all together, these results suggest a novel mechanism of regulation shared by icest icest and closely related ices, which we identified by analysis of recently sequenced genomes of firmicutes. this is the first report of a partial shutdown of the activity of an ice executed by a strain belonging to its primary host species. the sharp increase of icest copy number suggests an induction of replication; such conditional intracellular replication may be common among ices. 