BACKGROUND
in the early 1980s large unstable chromosomal regions carrying virulence-associated genes were identified in uropathogenic e. coli  <cit> .
later, such large unstable chromosomal regions were designated pathogenicity islands   <cit> .
a constantly increasing number of similar genetic elements detected in many pathogenic and non-pathogenic microorganisms led to the definition of a family of related genetic elements, termed genomic islands , whose members share characteristic features  <cit> .
although pais, a subgroup of geis, are in several cases superficially similar, they structurally differ with respect to the encoded virulence factors, the size and the presence of different mobile and accessory elements.
due to the presence of mobility genes  or the occurrence of recombination processes or point mutations, pais constantly undergo structural changes  <cit> .
upon acquisition and chromosomal insertion, islands together with additional large regions of flanking chromosomal sequence context can be transferred by conjugation and homologous recombination and thus contribute to genome plasticity and the simultaneous transfer of multiple traits  <cit> .
nevertheless, pais are in many cases not stably integrated into the e. coli host chromosome and may be lost upon deletion.
this process can be studied by island probing  <cit> .
the influence of different environmental conditions on the stability of five pais of upec strain  <dig> has already been investigated before  <cit>  indicating that pai i <dig>  pai ii <dig>  pai iii <dig>  and pai v <dig> delete with frequencies between 10- <dig> and 10- <dig>  while loss of pai iv <dig> could not be detected.
in upec strain  <dig>  pai deletion is catalyzed by a p4-like bacteriophage integrase which is encoded on the respective island  <cit> .
similar deletion frequencies  were also reported for pais of repec strain 84/110- <dig> and s. flexneri 2a  <cit> .
higher deletion frequencies  have, however, been observed for o-islands  <dig> and  <dig> in enterohemorrhagic e. coli  o157:h <dig> isolates  <cit> .
circular intermediate  formation in the cytoplasm of upec strain  <dig> was demonstrated for pai ii <dig> and pai iii <dig>  since none of these two islands apparently contain an origin of replication, it has been hypothesized that cis are lost upon cell division unless they reintegrate into the chromosome.
furthermore, horizontal gene transfer  of such circularized pais may occur with the help of bacteriophages or conjugative plasmids  <cit> .
a close functional association between pais and bacteriophages was reported for several bacterial pathogens.
in v. cholerae, the entire  <dig> -kb vibrio pathogenicity island  can be transfered by the general transducing phage cp-t <dig>  <cit> .
the "high pathogenicity island " of yersinia pseudotuberculosis has been shown to be transfered by a bacteriophage  <cit> .
the so-called staphylococcus aureus pathogenicity islands  can excise and replicate upon induction by other resident s. aureus bacteriophages and then packed into phage-like particles which can be transferred to recipient cells  <cit> .
sapi transfer by transduction can even occur between representatives of different species.
the intra- and interspecies transfer was demonstrated for the sapi- <dig> element which could be transferred into a variety of different recipients  <cit> .
the identification of self-replicating plasmid-like states of the excised sapi element, however, is also reminiscent of plasmid-like ancestors  <cit> .
bacteriophage-mediated transfer is limited by the amount of dna that can be packed into the phage capsid, but in some cases it can expand beyond  <dig> kb  <cit> .
as multiple island-like genomic regions in other bacteria exhibit features of degenerate prophages as well, there may be the possibility to mobilize these islands by other phages.
the discovery of integrative conjugative elements  and related genetic entities suggests another mechanism of pai transfer  <cit> .
with the help of excisionases and integrases pais and related integrative mobilisable elements are able to site-specifically delete from or integrate into the chromosome.
after deletion they are able to replicate and can also be transmitted into a new host by their own conjugative machinery.
a variant of the "high pathogenicity island"  has been described in e. coli strain ecor <dig> to contain a 35-kb sequence with striking homology to conjugative plasmids  <cit> .
the identification of this ice-ec <dig> carrying a functional transfer determinant suggests that conjugative transfer may have played a role in the spread of the hpi, and possibly also in the transmission of other pais.
the spread of the non-selftransmissible but mobilisable antibiotic resistance gene cluster of the salmonella genomic island  <dig>  also supports the existence of a conjugal transfer mechanism for pais as well as interstrain pai transfer observed in pseudomonas aeruginosa, enterococcus faecalis and streptococcus thermophilus  <cit> .
type iv secretion systems  have been shown to mediate the horizontal transfer of such dna elements in a broad range of bacteria  <cit> .
alternatively, mobilisation of circular intermediates of islands and related genetic elements has been described  <cit> .
to study whether archetypal pais of e. coli which usually lack traits that enable their distribution such as origins of replication and tra genes could be generally mobilised by a helper plasmid, we investigated the transferability of pai ii <dig>  the largest pai  of upec strain  <dig>  into an e. coli k- <dig> recipient and back into a pai ii536-negative mutant of strain  <dig>
RESULTS
transfer of the entire pai ii <dig> from upec strain  <dig> into e. coli k-12
altogether,  <dig> mating experiments were carried out at 20°c and 37°c.
plating of conjugation batches with e. coli strains 536-19/1mob  and sy327λpir  resulted in high numbers of chloramphenicol  and nalidixic acid -resistant colonies and  <dig> resulting haemolytic clones were further investigated.
screening of clones that grew on the cm-nal selective blood agar medium with the e. coli k-12- and k <dig> capsule-specific pcrs, however, revealed that only  <dig> %  of them were true e. coli k- <dig> transconjugants, whereas the rest proved to be spontaneous nalidixic acid resistant mutants of strain  <dig>  these clones were further analysed with four pai ii536-specific pcrs  to determine whether the complete pai ii <dig> had been transferred.
<dig> %  of the  <dig> transconjugants acquired the complete island and  <dig> %  of the haemolytic transconjugant clones have only been partially transferred to the recipient strain.
the mating temperature slightly affected the proportions of the different types of pai transfer.
at 20°c,  <dig> %  of the transconjugants carried the chromosomally inserted pai ii <dig> construct,  <dig> %  had circular intermediates, and  <dig> %  resulted from partial pai ii <dig> transfer.
upon mating at 37°c,  <dig> %  of pai ii <dig> were chromosomally inserted,  <dig> %  were circular intermediates, and  <dig> %  were only partially transferred.
the differences observed between the different types of transconjugants obtained at 20°c and 37°c were not significant.
transfer frequencies were between  <dig> × 10- <dig> and  <dig>  × 10- <dig> , depending on the mating temperature  as well as on the ratio of donor and recipient cells .
the mean transfer frequency at both temperatures was always higher with a donor: recipient ratio of 9: <dig> relative to a 3: <dig> ratio.
the differences observed were, however, not significant .
<dig> and  <dig> independent conjugation experiments were performed for the mobilisation and remobilisation experiment, respectively.
plasmid rp <dig> was used as a helper plasmid for mobilisation of the excised pai ii <dig> construct from e. coli  <dig> into recipient e. coli sy <dig>
pai ii <dig> integrates site-specifically into the e. coli k- <dig> chromosome at the trna gene leux
upon conjugation, the transferred circularised form of the pai ii <dig> derivative can integrate into the recipient's chromosome.
additionally, the recipient strain sy327λpir also enables episomal replication of the transferred ci.
analysis by pcr of the transconjugants carrying the complete pai ii <dig> derivative allowed to distinguish between chromosomally inserted and episomal circular forms of the pai ii <dig> construct.
episomal cis could not be detected in the clones with the chromosomally inserted pai ii <dig> derivative.
as exemplarily shown for clones  <dig>   <dig>  and  <dig>  the orientation of the site-specifically integrated pai ii <dig> within the chromosome was determined by using combinations of the four primer pairs indicated in figure  <dig>  in these three clones as well as in donor strain  <dig>  pcr screening products could only be obtained using primer pairs  <dig> and  <dig>  which amplify the ends of pai ii <dig> with the adjacent core genome context.
primer pair  <dig> amplifies the empty leux locus in the core genome context and gave only a pcr product in the recipient strain sy <dig>  accordingly, pai ii <dig> has been inserted into the leux gene of the e. coli sy <dig> chromosome in the identical orientation as in the donor chromosome .
genomic restriction patterns of representative transconjugants, carrying either the chromosomally inserted pai ii <dig> derivative or its episomal ci, were compared to each other and to those of the donor and recipient strain by pfge in order to assess their genomic homogeneity .
generally, the restriction patterns of the transconjugants were very similar to that of recipient strain sy327λpir.
the pfge patterns of the selected transconjugants which carried the transferred pai ii <dig> in their chromosome exhibited only minor differences among each other.
similarly, the restriction patterns of the clones containing the stable episomal ci of pai ii <dig> were identical.
both groups of transconjugants could be clearly distinguished upon the presence of a ~400-kb and a ~530-kb restriction fragment in those recipient clones with a stable cytoplasmic pai ii <dig> ci which were absent from recipients in which chromosomal integration of the island occurred.
instead, a restriction fragment of about  <dig> kb was visible in the latter clones .
this larger restriction fragment may comprise the 530-kb restriction fragment after chromosomal insertion of the transferred pai ii <dig>  construct.
these data demonstrate that pai ii <dig> can be mobilized upon excision from the chromosome by helper plasmids into suitable recipient strains.
upon transfer, the majority of cis integrates site-specifically into the recipient's chromosome at the leux locus or remains as an episomal ci.
remobilisation of the transferred pai ii <dig> into e. coli strain 536-21
since two types of transconjugants resulted from the pai ii <dig> mobilisation, two types of remobilisation experiments were performed: k- <dig> strains harbouring either the ci or the chromosomally inserted pai ii <dig> were used as donors.
since the recipient strain 536- <dig> does not express the π-protein, only chromosomal integration of pai ii <dig> into the leux gene was observed in all transconjugants.
there was a marked difference in the conjugation efficiency between the remobilisation of the circular and the integrated forms.
in those cases where strain sy327- <dig> carrying an episomal ci of pai ii <dig> was used as donor, average pai transfer was about 100- to 1000-fold more efficient with transfer rates of  <dig>  × 10- <dig> at 37°c and  <dig>  × 10- <dig> at 20°c, respectively.
however, if sy327- <dig> served as a donor, where pai ii <dig> was integrated into the chromosome, the average efficiency of transfer was  <dig> × 10- <dig> and  <dig>  × 10- <dig>  at 37°c and 20°c, respectively .
these results support that the mobilised pai and the rp <dig> plasmid include all the factors required for excision of the chromsomally inserted pai as well as for its efficient transfer.
discussion
horizontal gene transfer  plays an important role during prokaryotic evolution.
exchange and accumulation of a variety of fitness or virulence factors frequently carried on mobile genetic elements contributes to evolution of different pathogens and pathotypes from non- or less pathogenic variants  <cit> .
one perfect environment for this evolutionary process is the mammalian gut with its large bacterial density which offers the possibility of close cell-to-cell contacts between closely or even remotely related bacteria.
in this way, members of the gut flora, such as e. coli, may also increase their pathogenic potential and may evolve from commensals into e.g.
extraintestinal pathogens.
e. coli may, nevertheless, also exist outside of the gut, e.g.
in the environment having the possibility to exchange genetic information with other bacteria.
high bacterial cell densities could be observed, e.g.
in bacterial biofilms, an important bacterial lifestyle in the environment.
the pai ii <dig> transfer at 20°c indicates that e. coli can exchange pais not only upon growth at human body temperature but also at a temperature which is closer to the ambient temperature in the environment.
for the transfer of pais, different mechanisms have been postulated.
for example, the presence of phage-related sequences on most pais suggests a key role of bacteriophages in hgt, and, indeed, a transfer by transducing phages has been reported for vpi and sapi <dig>  <cit> .
flanking direct repeat sequences  and an active bacteriophage integrase play also an important role in the excision process of e. coli 536-specific pais  <cit> , which is essential for a subsequent transfer.
alternatively, pais can be transfered by conjugation.
the hpi of e. coli strain ecor <dig> with its flanking drs, an integrase gene and the right border region  encoding a functional mating pair formation system and a dna-processing region, fulfills all structural criteria of integrative and conjugative elements, ice  <cit> .
although neither conserved repabc genes, other indications of a plasmid replicon, nor mobilisation have been detected, this hpi variant supports the hypothesis that pai transfer can also occur by conjugal transfer  <cit> .
furthermore, high partial similarity between different polyketide biosynthesis determinants located on islands such as the hpi and the colibactin island of extraintestinal pathogenic e. coli, ices and different enterobacterial plasmids have been previously described.
the presence of these polyketide determinants in different enterobacterial species and their localisation on different mobile genetic elements further support the idea that different chromosomal and episomal elements can recombine and thus due to hgt promote bacterial genome plasticity  <cit> .
additionally, self-transmissible conjugative elements can mobilize other genomic dna regions in cis or in trans.
the conjugative plasmid rp <dig>  for example, can mediate transfer of mobilizable plasmids which code for an origin of transfer , a relaxase and nicking accessory proteins for interaction with orit.
a conjugative element then provides the mating pair formation functions for transfer  <cit> .
large-scale dna transfer followed by homologous recombination can also be involved in the distribution of chromosomally inserted pathogenicity islands.
different hpi-transfer events have been detected in e. coli, in which not only the hpi itself but also flanking regions of the genomic backbone have been transfered.
schubert and colleagues demonstrated that the conjugative f plasmid can transfer and insert the hpi into the recipient chromosome by homologous recombination of flanking dna regions.
upon chromosomal integration of an f plasmid, the recipient genome acquires an orit and thereby becomes mobilisable.
resulting so-called "high frequency of recombination"  strains can transfer large parts of their chromosomes at high frequency  <cit> .
pai deletion has been described for upec strain  <dig> and other pathogenic bacteria  <cit>  as well as the occurrence of circular intermediates upon pai excision of  <cit>  suggesting that the latter could be formed during conjugal or phage-mediated transfer.
using a conjugative helper plasmid, transfer of a ci was also verified for the 43-kb salmonella genomic island  <dig>   <cit> .
in addition, the 35-kb hpi of yersinia enterocolitica could be mobilised  <cit>  when a modified rp <dig> plasmid was used as a shuttle vector during the transfer experiments.
several cases of plasmid mobilisation as a major mechanism for horizontal gene transfer of pais have been described  <cit> .
with the pai ii <dig> construct used in this study, we were able to transfer this ~107-kb dna region in the presence of the unmodified rp <dig> plasmid and thereby demonstrated that pai ii <dig> is mobilisable, but not self-transmissible.
to increase the stability of the large pai ii536-specific ci and thus the transfer frequency, we also integrated an origin of replication into this pai.
in this respect, our model construct is artificial, but exhibits similar features of some ices including the hpiecor <dig>  in the latter case, the origin of replication seems to be inactivated by insertion of an is <dig> homologue  <cit> .
this may explain why hpiecor <dig> is not transferable although ci formation of this island was shown in the same study.
whereas plasmids replicate autonomously, ices are generally thought to be incapable of autonomous replication.
instead, their replication depends on that of host chromosome  <cit> .
some ice and ice-like elements, however, have been reported to be capable of autonomous replication  <cit> .
in the light f plasmid-mediated mobilization of the hpi  <cit> , it would, nevertheless, also be interesting to analyse in the future if a pai ii <dig> construct, which is not a self-replicating entity, but only carries an orit, could be mobilized upon provision of the appropriate conjugative machinery in trans on a plasmid.
the primary aim of our study was to demonstrate the transferability of a large archetypal island of upec strain  <dig> as this pai can be excised site-specifically from the chromosome by its cognate integrase.
on the other hand, we also tested conditions which may affect the transfer of an excised circular pai intermediate.
the frequency of pai transfer in the mobilisation experiments was low .
we postulate that the efficiency of pai ii <dig> transfer depends on several factors including the growth temperature, integrase activity, the size, and the chromosomal or episomal state of the pai.
in spite of the large size of pai ii <dig>  complete transfer occurred at a high rate.
<dig> % of the transconjugants received the complete 107-kb pai ii <dig> construct.
the activity of the pai-encoded integrase can contribute to the transfer efficiency by affecting the pai excision as well as the integration frequency.
the remobilisation efficiency was three log scales higher with a stable episomal ci compared to an integrated pai, indicating that a more active integrase may increase the chance of transfer by frequent induction of pai-excision from the chromosome .
pai ii <dig> transfer rates at 20°c and 37°c were not significantly different.
besides the gut, e. coli also faces the environment as a natural habitat since the bacteria are excreted each day in considerable amounts.
as a part of naturally occurring biofilms in sewage or drinking water systems, they are exposed to stimuli described above, i.e.
low temperature and high density of cells, what might explain their ability to efficiently exchange genetic elements also under these conditions.
in accordance with previously published results  <cit> , the mobilisation and remobilisation experiments corroborated that the p4-like integrase of pai ii <dig> is highly specific.
in both strain backgrounds, sy327λpir and 536- <dig>  the pai ii <dig> was found only to be inserted into the leux locus thereby restoring the complete trna gene in the latter strain.
this result demonstrated that leux is the preferred chromosomal integration site of pai ii <dig>  site-specific chromosomal integration of pais has already been described before.
however, if multiple isoacceptor trna genes exist, chromosomal insertion may occur at all the available isoacceptor trna loci.
the hpi of y. pestis is usually associated with the asnt trna locus, but in y. pseudotuberculosis the hpi can insert into any of the three chromosomal asn trna loci  <cit> .
the same phenomenon has been observed as well, e.g.
with lee pais  <cit>  and the papi- <dig> island of p. aeruginosa  <cit> .
the lack of genes required for mobilisation and/or transfer on the archetypal pais of upec strains such as e. coli  <dig> has been considered to reflect an advanced stage of "homing" of these islands, i.e.
an ongoing process of stabilisation of such chromosomal regions resulting from the selective inactivation and loss of corresponding genes  <cit> .
consequently, horizontal transfer of such islands, although they can be efficiently excised from the chromosome, could not be detected so far and the mechanism of acquisition remains speculative.
this study further supports the important role of mobilisation and conjugation for transfer and dissemination of genomic islands and indicates that loss of mobilisation and transfer genes promotes stabilisation of horizontally acquired genetic elements in the recipient genome.
CONCLUSIONS
we provide evidence that a 107-kb chromosomal pai derivative of upec can be mobilised into other e. coli recipient strains.
this transfer was dependent on the presence of a helper plasmid and accessory transfer genes.
the new host with the mobilisable pai ii <dig> could also serve as donor passing on this pai to other recipients.
these results underline that in a suitable genetic background dissemination of large genomic regions such as pais by conjugal transfer contributes to genome plasticity of e. coli and the evolution of bacterial pathogens.
stabilisation of beneficial genetic information localised on mobile genetic elements can be achieved by selective loss of transfer or mobilisation functions encoded by these elements.

 in the early 1980s large unstable chromosomal regions carrying virulence-associated genes were identified in uropathogenic e. coli. later, such large unstable chromosomal regions were designated pathogenicity islands. a constantly increasing number of similar genetic elements detected in many pathogenic and non-pathogenic microorganisms led to the definition of a family of related genetic elements, termed genomic islands, whose members share characteristic features. although pais, a subgroup of geis, are in several cases superficially similar, they structurally differ with respect to the encoded virulence factors, the size and the presence of different mobile and accessory elements. due to the presence of mobility genes or the occurrence of recombination processes or point mutations, pais constantly undergo structural changes. upon acquisition and chromosomal insertion, islands together with additional large regions of flanking chromosomal sequence context can be transferred by conjugation and homologous recombination and thus contribute to genome plasticity and the simultaneous transfer of multiple traits. nevertheless, pais are in many cases not stably integrated into the e. coli host chromosome and may be lost upon deletion. this process can be studied by island probing. the influence of different environmental conditions on the stability of five pais of upec strain has already been investigated before indicating that pai i pai ii pai iii and pai v delete with frequencies between 10- and 10- while loss of pai iv could not be detected. in upec strain pai deletion is catalyzed by a p4-like bacteriophage integrase which is encoded on the respective island. similar deletion frequencies were also reported for pais of repec strain 84/110- and s. flexneri 2a. higher deletion frequencies have, however, been observed for o-islands and in enterohemorrhagic e. coli o157:h isolates. circular intermediate formation in the cytoplasm of upec strain was demonstrated for pai ii and pai iii since none of these two islands apparently contain an origin of replication, it has been hypothesized that cis are lost upon cell division unless they reintegrate into the chromosome. furthermore, horizontal gene transfer of such circularized pais may occur with the help of bacteriophages or conjugative plasmids. a close functional association between pais and bacteriophages was reported for several bacterial pathogens. in v. cholerae, the entire -kb vibrio pathogenicity island can be transfered by the general transducing phage cp-t. the "high pathogenicity island " of yersinia pseudotuberculosis has been shown to be transfered by a bacteriophage. the so-called staphylococcus aureus pathogenicity islands can excise and replicate upon induction by other resident s. aureus bacteriophages and then packed into phage-like particles which can be transferred to recipient cells. sapi transfer by transduction can even occur between representatives of different species. the intra- and interspecies transfer was demonstrated for the sapi- element which could be transferred into a variety of different recipients. the identification of self-replicating plasmid-like states of the excised sapi element, however, is also reminiscent of plasmid-like ancestors. bacteriophage-mediated transfer is limited by the amount of dna that can be packed into the phage capsid, but in some cases it can expand beyond kb. as multiple island-like genomic regions in other bacteria exhibit features of degenerate prophages as well, there may be the possibility to mobilize these islands by other phages. the discovery of integrative conjugative elements and related genetic entities suggests another mechanism of pai transfer. with the help of excisionases and integrases pais and related integrative mobilisable elements are able to site-specifically delete from or integrate into the chromosome. after deletion they are able to replicate and can also be transmitted into a new host by their own conjugative machinery. a variant of the "high pathogenicity island" has been described in e. coli strain ecor to contain a 35-kb sequence with striking homology to conjugative plasmids. the identification of this ice-ec carrying a functional transfer determinant suggests that conjugative transfer may have played a role in the spread of the hpi, and possibly also in the transmission of other pais. the spread of the non-selftransmissible but mobilisable antibiotic resistance gene cluster of the salmonella genomic island also supports the existence of a conjugal transfer mechanism for pais as well as interstrain pai transfer observed in pseudomonas aeruginosa, enterococcus faecalis and streptococcus thermophilus. type iv secretion systems have been shown to mediate the horizontal transfer of such dna elements in a broad range of bacteria. alternatively, mobilisation of circular intermediates of islands and related genetic elements has been described. to study whether archetypal pais of e. coli which usually lack traits that enable their distribution such as origins of replication and tra genes could be generally mobilised by a helper plasmid, we investigated the transferability of pai ii the largest pai of upec strain into an e. coli k- recipient and back into a pai ii536-negative mutant of strain  transfer of the entire pai ii from upec strain into e. coli k-12 altogether, mating experiments were carried out at 20°c and 37°c. plating of conjugation batches with e. coli strains 536-19/1mob and sy327λpir resulted in high numbers of chloramphenicol and nalidixic acid -resistant colonies and resulting haemolytic clones were further investigated. screening of clones that grew on the cm-nal selective blood agar medium with the e. coli k-12- and k capsule-specific pcrs, however, revealed that only % of them were true e. coli k- transconjugants, whereas the rest proved to be spontaneous nalidixic acid resistant mutants of strain these clones were further analysed with four pai ii536-specific pcrs to determine whether the complete pai ii had been transferred. % of the transconjugants acquired the complete island and % of the haemolytic transconjugant clones have only been partially transferred to the recipient strain. the mating temperature slightly affected the proportions of the different types of pai transfer. at 20°c, % of the transconjugants carried the chromosomally inserted pai ii construct, % had circular intermediates, and % resulted from partial pai ii transfer. upon mating at 37°c, % of pai ii were chromosomally inserted, % were circular intermediates, and % were only partially transferred. the differences observed between the different types of transconjugants obtained at 20°c and 37°c were not significant. transfer frequencies were between × 10- and × 10-, depending on the mating temperature as well as on the ratio of donor and recipient cells. the mean transfer frequency at both temperatures was always higher with a donor: recipient ratio of 9: relative to a 3: ratio. the differences observed were, however, not significant. and independent conjugation experiments were performed for the mobilisation and remobilisation experiment, respectively. plasmid rp was used as a helper plasmid for mobilisation of the excised pai ii construct from e. coli into recipient e. coli sy pai ii integrates site-specifically into the e. coli k- chromosome at the trna gene leux upon conjugation, the transferred circularised form of the pai ii derivative can integrate into the recipient's chromosome. additionally, the recipient strain sy327λpir also enables episomal replication of the transferred ci. analysis by pcr of the transconjugants carrying the complete pai ii derivative allowed to distinguish between chromosomally inserted and episomal circular forms of the pai ii construct. episomal cis could not be detected in the clones with the chromosomally inserted pai ii derivative. as exemplarily shown for clones and the orientation of the site-specifically integrated pai ii within the chromosome was determined by using combinations of the four primer pairs indicated in figure in these three clones as well as in donor strain pcr screening products could only be obtained using primer pairs and which amplify the ends of pai ii with the adjacent core genome context. primer pair amplifies the empty leux locus in the core genome context and gave only a pcr product in the recipient strain sy accordingly, pai ii has been inserted into the leux gene of the e. coli sy chromosome in the identical orientation as in the donor chromosome. genomic restriction patterns of representative transconjugants, carrying either the chromosomally inserted pai ii derivative or its episomal ci, were compared to each other and to those of the donor and recipient strain by pfge in order to assess their genomic homogeneity. generally, the restriction patterns of the transconjugants were very similar to that of recipient strain sy327λpir. the pfge patterns of the selected transconjugants which carried the transferred pai ii in their chromosome exhibited only minor differences among each other. similarly, the restriction patterns of the clones containing the stable episomal ci of pai ii were identical. both groups of transconjugants could be clearly distinguished upon the presence of a ~400-kb and a ~530-kb restriction fragment in those recipient clones with a stable cytoplasmic pai ii ci which were absent from recipients in which chromosomal integration of the island occurred. instead, a restriction fragment of about kb was visible in the latter clones. this larger restriction fragment may comprise the 530-kb restriction fragment after chromosomal insertion of the transferred pai ii construct. these data demonstrate that pai ii can be mobilized upon excision from the chromosome by helper plasmids into suitable recipient strains. upon transfer, the majority of cis integrates site-specifically into the recipient's chromosome at the leux locus or remains as an episomal ci. remobilisation of the transferred pai ii into e. coli strain 536-21 since two types of transconjugants resulted from the pai ii mobilisation, two types of remobilisation experiments were performed: k- strains harbouring either the ci or the chromosomally inserted pai ii were used as donors. since the recipient strain 536- does not express the π-protein, only chromosomal integration of pai ii into the leux gene was observed in all transconjugants. there was a marked difference in the conjugation efficiency between the remobilisation of the circular and the integrated forms. in those cases where strain sy327- carrying an episomal ci of pai ii was used as donor, average pai transfer was about 100- to 1000-fold more efficient with transfer rates of × 10- at 37°c and × 10- at 20°c, respectively. however, if sy327- served as a donor, where pai ii was integrated into the chromosome, the average efficiency of transfer was × 10- and × 10- at 37°c and 20°c, respectively. these results support that the mobilised pai and the rp plasmid include all the factors required for excision of the chromsomally inserted pai as well as for its efficient transfer. discussion horizontal gene transfer plays an important role during prokaryotic evolution. exchange and accumulation of a variety of fitness or virulence factors frequently carried on mobile genetic elements contributes to evolution of different pathogens and pathotypes from non- or less pathogenic variants. one perfect environment for this evolutionary process is the mammalian gut with its large bacterial density which offers the possibility of close cell-to-cell contacts between closely or even remotely related bacteria. in this way, members of the gut flora, such as e. coli, may also increase their pathogenic potential and may evolve from commensals into e.g. extraintestinal pathogens. e. coli may, nevertheless, also exist outside of the gut, e.g. in the environment having the possibility to exchange genetic information with other bacteria. high bacterial cell densities could be observed, e.g. in bacterial biofilms, an important bacterial lifestyle in the environment. the pai ii transfer at 20°c indicates that e. coli can exchange pais not only upon growth at human body temperature but also at a temperature which is closer to the ambient temperature in the environment. for the transfer of pais, different mechanisms have been postulated. for example, the presence of phage-related sequences on most pais suggests a key role of bacteriophages in hgt, and, indeed, a transfer by transducing phages has been reported for vpi and sapi. flanking direct repeat sequences and an active bacteriophage integrase play also an important role in the excision process of e. coli 536-specific pais, which is essential for a subsequent transfer. alternatively, pais can be transfered by conjugation. the hpi of e. coli strain ecor with its flanking drs, an integrase gene and the right border region encoding a functional mating pair formation system and a dna-processing region, fulfills all structural criteria of integrative and conjugative elements, ice. although neither conserved repabc genes, other indications of a plasmid replicon, nor mobilisation have been detected, this hpi variant supports the hypothesis that pai transfer can also occur by conjugal transfer. furthermore, high partial similarity between different polyketide biosynthesis determinants located on islands such as the hpi and the colibactin island of extraintestinal pathogenic e. coli, ices and different enterobacterial plasmids have been previously described. the presence of these polyketide determinants in different enterobacterial species and their localisation on different mobile genetic elements further support the idea that different chromosomal and episomal elements can recombine and thus due to hgt promote bacterial genome plasticity. additionally, self-transmissible conjugative elements can mobilize other genomic dna regions in cis or in trans. the conjugative plasmid rp for example, can mediate transfer of mobilizable plasmids which code for an origin of transfer, a relaxase and nicking accessory proteins for interaction with orit. a conjugative element then provides the mating pair formation functions for transfer. large-scale dna transfer followed by homologous recombination can also be involved in the distribution of chromosomally inserted pathogenicity islands. different hpi-transfer events have been detected in e. coli, in which not only the hpi itself but also flanking regions of the genomic backbone have been transfered. schubert and colleagues demonstrated that the conjugative f plasmid can transfer and insert the hpi into the recipient chromosome by homologous recombination of flanking dna regions. upon chromosomal integration of an f plasmid, the recipient genome acquires an orit and thereby becomes mobilisable. resulting so-called "high frequency of recombination" strains can transfer large parts of their chromosomes at high frequency. pai deletion has been described for upec strain and other pathogenic bacteria as well as the occurrence of circular intermediates upon pai excision of suggesting that the latter could be formed during conjugal or phage-mediated transfer. using a conjugative helper plasmid, transfer of a ci was also verified for the 43-kb salmonella genomic island. in addition, the 35-kb hpi of yersinia enterocolitica could be mobilised when a modified rp plasmid was used as a shuttle vector during the transfer experiments. several cases of plasmid mobilisation as a major mechanism for horizontal gene transfer of pais have been described. with the pai ii construct used in this study, we were able to transfer this ~107-kb dna region in the presence of the unmodified rp plasmid and thereby demonstrated that pai ii is mobilisable, but not self-transmissible. to increase the stability of the large pai ii536-specific ci and thus the transfer frequency, we also integrated an origin of replication into this pai. in this respect, our model construct is artificial, but exhibits similar features of some ices including the hpiecor in the latter case, the origin of replication seems to be inactivated by insertion of an is homologue. this may explain why hpiecor is not transferable although ci formation of this island was shown in the same study. whereas plasmids replicate autonomously, ices are generally thought to be incapable of autonomous replication. instead, their replication depends on that of host chromosome. some ice and ice-like elements, however, have been reported to be capable of autonomous replication. in the light f plasmid-mediated mobilization of the hpi, it would, nevertheless, also be interesting to analyse in the future if a pai ii construct, which is not a self-replicating entity, but only carries an orit, could be mobilized upon provision of the appropriate conjugative machinery in trans on a plasmid. the primary aim of our study was to demonstrate the transferability of a large archetypal island of upec strain as this pai can be excised site-specifically from the chromosome by its cognate integrase. on the other hand, we also tested conditions which may affect the transfer of an excised circular pai intermediate. the frequency of pai transfer in the mobilisation experiments was low. we postulate that the efficiency of pai ii transfer depends on several factors including the growth temperature, integrase activity, the size, and the chromosomal or episomal state of the pai. in spite of the large size of pai ii complete transfer occurred at a high rate. % of the transconjugants received the complete 107-kb pai ii construct. the activity of the pai-encoded integrase can contribute to the transfer efficiency by affecting the pai excision as well as the integration frequency. the remobilisation efficiency was three log scales higher with a stable episomal ci compared to an integrated pai, indicating that a more active integrase may increase the chance of transfer by frequent induction of pai-excision from the chromosome. pai ii transfer rates at 20°c and 37°c were not significantly different. besides the gut, e. coli also faces the environment as a natural habitat since the bacteria are excreted each day in considerable amounts. as a part of naturally occurring biofilms in sewage or drinking water systems, they are exposed to stimuli described above, i.e. low temperature and high density of cells, what might explain their ability to efficiently exchange genetic elements also under these conditions. in accordance with previously published results, the mobilisation and remobilisation experiments corroborated that the p4-like integrase of pai ii is highly specific. in both strain backgrounds, sy327λpir and 536- the pai ii was found only to be inserted into the leux locus thereby restoring the complete trna gene in the latter strain. this result demonstrated that leux is the preferred chromosomal integration site of pai ii site-specific chromosomal integration of pais has already been described before. however, if multiple isoacceptor trna genes exist, chromosomal insertion may occur at all the available isoacceptor trna loci. the hpi of y. pestis is usually associated with the asnt trna locus, but in y. pseudotuberculosis the hpi can insert into any of the three chromosomal asn trna loci. the same phenomenon has been observed as well, e.g. with lee pais and the papi- island of p. aeruginosa. the lack of genes required for mobilisation and/or transfer on the archetypal pais of upec strains such as e. coli has been considered to reflect an advanced stage of "homing" of these islands, i.e. an ongoing process of stabilisation of such chromosomal regions resulting from the selective inactivation and loss of corresponding genes. consequently, horizontal transfer of such islands, although they can be efficiently excised from the chromosome, could not be detected so far and the mechanism of acquisition remains speculative. this study further supports the important role of mobilisation and conjugation for transfer and dissemination of genomic islands and indicates that loss of mobilisation and transfer genes promotes stabilisation of horizontally acquired genetic elements in the recipient genome.  we provide evidence that a 107-kb chromosomal pai derivative of upec can be mobilised into other e. coli recipient strains. this transfer was dependent on the presence of a helper plasmid and accessory transfer genes. the new host with the mobilisable pai ii could also serve as donor passing on this pai to other recipients. these results underline that in a suitable genetic background dissemination of large genomic regions such as pais by conjugal transfer contributes to genome plasticity of e. coli and the evolution of bacterial pathogens. stabilisation of beneficial genetic information localised on mobile genetic elements can be achieved by selective loss of transfer or mobilisation functions encoded by these elements. 
a substantial amount of data has been accumulated supporting the important role of genomic islands - including pathogenicity islands - in bacterial genome plasticity and the evolution of bacterial pathogens. their instability and the high level sequence similarity of different islands suggest an exchange of pais between strains of the same or even different bacterial species by horizontal gene transfer. transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands. our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as pais. this locus is the chromosomal integration site of pai ii in upec strain from the e. coli k- recipient, the chromosomal pai ii construct as well as the cis could be successfully remobilised and inserted into leux in a pai ii deletion mutant of e. coli 
 a substantial amount of data has been accumulated supporting the important role of genomic islands - including pathogenicity islands - in bacterial genome plasticity and the evolution of bacterial pathogens. their instability and the high level sequence similarity of different islands suggest an exchange of pais between strains of the same or even different bacterial species by horizontal gene transfer. transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.  to study mobilisation of such large genomic regions in prototypic uropathogenic e. coli strain pai ii was supplemented with the mobrp region, an origin of replication, an origin of transfer and a chloramphenicol resistance selection marker. in the presence of helper plasmid rp conjugative transfer of the 107-kb pai ii construct occured from strain into an e. coli k- recipient. in transconjugants, pai ii existed either as a cytoplasmic circular intermediate or integrated site-specifically into the recipient's chromosome at the leux trna gene. this locus is the chromosomal integration site of pai ii in upec strain from the e. coli k- recipient, the chromosomal pai ii construct as well as the cis could be successfully remobilised and inserted into leux in a pai ii deletion mutant of e. coli  our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as pais. stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands. 