Horizontal gene transfer (HGT) 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 [8, 45]. 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 II536 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 SaPI1 [20, 26, 27]. Flanking direct repeat sequences (DRs) 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 ECOR31 with its flanking DRs, an integrase gene and the right border region (RB-HPIECOR31) encoding a functional mating pair formation system and a DNA-processing region, fulfills all structural criteria of integrative and conjugative elements, ICE [29, 31, 33]. 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 (co-)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 RP4, for example, can mediate transfer of mobilizable plasmids which code for an origin of transfer (oriT), 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" (Hfr) strains can transfer large parts of their chromosomes at high frequency .
PAI deletion has been described for UPEC strain 536 and other pathogenic bacteria [10, 14, 17, 48–50] as well as the occurrence of circular intermediates upon PAI excision of [12, 23, 26, 30, 33, 35, 36, 50] 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 1 (SGI1) . In addition, the 35-kb HPI of Yersinia enterocolitica could be mobilised  when a modified RP4 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 [42–44].
With the PAI II536 construct used in this study, we were able to transfer this ~107-kb DNA region in the presence of the unmodified RP4 plasmid and thereby demonstrated that PAI II536 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 HPIECOR31. In the latter case, the origin of replication seems to be inactivated by insertion of an IS630 homologue . This may explain why HPIECOR31 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 [53–57]. In the light F plasmid-mediated mobilization of the HPI , it would, nevertheless, also be interesting to analyse in the future if a PAI II536 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 536 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 (between 10-8 and 10-9). We postulate that the efficiency of PAI II536 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 II536, complete transfer occurred at a high rate. 93.1% of the transconjugants received the complete 107-kb PAI II536 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 (Table 1). PAI II536 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 II536 is highly specific. In both strain backgrounds, SY327λpir and 536-21, the PAI II536 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 II536. 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-1 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 536 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 [5, 32]. 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.