Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitrosupport the role of conjugation for horizontal transfer of genomic islands

In the early 1980s large unstable chromosomal regions carrying virulence-associated genes were identified in uropathogenic E. coli [1]. Later, such large unstable chromosomal regions were designated pathogenicity islands (PAIs) [2–4]. 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 (GEIs), whose members share characteristic features [5–7]. 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 (integrases, transposases, IS elements) or the occurrence of recombination processes or point mutations, PAIs constantly undergo structural changes [4, 8–12]. 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 [13].

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 [10, 14–16]. The influence of different environmental conditions on the stability of five PAIs of UPEC strain 536 has already been investigated before [17] indicating that PAI I₅₃₆, PAI II₅₃₆, PAI III₅₃₆, and PAI V₅₃₆ delete with frequencies between 10^-5 and 10^-6, while loss of PAI IV₅₃₆ could not be detected. In UPEC strain 536, PAI deletion is catalyzed by a P4-like bacteriophage integrase which is encoded on the respective island [18]. Similar deletion frequencies (10^-5 - 10^-6) were also reported for PAIs of REPEC strain 84/110-1 and S. flexneri 2a [12, 19]. Higher deletion frequencies (10^-3 - 10^-4) have, however, been observed for O-islands 43 and 48 in enterohemorrhagic E. coli (EHEC) O157:H7 isolates [14]. Circular intermediate (CI) formation in the cytoplasm of UPEC strain 536 was demonstrated for PAI II₅₃₆ and PAI III₅₃₆. 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 (HGT) of such circularized PAIs may occur with the help of bacteriophages or conjugative plasmids [17].

A close functional association between PAIs and bacteriophages was reported for several bacterial pathogens. In V. cholerae, the entire 39.5-kb Vibrio Pathogenicity Island (VPI) can be transfered by the general transducing phage CP-T1 [20]. The "high pathogenicity island (HPI)" of Yersinia pseudotuberculosis has been shown to be transfered by a bacteriophage [21]. The so-called Staphylococcus aureus pathogenicity islands (SaPIs) 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 [22–24]. SaPI transfer by transduction can even occur between representatives of different species. The intra- and interspecies transfer was demonstrated for the SaPI-2 element which could be transferred into a variety of different recipients [22, 25, 26]. The identification of self-replicating plasmid-like states of the excised SaPI element, however, is also reminiscent of plasmid-like ancestors [22]. 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 100 kb [27, 28]. 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 (ICEs) and related genetic entities suggests another mechanism of PAI transfer [29–32]. 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" (HPI) has been described in E. coli strain ECOR31 to contain a 35-kb sequence with striking homology to conjugative plasmids [33]. The identification of this ICE-EC1 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 1 (SGI1) 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 [34–36]. Type IV secretion systems (T4SSs) have been shown to mediate the horizontal transfer of such DNA elements in a broad range of bacteria [32, 37–40]. Alternatively, (co-)mobilisation of circular intermediates of islands and related genetic elements has been described [23, 41–44].

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 (co-)mobilised by a helper plasmid, we investigated the transferability of PAI II₅₃₆, the largest PAI (102.2 kb) of UPEC strain 536, into an E. coli K-12 recipient and back into a PAI II₅₃₆-negative mutant of strain 536.

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 [18], 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-HPI_ECOR31) 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 [33]. 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 [46]. 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 [47].

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 [13].

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) [30]. In addition, the 35-kb HPI of Yersinia enterocolitica could be mobilised [51] 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 HPI_ECOR31. In the latter case, the origin of replication seems to be inactivated by insertion of an IS630 homologue [33]. This may explain why HPI_ECOR31 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 [52]. 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 [13], 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 [18], 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 [58]. The same phenomenon has been observed as well, e.g. with LEE PAIs [12] and the PAPI-1 island of P. aeruginosa [36].

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.

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 II₅₃₆ 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.