Mobilizable genomic islands: going mobile with oriT mimicry

Abstract

Many bacterial chromosomes contain genomic islands, large DNA segments that became incorporated into the chromosome following their horizontal transmission. However, the mechanisms that mediate the lateral transfer of genomic islands are for the most part unknown. In this issue of Molecular Microbiology, Daccord et al describe a new type of transmissible genomic island that can be mobilized by co-resident integrating conjugative elements (ICEs). These ‘mobilizable genomic islands’ (MGIs) require many ICE-encoded factors for their transmission, including transcription activators to induce MGI excision, the relaxase to initiate transfer at the MGI oriT and the conjugation machinery to transport MGI DNA to recipient cells. However, MGIs encode their own integrases, which enable their recombination with the chromosome of a new host, as well as a variety of other genes that do not have functions related to mobility. The MGI oriT, which resembles the ICE oriT, can also serve as a site for initiation of ICE-mediated conjugative transfer of large regions of chromosomal DNA. Overall, these findings raise the possibility that mobilization of chromosomal DNA from cyptic oriTs within genomic islands or elsewhere on the chromosome could be more common place than has been previously appreciated.

With more than 700 bacterial genome sequences now available, it is clear that most bacterial chromosomes are mosaics, composed of DNA obtained via horizontal gene transfer as well as vertical inheritance. Horizontally acquired sequences typically include integrated mobile elements, such as prophages and integrating conjugative elements (ICEs, aka conjugative transposons). Many chromosomes also contain ‘genomic islands’ (GIs), DNA segments that do not encode the machinery for self-mobilization like phages and ICEs, but nevertheless are thought to be (or to have been) mobile DNA elements because they differ in G+C content from the surrounding chromosome (Boyd et al., 2009, Dobrindt et al., 2004, Juhas et al., 2009). Furthermore, GIs are often flanked by direct repeats, and they encode recombinases that likely enable their integration into the host chromosome. GIs can be large and frequently encode sets of proteins that enable the host bacterium to occupy a new niche. For example, the TCP pathogenicity island (a kind of GI that encodes virulence determinants) enables Vibrio cholerae, the agent of cholera, to colonize the human small intestine (Herrington et al., 1988, Kovach et al., 1996). Since GIs are not self-transmissible, a key question regarding these horizontally transfered DNA segments has been how are they mobilized? In this issue of Molecular Microbiology, Burrus and colleagues identify and describe mobilizable GIs (MGIs) in several vibrio species. MGIs are satellite elements that depend on ICEs for their transfer (Daccord et al., 2010). They show that MGIs, by mimicking a co-resident ICE’s origin of transfer (oriT), can exploit ICE-encoded mobilization and transfer functions to enable their conjugative transfer to other bacteria. Their work also raises the exciting possibility that similar mimicry of oriT sequences could explain the lateral transfer of up to megabase segments of chromosomal DNA.

Conjugative DNA transfer is a multistep process that begins when a plasmid or ICE encoded relaxase (often called TraI), recognizes and nicks an oriT, a cis-acting site, yielding a single-stranded DNA break (de la Cruz et al., 2009). Subsequently, single-stranded DNA is exported to the recipient cell via plasmid or ICE-encoded conjugation machinery through a mating pore. Once transferred to the recipient cell, plasmids replicate as extrachromosomal elements, whereas ICEs integrate into the host chromosome in a process that requires an ICE-encoded recombinase (Wozniak & Waldor, 2010). Daccord et al noticed that sequences similar to the oriT of SXT, an ICE that is found in gram-negative bacteria, including several species of pathogenic vibrios, were found in the chromosomes of 2 recently sequenced vibrios (Daccord et al., 2010). Analyses of the sequences surrounding these oriT_SXT-like sequences revealed that they were part of GIs whose sequences were not related to SXT. Both GIs encoded a conserved putative recombinase (Int_MGI), but they also had distinct gene contents. A third related GI was identified by probing a collection of strains with the int_MGI sequence; all 3 MGIs were located in the same locus in their respective genomes.

Daccord et al found that SXT-encoded proteins could recognize and mediate the transfer of an ordinarily non-mobilizable vector if it contained oriT_MGI. Furthermore, the MGIs themselves were mobilizable in a SXT-dependent fashion when they were present in the same donor cell (Figure 1). Importantly, MGI transfer was dependent on oriT_MGI as well as the SXT genes traI and mobI, which encode the putative SXT relaxase and a factor required for relaxase activity respectively (Ceccarelli et al., 2008); however, SXT gene products that mediate the element’s integration into and excision from the chromosome were not required for MGI transfer. Int_MGI, the MGI recombinase, was critical for MGI excision as well the integration of MGI DNA into the recipient chromosome. Together, their findings suggest that excised circularized MGI DNA becomes a substrate for the ICE conjugation machinery because their oriTs are recognized and cleaved by ICE relaxases (Figure 1).

Figure 1.

Schematic of SXT mobilization of an MGI. A. SXT encoded transcription factors (reg) induce expression of Int_MGI. B. This recombinase enables the MGI to excise from the chromosome and circularize. TraI and MobI, the SXT encoded relaxase and accessory factor generate single-stranded MGI DNA which is transported via the SXT-encoded conjugation machinery (mpf). C. In the recipient cell, Int_MGI enables MGI DNA to recombine with chromosomal DNA. TraI and MobI can also initiate transfer of chromosomal DNA from oriT_MGI (not shown).

ICEs are not only capable of mobilizing themselves and MGIs. SXT is known to be capable of mobilizing large fragments of chromosomal DNA in cis, via a process where transfer of chromosomal DNA initiates from the ICE’s oriT (Hochhut et al., 2000). Daccord et al found that ICEs can also mediate transfer of segments of chromosomal DNA in trans, via a process where transfer of chromosomal DNA initiates from the MGI’s oriT. Since oriT_MGI and oriT_SXT are far apart in the chromosome, ICE mediated transfer of chromosomal DNA in cis, from oriT_SXT, or in trans from oriT_MGI can in principal enable horizontal transmission of most of the chromosome.

The dependence of MGIs on ICEs for their horizontal transmission is not limited to the ICE-encoded MobI/TraI and conjugation machinery. MGIs also depend on SXT transcriptional activators, which are thought to enable expression of int_MGI. Without Int_MGI, MGIs would be incapable of excision and would therefore be locked into the host chromosome. Expression of the SXT activators is elevated by the host SOS response to DNA damage (Beaber et al., 2004, Beaber & Waldor, 2004). The authors found that the frequency of MGI transfer is increased more than 100-fold by the host SOS response, at least in part because MGI excision was enhanced in this setting. Thus, MGIs take advantage of ICE-encoded regulatory machinery to control their transmission.

Overall, MGIs can be thought of as satellite conjugative elements, which depend on a co-resident ICE for their transmission. Several satellite phages, such as E. coli P4 (Liu et al., 1998) and V. cholerae RS1 (Davis et al., 2002), whose DNA is packaged into virions encoded by co-resident prophages (P2 and CTXφ for P4 and RS1, respectively), have been described. However, satellite conjugative elements are unusual, although NBUs (Non-replicating Bacteriodes Units) are another type of satellite conjugative element (Salyers et al., 1995). NBUs are excised and mobilized by Bacteriodes conjugative transposons, but in contrast to MGIs, NBUs do not depend on the relaxase encoded by the conjugative transposon. Transmission of both types of satellite conjugative elements is stimulated by conditions that induce transfer of the respective parent ICE (Song et al., 2009, Daccord et al., 2010).

Finally, as Burrus and colleagues suggest, their discovery that mimicry of oriT sequences can provide a molecular basis for mobilization of chromosomal DNA by ICEs may have broad implications for genome evolution. Mobilization of chromosomal DNA from cyptic oriTs within GIs or elsewhere on the chromosome could be much more common place than has been previously appreciated. Conjugative plasmids as well as ICEs could mobilize chromosomal DNA from cryptic chromosomal oriTs as described by Meyer (Meyer, 2009). Since MGIs require many ICE-encoded functions for excision and conjugative transfer, it seems likely that these 2 elements have co-evolved. However, less intimate connections between conjugative elements and cryptic chromosomal oriTs seem possible. Since ICEs or plasmids can enable transfer of chromosomal DNA from cyptic oriTs without being transmitted themselves, it may be difficult to assess the true contribution of oriT mimicry to genome evolution.