Mobilization of IMEs Integrated in the oriT of ICEs Involves Their Own Relaxase Belonging to the Rep-Trans Family of Proteins

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes.

Multiple plasmid origin-of-transfer substrates enable the spread of natural antimicrobial resistance to human pathogens

Antimicrobial resistance poses a great danger to humanity, in part due to the widespread horizontal transfer of plasmids via conjugation. Modeling of plasmid transfer is essential to uncovering the fundamentals of resistance transfer and for development of predictive measures to limit the spread of resistance. However, a major limitation in the current understanding of plasmids is the inadequate characterization of the DNA transfer mechanisms, which conceals the actual potential for plasmid transfer in nature. Here, we consider that the plasmid-borne origin-of-transfer substrates encode specific DNA structural properties that can facilitate finding these regions in large datasets, and develop a DNA structure-based alignment procedure for typing the transfer substrates that outperforms mere sequence-based approaches. We identify thousands of yet undiscovered DNA transfer substrates, showing that actual plasmid mobility can in fact be 2-fold higher and span almost 2-fold more host species than is currently known. Over half of all mobile plasmids contain the means to transfer between different mobility groups, which links previously confined host ranges across ecological habitats into a robust plasmid transfer network. We show that this network in fact serves to transfer antimicrobial resistance from the environmental genetic reservoirs to human pathogens, which might be an important driver of the observed rapid resistance development in humans and thus an important point of focus for future prevention measures.

Comparative analysis of MOBQ4 plasmids demonstrates that MOBQ is a cis-acting-enriched relaxase protein family

ABSTRACTA group of small mobilizable plasmids is increasingly being reported in epidemiology surveys of enterobacteria. Some of them encode colicins, while others are cryptic. All of them encode a relaxase belonging to a previously non-described group of the MOBQ class, MOBQ4. While highly similar in their mobilization module, two families with unrelated replicons can be distinguished, MOBQ41 and MOBQ42. Members of both groups were compatible between them and stably maintained in E. coli. MOBQ4 plasmids were mobilized by conjugation. They contain two transfer genes, mobA coding for the MOBQ4 relaxase and mobC, which was non-essential but enhanced the plasmid mobilization frequency. The origin of transfer was located between these two divergently transcribed mob genes. MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative transmission. No interference in mobilization was observed when both MOBQ41 and MOBQ42 were present in the same donor cell. Remarkably, MOBQ4 relaxases exhibited a cis-acting preference for their oriTs, a feature already observed in other MOBQ plasmids. These findings indicate that MOBQ4 plasmids can efficiently spread among enterobacteria aided by coresident IncI1, IncK and IncL/M plasmids, while ensuring their self-dissemination over highly-related elements.IMPORTANCEPlasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. A group of plasmids, called mobilizable, is able to disseminate aided by helper conjugative plasmids. Here, we studied a group of phylogenetically-related mobilizable plasmids, MOBQ4, commonly found in clinically-relevant enterobacteria, uncovering the helper plasmids responsible for their dissemination. We found that the two plasmid species encompassed in the MOBQ4 group can coexist and transfer orthogonally, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

Autonomous Replication of the Conjugative Transposon Tn916

ABSTRACTIntegrative and conjugative elements (ICEs), also known as conjugative transposons, are self-transferable elements that are widely distributed among bacterial phyla and are important drivers of horizontal gene transfer. Many ICEs carry genes that confer antibiotic resistances to their host cells and are involved in the dissemination of these resistance genes. ICEs reside in host chromosomes but under certain conditions can excise to form a plasmid that is typically the substrate for transfer. A few ICEs are known to undergo autonomous replication following activation. However, it is not clear if autonomous replication is a general property of many ICEs. We found that Tn916, the first conjugative transposon identified, replicates autonomously via a rolling-circle mechanism. Replication of Tn916was dependent on the relaxase encoded byorf20of Tn916. The origin of transfer of Tn916,oriT(916), also functioned as an origin of replication. Using immunoprecipitation and mass spectrometry, we found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916likely interact in a complex and that the Tn916relaxase contains a previously unidentified conserved helix-turn-helix domain in its N-terminal region that is required for relaxase function and replication. Lastly, we identified a functional single-strand origin of replication (sso) in Tn916that we predict primes second-strand synthesis during rolling-circle replication. Together these results add to the emerging data that show that several ICEs replicate via a conserved, rolling-circle mechanism.IMPORTANCEIntegrative and conjugative elements (ICEs) drive horizontal gene transfer and the spread of antibiotic resistances in bacteria. ICEs reside integrated in a host genome but can excise to create a plasmid that is the substrate for transfer to other cells. Here we show that Tn916, an ICE with broad host range, undergoes autonomous rolling-circle replication when in the plasmid form. We found that the origin of transfer functions as a double-stranded origin of replication and identified a single-stranded origin of replication. It was long thought that ICEs do not undergo autonomous replication. Our work adds to the evidence that ICEs replicate autonomously as part of their normal life cycle and indicates that diverse ICEs use the same replicative mechanism.