Diversity of integrating conjugative elements in actinobacteria: Coexistence of two mechanistically different DNA-translocation systems

Abstract

Conjugation is certainly the most widespread and promiscuous mechanism of horizontal gene transfer in bacteria. During conjugation, DNA translocation across membranes of two cells forming a mating pair is mediated by two types of mobile genetic elements: conjugative plasmids and integrating conjugative elements (ICEs). The vast majority of conjugative plasmids and ICEs employ a sophisticated protein secretion apparatus called type IV secretion system to transfer to a recipient cell. Yet another type of conjugative DNA translocation machinery exists and to date appears to be unique to conjugative plasmids and ICEs of the Actinomycetales order, a sub-group of high G + C Gram-positive bacteria. This conjugative system is reminiscent of the machinery that allows segregation of chromosomal DNA during bacterial cell division and sporulation, and relies on a single FtsK-homolog protein to translocate double-stranded DNA molecules to the recipient cell. Recent thorough sequence analyses reveal that while this latter strategy appears to be used by the majority of ICEs in Actinomycetales, the former is also predicted to be important in exchange of genetic material in actinobacteria.

Figure 1. Conjugative transfer models of ICEs from the two superfamilies. (A): (1) In the donor cell, ICE excision from the chromosome results from site-specific recombination between the attL and attR sites. Following excision, the relaxase (Mob), which is part of a multiprotein complex called relaxosome, recognizes the origin of transfer (oriT). (2) The Mob protein generates a nick in one strand and becomes covalently bound to the 5′ end of the nicked strand. (3) While the single-stranded nucleoprotein complex is displaced by ongoing rolling-circle (RC) replication, it interacts with the type IV coupling protein (T4CP) which generates the energy for its translocation through a dedicated type IV secretion system (T4SS). (4) Once transferred in the recipient cell, the Mob protein ligates the single-stranded DNA molecule and the complementary strand is synthesized. (5) Integration in the recipient cell’s chromosome is mediated by recombination between the attP site on the circular ICE and the chromosomal attB site. (B): (1) Like ICEs, AICEs excise from the chromosome by site-specific recombination. (2) The excised circular AICE then replicates by RC replication and reintegrate into the chromosome and/or transfer to a recipient cell by conjugation. (3) The transfer protein Tra recognizes the AICE cis-acting locus (clt) and mediates the transfer of the double-stranded AICE by forming a pore (Tra hexamer) in the lipid bilayer and the use of its ATPase activity. (4) The circular AICE integrates into the chromosome by site-specific recombination as described above. Alternatively, integration into the chromosome of the recipient cell could be preceded by an additional step in which RC replication would occur.

Figure 2. Taxonomic distribution of AICEs. Percent of the 140 AICEs detected in complete (130) and draft (145) actinobacterial genomes is indicated for each clade. Numbers in parentheses represent the number of genomes analyzed for each clade. The clades for which no AICEs could be found are not shown [Acidimicrobidae (1) Coriobacteridae (15), Rubrobacteridae (2), Glycomycineae (1), Kineosporiineae (1), unclassified actinobacteria (1)]. The four AICEs detected in the analysis of 176 actinobacterial plasmids are not included.

Figure 3. Phylogenetic analysis of the actinobacterial T4SS-type ICE tyrosine integrases. The phylogenetic relatedness of the tyrosine integrases encoded by 17 T4SS-type ICEs (black) with those of the 9 AICEs tyrosine-integrase subfamilies (green) is represented. For simplification, only the 9 proteins encoded by the AICEs of each eponymous subfamily were used for this analysis. The relatedness of the tyrosine integrases of Intca3128 and Nbcg01645 with those of pMR2 and pSLS, respectively, is emphasized (gray shading).