Uncovering the prevalence and diversity of integrating conjugative elements in actinobacteria

Abstract

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.

Figure 1. Schematic representation of canonical AICEs modules and corresponding protein combinations.

Possible combination of integration/excision, replication and conjugative transfer modules are represented. For each functional module, specific domain-containing proteins where searched for as follow: Tra (FtsK/SpoIIIE, PF01580), Int-Tyr (Phage integrase, PF00589), Int-Ser (Recombinase, PF07508), RepSA (this study), RepAM (DUF3631, PF12307) and Prim-pol (Prim-pol, PF09250). AICEs Prim-pol proteins are encoded along with putative replication proteins (RepPP) or can be associated with RepAM replication initiator proteins.

Figure 2. Distribution of AICEs in completed genomes.

(A) Distribution per suborder of actinobacteria. (B) Distribution per environmental niche of the original host. (C) Distribution as a function of genome size. (A and B) Median, standard deviation (rectangles) and extreme values (brackets) are shown. (B and C) Only the Actinomycetales are shown.

Figure 3. Phylogenetic trees of the site-specific recombinases found in AICEs.

(A) Serine recombinases. (B) Tyrosine recombinases. Sub-families were defined as clades containing at least 4 members with a distance cut-off of 1 and with a >80 bootstraps support. Label colors indicate the suborders of Actinomycetales within which the AICEs were found (see legend). Colors in the outer wheel in panel A and the dark green strip in panel B indicate the type of tRNA genes into which each AICE is integrated (see Table S1 for details). Black dots in panel A for Ssrg02924 and Sslg03435 indicate that while no tRNA-encoding gene was detected as their integration sites, the att sites nearest to their respective int genes were identical to the ones of their closest relatives, i.e. pSAM2 and Ssbg00935, respectively.

Figure 4. Evolutionary relationship of RepSA-like rolling-circle initiator proteins found in AICEs.

(A) Phylogenetic tree of RepSA proteins. Sub-families were defined as clades containing at least 4 members with a distance cut-off of 1 and with a >80 bootstraps support. Label colors indicate the suborders of Actinomycetales within which the AICEs were found (see legend). (B) Comparison of the conserved motifs found in RepSA-like proteins from AICEs with the conserved motifs found in the catalytic domain of rolling-circle initiator proteins of plasmids and single-stranded DNA coliphages. The consensus sequences shown are as previously reported by Del Solar et al. . Motif 2 (HUH or His-hydrophobic-His) corresponds to the putative divalent cation-binding site and motif 3 contains the tyrosine catalytic residue. Mmu2220, Mmul0108 and Avis01496 were not included in the alignment used to generate the logo sequence of the RepSA^SAM2 conserved motifs.

Figure 5. Phylogenetic trees of putative replication-associated proteins identified in putative AICEs.

(A) RepAM (DUF3631) domain-containing proteins. (B) Prim-Pol (bifunctional DNA primase/polymerase) domain-containing proteins Label colors indicate the suborders of Actinomycetales within which the AICEs were found (see legend). The color strip in panel B indicates the type of associated replication protein: dark blue, RepAM; light blue, RepPP.

Figure 6. Phylogenetic tree of proteins containing an FtsK/SpoIIIE domain (Tra) found in AICEs.

Sub-families were defined as clades containing at least 4 members with a distance cut-off of 2 and with a >80 bootstraps support. Label colors indicate the suborders of Actinomycetales within which the AICEs were found (see legend). Outer color strips indicate AICEs encoding Prim-Pol (red), RepAM (dark blue), RepPP (light blue), and RepSA^SAM2 (green), RepSA^MR2 (light green) and other RepSA-like (dark green) replication proteins. Black dots indicate AICEs encoding serine site-specific serine recombinases.

Figure 7. Genetic organization of putative AICEs identified in 6 Frankia genomes.

The name, size and type of tRNA gene within which AICEs are inserted are indicated. Colour coding: orange, recombination; red, recombination directionality; yellow and gray, replication; blue, intermycelial transfer; lime green, regulation; dark green, transcriptional regulator LacI; pink, IS elements; mauve and fuchsia, conserved hypothetical proteins; dashed black arrows, ORFs with G+C contents <55%. Arrows indicate the noncoding sequence regions that were deleted in order to simplify the schematic representation of Fean6303. pept, peptidase C14 caspase catalytic subunit P20; HD, metal dependent phosphohydrolase; hap, Peptidase S1 and S6 chymotrypsin/Hap; LigT, 2′–5′ RNA-ligase; PhyH, phytanoyl-CoA dioxygenase; MTase_11, methyltransferase, domain MTase_11; NodU, carbamoyltransferase; PurN, phosphoribosylglycinamide formyltransferase; akr, aldo/keto reductase; amt, aminotransferase class I and II ; P450, cytochrome P450; hap, 2-alkenal reductase; NB-ARC, NB-ARC domain protein; p-kin, putative protein kinase; pcmt, protein-L-isoaspartate(D-aspartate) O-methyltransferase; php, PHP -like protein; MT_19, putative S-adenosyl-L-methionine-dependent methyltransferase, pks, Putative modular polyketide synthase; cbiA, CobQ/CobB/MinD/ParA nucleotide binding domain protein; hth, helix-turn-helix domain protein; phz, phenazine biosynthesis PhzC/PhzF protein; nbp, nucleotide-binding protein; ssb, single-strand binding protein; C2H2-lp, zinc finger - C2H2-like protein; if-2, translation initiation factor IF-2; whiB, transcription factor WhiB; cutA1, divalent ion tolerance protein CutA1; C5-MT, C-5 cytosine-specific DNA methylase; NTP-ase, putative signal transduction protein with Nacht domain – NTPase; DNA_pol3_beta_2, DNA polymerase III beta subunit, central domain.

Figure 8. Detection of excision of putative Frankia AICEs in vitro and in planta.

Formation of attP sites was detected by nested PCR in circular excised elements. (A) Detection in vitro. 1, Fean1457; 2, Fean5323; 3, Fean5518; 4, Fean5534; 5, Fean5518–5534; 6, Fean6303; 7, Fean6303–6336; 8, Faln1739; 9, Faln5456; 10, Fcci0407; 11, Fcci1033; 12, Faln2929; 13, Fcci3388. (B) Detection in planta. 1, Faln1739; 2, Faln5456. M: marker DNA fragments.