Comparative analysis of integrative and conjugative mobile genetic elements in the genus Mesorhizobium

Abstract

Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICEMspSym^AA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSym^R7A, with each carrying conserved genes for conjugative pilus formation (trb), excision (rdfS), DNA transfer (rlxS) and regulation (fseA). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with diverse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK. Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICEMlSym^R7A.

Keywords: ICE; IME; bacterial evolution; conjugation; integrase; integrative and conjugative elements; legumes; mobilization; recombinase; symbiosis; tripartite ICE.

Fig. 1.

Representative gene maps of identified conjugation and mobilization regions. For each family of MOB relaxase, a representative element of each type (ICE, IME, conjugative or mobilizable plasmid) is shown and the number of elements of the same type found in the 41 genomes is indicated. The gene map of the conjugative genes and relaxase locus displayed refers to the representative element: in black conserved genes predicted to have a role in conjugation, blue and green boxes depict T4CP and relaxase genes, respectively. Relaxases were named after their family, unless previously named. Replication modules carried by plasmids and integration sites occupied by ICEs or IMEs are reported, with * indicating att sites were not identified for some integrative elements.

Fig. 2.

ICE phylogeny and regulatory-gene organisation. (a) PhyML tree based on the concatenation of alignments of 15 single-copy backbone genes, scale bar indicates substitutions per site. (b) Organization of regulatory genes colour-coded as in c, lighter boxes are likely pseudogenes, numbers in traR and traI indicate the relatedness between the copies as described in main text, * indicates the gene is truncated. (c) Regulatory network activating ICE transfer in ICEMlSym^R7A and ICEMcSym¹²⁷¹.

Fig. 3.

Recombination detected between and among ICEs, IMEs and plasmids. Cytoscape network produced with recombination analysis between ICE, IME and plasmid genes. Elements associated with brown circles are ICEs and plasmids lacking symbiosis genes, ICESyms are green while IMEs and the mobilizable plasmid pWSM1497 are pink. The coloured regions indicate highlight ICESyms associated with Biserrula (blue), Lotus (green) and Cicer (lilac).The tripartite ICEs are circled with a dashed line. Arrows between elements start from the query sequence used in Alfy nine and point to the element within which homologous regions were detected. Arrow width is proportional to the additive length of the sequences exhibiting homology.

Fig. 4.

Chimaeric ICESym loci. Genes coding for QseC proteins are coloured in azure, qseM is displayed in red, fseA in brown, traI in purple, traR in yellow, and insertion sequences in white. Black indicates backbone genes, other genes are shown in grey, light-grey boxes indicate pseudogenes. Blue-shaded areas between genes on different ICESyms indicate homologous regions and the pairwise nucleotide identity is indicted. The triangle indicates tripartite ICE. (a) Comparison of the quorum-sensing-gene loci on ICEMjSym^MAFF303099, ICEMlSym^NZP2037 and ICEMlSym^R7A. (b) Comparison of the quorum-sensing-gene loci on ICEMcSym^M1D-2, ICEMsp.Sym^M4B and ICEMsp.Sym^M1A. (c) Comparison of the fix-gene loci on ICEMlSym^NZP2037, ICEMlSym^NZP2042 and ICEMlSym^R7A.