Comprehensive in silico survey of the Mycolicibacterium mobilome reveals an as yet underexplored diversity

Abstract

The mobilome plays a crucial role in bacterial adaptation and is therefore a starting point to understand and establish the gene flow occurring in the process of bacterial evolution. This is even more so if we consider that the mobilome of environmental bacteria can be the reservoir of genes that may later appear in the clinic. Recently, new genera have been proposed in the family Mycobacteriaceae, including the genus Mycolicibacterium, which encompasses dozens of species of agricultural, biotechnological, clinical and ecological importance, being ubiquitous in several environments. The current scenario in the Mycobacteriaceae mobilome has some bias because most of the characterized mycobacteriophages were isolated using a single host strain, and the few plasmids reported mainly relate to the genus Mycobacterium. To fill in the gaps in these issues, we performed a systematic in silico study of these mobile elements based on 242 available genomes of the genus Mycolicibacterium. The analyses identified 156 putative plasmids (19 conjugative, 45 mobilizable and 92 non-mobilizable) and 566 prophages in 86 and 229 genomes, respectively. Moreover, a contig was characterized by resembling an actinomycete integrative and conjugative element (AICE). Within this diversity of mobile genetic elements, there is a pool of genes associated with several canonical functions, in addition to adaptive traits, such as virulence and resistance to antibiotics and metals (mercury and arsenic). The type-VII secretion system was a common feature in the predicted plasmids, being associated with genes encoding virulent proteins (EsxA, EsxB, PE and PPE). In addition to the characterization of plasmids and prophages of the family Mycobacteriaceae, this study showed an abundance of these genetic elements in a dozen species of the genus Mycolicibacterium.

Keywords: AICE; Mycobacterium; T7SS-PE/PPE; antibiotic and metal resistance; plasmid; prophage.

Fig. 1.

Characterization of Mycolicibacterium mobile elements. (a) Number of plasmids distributed in the genomes; (b) size distribution of the predicted plasmid according to their topology; (c) number of predicted plasmids identified in Mycolicibacterium species.

Fig. 2.

Mycolicibacterium phylogenetic tree based on seven concatenated core genes of the genus (~8 kb): RNA polymerase sigma factor (hrdB-like), adenylate kinase (adk-like), 50S ribosomal subunit L19 (rplS-like), glycogen accumulation regulator (garA-like), riboflavin synthase (ribE-like), glutamine-hydrolysing carbamoyl-phosphate synthase small subunit (carA-like), and UMP kinase (pyrH-like). Genomes presenting the predicted plasmids and the known plasmids are marked with red and green squares, respectively. Green circles at branches indicate bootstrap values >80 %.

Fig. 3.

Heatmap showing groups of related plasmids. The pair of plasmids with a value of 1 (red) simultaneously present gANI≥96.5 and AF≥0.6. This analysis included all known Mycobacteriaceae plasmids (Table S3), but only those related to the predicted plasmids are displayed.

Fig. 4.

Mobility features of the predicted plasmids. (a) Number of plasmids versus their sequence size (kb) according to plasmid mobility. (b) Phylogeny of MOB_F relaxases encoded by the predicted plasmids. The blue and red branches represent the G1 and G2 relaxase groups, respectively. The green labels represent the plasmids that encode two relaxase genes. (c) Size of the relaxase proteins encoded by the predicted plasmids.

Fig. 5.

Functional characterization of the proteins encoded by the mobile elements. The number of proteins of the predicted plasmids (a) and prophages (b) presenting the most prevalent PF domains and GO terms.

Fig. 6.

Maximum-likelihood tree of ESX loci encoded by Mycobacteriaceae plasmids. The reference sequences of chromosomal and plasmid origin are indicated and coloured in green and red, respectively.

Fig. 7.

The number of prophages distributed in the genomes.

Fig. 8.

The bipartite network of the gene content of the prophages. Coloured circles and grey diamonds represent the genomes and the genes, respectively. Edges connect the genomes to the genes carried by them. The green and yellow circles represent the reference mycobacteriophages and the predicted prophages, respectively. Mycobacteriophages belonging to cluster A are delimited by the red circle.