PMC

Distribution of relaxase families within each T4SS type.

Clade distribution of plasmid relaxases, T4SSs, and mobility class.

Distribution of conjugative, mobilizable, and nonconjugative plasmids according to plasmid size (curves were created from a polynomial interpolation of the histograms of each class).

Distribution of relaxase families according to plasmid size. Each bar shows the abundance of each relaxase family for the given plasmid size range. The rightmost bar represents the overall distribution of relaxases in all plasmids.

Distribution of T4SS types in proteobacterial plasmids according to plasmid size. Each bar shows the abundance of each T4SS family for the given plasmid size range. The rightmost bar represents the overall distribution of T4SSs in all plasmids.

Frequency with which key elements of plasmid mobility are present in plasmid genomes of different clades. The figure indicates the abundance of relaxases (MOB) in all clades and MPF types in proteobacteria. It also indicates the overall classification of plasmids in terms of mobility.

Distribution of the 1,730 GenBank plasmids by taxon. The large bars represent, for a given clade, the proportion of plasmids sequenced along with prokaryotic genomes (dark color) and sequenced independently of the host (light color). Bars are topped by the numbers of plasmids for the clades. The thin bars represent the proportion of plasmids of each clade among the plasmids sequenced with the prokaryotic host.

Presence of tRNA, rRNA, or protein-encoding genes best homologous to E. coli or B. subtilis essential genes in plasmids classified according to genome size. Small plasmids (<25 kb) rarely contain such genes, whereas very large plasmids (>400 kb) often contain them. We searched for homologues of essential B. subtilis genes in plasmids of firmicutes and for those of E. coli in the remaining plasmids. If the resident chromosome had a homologue more similar to the essential gene than the plasmid homologue, the plasmid hit was excluded from the analysis.

(A) Schematic view of the genetic constitution of transmissible plasmids. Self-transmissible or conjugative plasmids code for the four components of a conjugative apparatus: an origin of transfer (oriT) (violet), a relaxase (R) (red), a type IV coupling protein (T4CP) (green), and a type IV secretion system (T4SS) (blue). The T4SS is, in fact, a complex of 12 to 30 proteins, depending on the system (see text). Mobilizable plasmids contain just a MOB module (with or without the T4CP) and need the MPF of a coresident conjugative plasmid to become transmissible by conjugation. (B) Scheme of some essential interactions in the process of conjugation. The relaxase cleaves a specific site within oriT, and this step starts conjugation. The DNA strand that contains the relaxase protein covalently bound to its 5′ end is displaced by an ongoing conjugative DNA replication process. The relaxase interacts with the T4CP and then with other components of the T4SS. As a result, it is transported to the recipient cell, with the DNA threaded to it. Subsequently, the DNA is pumped into the recipient by the ATPase activity of the T4CP ().

Phylogenetic analysis of the T4CP family (left) compared to the T4SS main ATPase (VirB4 and TraU) (right). The middle panels indicate the phylogenetic origin of the plasmids harboring the T4CPs and the T4SSs. The phylogenetic trees were cartooned; i.e., tips were transformed into triangles whose height is proportional to the number of proteins and its depth is proportional to the most-deep-branching element, for clarity, when the groups were consistent and when support for the branches was high. When some large triangles included a few elements that do not have the same relaxase family, they are indicated by numbers [e.g., 41(5) indicates that out of 41 proteins, 5 have a different classification]. Colors in the tree cartoons correspond to families of relaxases. Numbers in nodes correspond to the values of the aLRT statistical test of branch support (). The lateral panels represent the distribution of the prototypical T4SS in the trees. To reduce computational time, sequences with ≥95% identity were collapsed into a single sequence corresponding to the longest in the set. MUSCLE v3.6 () was used to make multiple alignments (default settings), and poorly aligned columns of the alignment were removed by visual inspection using Seaview (). Phyml v3.0 was used to compute the phylogenies using maximum likelihood [model WAG+Γ(6)+I] ().

Overall diagram of the analysis to identify and classify relaxases, T4CPs, and T4SSs. To classify T4SSs, we used selected genes from four well-known systems (A), which were then used as templates to search for homology (B). To cluster homologous proteins, we used all-against-all BLASTP () (E value of <1e−4) and the Markov cluster algorithm (MCL) () (after extensive searches, I = 1.12, using a log transformation of the BLASTP E values as edge weights). We identified VirB4s and T4CPs by BLASTP. These families being homologous, a disambiguation step was necessary. To discriminate between VirB4s and T4CPs, we used an expert annotated data set of these proteins, elaborated by the authors, which was used to query the MCL clusters resulting in a resolution between the two families (I = 1.16). Phylogenetic analyses were then used to remove spurious hits. To classify T4SSs into archetypes, we analyzed the indicated genes of plasmid F (MPF_F) (GenBank accession number NC_002483), plasmid Ti (MPF_T) (accession number NC_002377), plasmid R64 (MPF_I) (accession number NC_005014), and the genomic loci of ICEHin1056 (MPF_G) (accession number AJ627386). We made PSI-BLAST searches against the plasmid database (maximum of 30 iterations). Genes with convergent PSI-BLAST results were grouped into colocalized systems (defined as sets of genes <50 genes apart), and the distribution of each gene across all systems was calculated. For each system, we defined a set of marker genes that were nearly universally present in plasmids containing T4SSs and absent from plasmids lacking T4SSs (they were called “type-specific” genes and are shown in green to distinguish them from the remaining “nonspecific” genes, shown in gray). The final list of T4SSs resulted from the joint analysis of type-specific T4SS- and VirB4-containing plasmids. The automatic discovery of relaxases first used iterative BLASTP searches using the 1,730 plasmids and the 300 N-terminal amino acids for each of the six prototypical relaxases previously described (). The E values used were those for TrwC_R388 (MOB_F; 1e−8), TraI_R27 (MOB_H; 1e−4), TraI_RP4 (MOB_P; 1e−4), MobA_RSF1010 (MOB_Q; 1e−4), MobM_pMV158 (MOB_V; 1e−5), and MobC_CloDF13 (MOB_C; 1e−4). After completing a search, the hits with the lowest E values were used as queries to retrieve distantly related sequences. We used this set to query the MCL clusters, with each relaxase family being unambiguously assigned to a cluster, which were then manually validated.