A degenerate primer MOB typing (DPMT) method to classify gamma-proteobacterial plasmids in clinical and environmental settings

Abstract

Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from γ-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of γ-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call "Degenerate Primer MOB Typing" or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type.

Figure 1. DPMT validation for MOB_F relaxases.

A) Phylogenetic tree of MOB_F relaxases. Triangles at the end of the branches represent a compressed group of very similar relaxases (>95%). A solid black arrow points to the prototype plasmid for each subfamily. Arrows point to plasmids that experimentally amplified, in spite of containing at least one mismatch in the 12 nucleotides of the CORE sequence. Relaxases contained in our reference collection (Table 1) are denoted by an asterisk. Plasmids detectable by PBRT amplification , , , , , , , , are underlined. New relaxase sequences uncovered by DPMT are shown in red. B) Alignment of the relaxase motifs used to design the MOB_F degenerate primers. Colour code: red on yellow  =  invariant amino acids; blue on blue  =  strongly conserved; black on green  =  similar; green on white  =  weakly similar; black on white  =  not conserved. Black arrowheads point to the key residues that define the relaxase motifs. Different rectangles embrace the conserved amino acids used to infer the 3′ degenerate core of each oligonucleotide (F11-f, continuous black; F12-f, continuous dark grey; and F1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB_F11 (F11-f and F1-r). Lane 1, pSU1588; 2, pSU4280; 3, pSU10013; 4, pSU10014; 5, pSU10017; 6, pSU10018; 7, pSU10021; 8, pSU316; 9, pSU10022; 10, pSU10010; 11, R751; 12, pSU10028; 13, pSU10029; 14, pSU10056; 15, pSU10055; 16, pSU10001; 17, pSU10012; 18, pSU10011; 19, pSU10009; 20, pSU4601; 21, pSU10006; 22, pSU10007; 23, pSU10064; 24, pSU10059; 25, pSU10008; 26, pSU10039; 27, pSU10040; 28, pSU10041; 29, pSU10004; 30, pSU10003; 31, pSU10043; 32, pSU4830; 33, pSU10002; 34, negative control. Lane M, molecular mass marker, HyperLadder IV (Bioline). D) Amplicons obtained with primers for subfamily MOB_F12 (F12-f and F1-r). Lanes as in (C).

Figure 2. DPMT validation for MOB_P1 relaxases.

A) Phylogenetic tree of MOB_P1 relaxases. B) Alignment of the relaxase motifs used to design the MOB_P1 degenerate primers (P11-f, continuous black; P12-f, continuous dark grey; P131-f, continuous grey; P14-f, continuous light grey; and P1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB_P11 (P11-f and P1-r). D) Amplicons obtained with primers for subfamily MOB_P12 (P12-f and P1-r). E) Amplicons obtained with primers for subfamily MOB_P13 (P131-f and P1-r). F) Amplicons obtained with primers for subfamily MOB_P14 (P14-f and P1-r). Symbols, colour codes and lanes as in Figure 1.

Figure 3. DPMT validation for MOB_P3 and MOB_P4 relaxases.

A) Phylogenetic tree of MOB_P3 and MOB_P4 relaxase families. B) Alignment of the relaxase motifs used to design the MOB_P3 and MOB_P4 degenerate primers (P3-f+P3-r, continuous black; and P4-f+P4-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB_P31 (P3-f and P3-r). D) Amplicons obtained with primers for subfamily MOB_P42 (P4-f and P4-r). Symbols, colour codes and lanes as in Figure 1.

Figure 4. DPMT validation for MOB_P5 relaxases.

A) Phylogenetic tree of MOB_P5 relaxase family. B) Alignment of the relaxase motifs used to design the MOB_P5 degenerate primers (P51-f, continuous black; P52-f, continuous dark grey; P5-r, dashed black; and P53-f+P53-r, continuous grey) C) Amplicons obtained with primers for subfamily MOB_P51 (P51-f and P5-r). D) Amplicons obtained with primers for subfamily MOB_P52 (P52-f and P5-r). E) Amplicons obtained with primers for subfamily MOB_P53 (P53-f and P53-r). Symbols, colour codes and lanes as in Figure 1.

Figure 5. DPMT validation for MOB_Q relaxases.

A) Phylogenetic tree of MOB_Q relaxase family. B) Alignment of the relaxase motifs used to design the MOB_Q degenerate primers (Q11-f+Q11-r, continuous black; Q12-f+Q12-r, continuous dark grey; and Qu-f+Qu-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB_Q11 (Q11-f and Q11-r). D) Amplicons obtained with primers for subfamily MOB_Q12 (Q12-f and Q12-r). E) Amplicons obtained with primers for subfamily MOB_Qu (Qu-f and Qu-r). Symbols, colour codes and lanes as in Figure 1.

Figure 6. DPMT validation for MOB_H relaxases.

A) Phylogenetic tree of MOB_H relaxase family. B) Alignment of the relaxase motifs used to design the MOB_H degenerate primers (H11-f+H11-r, continuous black; H121-f+H121-r, continuous dark grey; and H2-f+H2-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB_H11 (H11-f and H11-r). D) Amplicons obtained with primers for subfamily MOB_H121 (H121-f and H121-r). E) Amplicons obtained with primers for subfamily MOB_H2 (H2-f and H2-r). Symbols, colour codes and lanes as in Figure 1.

Figure 7. DPMT validation for MOB_C relaxases.

A) Phylogenetic tree of MOB_C relaxase family. B) Alignment of the relaxase motifs used to design the MOB_C degenerate primers (C11-f+C11-r, continuous black; C12-f+C12-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB_C11 (C11-f and C11-r). D) Amplicons obtained with primers for subfamily MOB_C12 (C12-f and C12-r). Symbols, colour codes and lanes as in Figure 1.

Figure 8. Correspondence between MOB and Rep types.

A) Simplified phylogenetic representation of the five relaxase MOB families considered in this study. Coloured triangles represent the MOB subfamilies amplified by DPTM. Their width and depth correspond, respectively, to the abundance and phylogenetic diversity of their relaxase sequences (Table S1). B) The Inc groups contained within each MOB subfamily are indicated at the right, boxed in the same colour. When no Inc group is contained, the name of a prototype plasmid is given.