Modular evolution of TnGBSs, a new family of integrative and conjugative elements associating insertion sequence transposition, plasmid replication, and conjugation for their spreading - PubMed (original) (raw)
Modular evolution of TnGBSs, a new family of integrative and conjugative elements associating insertion sequence transposition, plasmid replication, and conjugation for their spreading
Romain Guérillot et al. J Bacteriol. 2013 May.
Abstract
Integrative and conjugative elements (ICEs) have a major impact on gene flow and genome dynamics in bacteria. The ICEs TnGBS1 and TnGBS2, first identified in Streptococcus agalactiae, use a DDE transposase, unlike most characterized ICEs, which depend on a phage-like integrase for their mobility. Here we identified 56 additional TnGBS-related ICEs by systematic genome analysis. Interestingly, all except one are inserted in streptococcal genomes. Sequence comparison of the proteins conserved among these ICEs defined two subtypes related to TnGBS1 or TnGBS2. We showed that both types encode different conjugation modules: a type IV secretion system, a VirD4 coupling protein, and a relaxase and its cognate oriT site, shared with distinct lineages of conjugative elements of Firmicutes. Phylogenetic analysis suggested that TnGBSs evolved from two conjugative elements of different origins by the successive recruitment of a transposition module derived from insertion sequences (ISs). Furthermore, TnGBSs share replication modules with different plasmids. Mutational analyses and conjugation experiments showed that TnGBS1 and TnGBS2 combine replication and transposition upstream promoters for their transfer and stabilization. Despite an evolutionarily successful horizontal dissemination within the genus Streptococcus, these ICEs have a restricted host range. However, we reveal that for TnGBS1 and TnGBS2, this host restriction is not due to a transfer incompatibility linked to the conjugation machineries but most likely to their ability for transient maintenance through replication after their transfer.
Figures
Fig 1
Distribution of conserved proteins among Tn_GBS1_-like ICEs and related conjugative plasmids. On the right, the presence of a gene encoding a protein similar to a Tn_GBS1_ protein is indicated by a filled box. The intensity of the blue color corresponds to the percent identities according to the indicated scale. For the related plasmids, the presence of a gene coding for a similar protein or a protein showing a similar domain is indicated by a gray box. The CDS map of Tn_GBS1_ is shown on the left. Genes are indicated by arrows. The gene encoding the DDE transposase, proteins involved in conjugation, and proteins involved in replication are colored in red, green, and orange, respectively. Boxes marked with a white line correspond to genes with a frameshift mutation or a sequencing error. ICE accession numbers are indicated in Table S3 in the supplemental material. Plasmid accession numbers are as follows: pMP118, NC_007930; pAW63, NC_010599; pFR55, NC_010283; pHTβ, AB183714; and pMG1, NC_011364.
Fig 2
Distribution of conserved proteins among Tn_GBS2_-like ICEs and related conjugative plasmids and ICEs depending on a phage-like integrase. On the right chart, the presence of a gene encoding a protein similar to a Tn_GBS2_ protein is indicated by a filled box. The intensity of the blue color corresponds to the percent identities according to the indicated scale. For the related plasmids, the presence of a gene coding for a similar protein or a protein showing a similar domain is indicated by a gray box. The CDS map of Tn_GBS2_ is shown on the left. Genes are indicated by arrows. The gene encoding the DDE transposase, proteins involved in conjugation, and proteins involved in replication are colored in red, green, and orange, respectively. Boxes marked with a white line correspond to genes with a frameshift mutation or a sequencing error. Tn_GBS2_-related ICE accession numbers are indicated in Table S3 in the supplemental material. Plasmid and ICE accession numbers are as follows: pTEF2, NC_004671.1; pCF10, AY855841.2; sex factor, NC_009004; pIL6, HM021331.1; Tn_5253_, EU351020.1; ICESde3396, EU142041; 89K pathogenicity island of S. suis, EU589333.1; and pBD-II, NC_017476.
Fig 3
Phylogenetic tree of the Tn_GBS_ transposases. Maximum likelihood (ML) using PhyML on the phylogeny.fr platform (20) was used to infer phylogenetic relationships. ML bootstrap support was determined using 100 bootstrap replicates. The sequence of the transposases of Lactobacillus crispatus, Lactobacillus helveticus, and Bacillus coagulans ISs were used to root the tree. The names correspond to the ICE name given in Table S3 in the supplemental material.
Fig 4
Sequence of the putative origins of replication of Tn_GBS1_ and Tn_GBS2_. (A) The putative origins of replication located downstream of the Tn_GBS1 repE_ homologs were aligned with validated or hypothetical origins of replication of Gram-positive plasmids. The validated replication initiation site of pAMβ1 is indicated by an arrow (23). (B) The putative origins of replication located within the repA coding region of Tn_GBS2_, Tn_Ssang2.1_, and Tn_Soral2.1_ were aligned with the oriR site of the S. aureus plasmid pSK41. The four directly repeated sequences (Rep box 1 to Rep box 4) correspond to Rep protein binding sites identified in the S. aureus plasmid pSK41 oriR region (27). Conserved bases are indicated in blue and nonconserved bases in red.
Fig 5
Replication of Tn_GBS1_ and Tn_GBS2_ correlates with its transfer efficiency. (A) The amount of circular forms and the efficiency of transfer were quantified in three independent experiments for Tn_GBS1_ and Tn_GBS2_. Ratios of circular forms to the total amount of ICEs (indicated with blue bars) were quantified by qPCR in the initial donor strain (D) in transconjugants just after selection (TC) and in transconjugants after serial passages on plates to allow the integration of the ICE into the chromosome (TCP). Purple bars represent the conjugation efficiency measured by the ratio of TC colonies to donor colonies using D, TC, and TCP as donor strains. (B) Quantification of the circular forms of Tn_GBS1_ and Tn_GBS2_. Tn_GBS1_ was quantified in strains GMP212 (BM110 Tn_GBS1_::erm), GMP208 (BM110 Tn_GBS1-repE_::pG1), GMP208 pJIM-repE (BM110 Tn_GBS1-repE_::pG1, complemented), and GMP211 (a BM110 Tn_GBS1_-repE+::pG1 transconjugant). Tn_GBS2_ was quantified in strains GMP201 (NEM316 Tn_GBS2_::erm), GMP207 (NEM316 Tn_GBS2-repA_::pG1), GMP207 pJIM-repA (NEM316 Tn_GBS2-repA_::pG1, complemented), and GMP209 (NEM316 Tn_GBS2-repA_+::pG1). Quantifications were performed in triplicate.
Fig 6
Tn_GBS oriT_ regions. (A) Tn_GBS1 oriT_ regions. The predicted origin of transfer of Tn_GBS1_ located upstream of the gbs0384 putative relaxase gene in Tn_GBS1_ was aligned with validated or hypothetical oriT sites of Gram-positive plasmids. The two inverted repeats are shown in green and blue, and the conserved nucleotides are in black and bold. (B) Tn_GBS2 oriT_ region. The conserved parts of the predicted origin of transfer of selected Tn_GBS2_-like ICEs located upstream of the gbs1121 relaxase gene in Tn_GBS2_ were aligned with the validated or hypothetical oriT sites of Gram-positive plasmids and ICEs. The inverted repeat is shown in blue, and the conserved sequence that includes the nick site is in bold. The demonstrated nick sites in pRS01 and pCF10 are indicated by an arrow.
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