Comparative ICE genomics: insights into the evolution of the SXT/R391 family of ICEs

Abstract

Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements.

Figure 1. Schematic of the ICE capture system.

Conjugation between a donor strain bearing a chromosomal ICE and a ΔprfC recipient strain harboring pIceCap, which contains attB, yields exconjugants that contain the transferred ICE integrated into pIceCap. Exconjugants were selected for using a marker on pIceCap and on the ICE. attR and attL represent the right and left ICE-chromosome junctions.

Figure 2. Structure of the genomes of 13 SXT/R391 ICEs.

(A) The upper line represents the set of core genes (thick arrows) and sequences common to all 13 SXT/R391 genomes analyzed. Hatched ORFs indicate genes involved in site-specific excision and integration (xis and int), error-prone DNA repair (rumAB), DNA recombination (bet and exo) or entry exclusion (eex). Dark gray ORFs correspond to genes involved in regulation (setCDR). Light gray ORFs represent genes encoding the conjugative transfer machinery, and white ORFs represent genes of unknown function. (B) Variable ICE regions are shown with colors according to the elements in which they were originally described SXT (blue), R391 (red), ICEPdaSpa1 (green), ICESpuPO1 (purple), ICEVchMex1 (yellow), ICEPalBan1 (orange), ICEVchInd5 (turquoise), ICEPmiUSA1 (olive), ICEVchBan9 (pink), ICEVflInd1 (light green). Thin arrows indicate the sites of insertion for each variable region and HS1–HS5 represent hotspots 1–5. Roman numerals indicate variable regions not considered true hotspots. Cm, chloramphenicol; Hg, mercury; Kn, kanamycin; Sm, streptomycin; Su, sulfamethoxazole; Tc, tetracycline; Tm, trimethoprim. * indicates that s073 is absent from ICEPdaSpa1. ^a ICEVchMoz10, which lacks dfrA1 in the integron structure, does not confer resistance to Tm. ^b The purple gene content of ICEVflInd1 was deduced from partial sequencing, PCR analysis and comparison with ICESpuPO1.

Figure 3. The boundaries of the 5 hotspots.

The boundaries between conserved and hotspot variable regions are shown. Black typeface indicates conserved sequence, while color indicates variable sequence. Numbers in parentheses indicate the number of intervening nucleotides. The thin dotted lines indicate continuations of variable DNA. Bold letters indicate a non-conserved base. (A) Hotspot 1, which is present between traJ and traL. Line 1: SXT, ICEVchInd4, ICEPalBan1; Line 2: R391, ICEPdaSpa1, ICEVchBan5, ICEVchInd5, ICEPmiUSA, ICESpuPO1, ICEVflInd, ICEVchMoz10, ICEVchBan9; Line 3: ICEVchMex1. (B) Hotspot 2, which is present between traA and s054. Line 1: SXT, ICEVchInd4, ICEPmiUSA, ICEVflInd, ICEVchInd5, ICEPalBan1, ICEVchBan5; Line 2: ICEPdaSpa1, ICEVchMoz10, ICEVchBan9; Line 3: R391; Line 4: ICEVchMex1; Line 5: ICESpuPO1. (C) Hotspot 3, which is present between s073 and traF. Line 1: SXT, ICEVchInd4; Line 2: ICEVchMex1, ICEVflInd; Line 3: ICEVchMoz10, ICEVchBan9, ICEVchInd5, ICEPalBan1, ICEVchBan5. Line 4: R391; Line 5: ICEPmiUSA; Line 6: ICESpuPO1; Line 7: ICEPdaSpa1. (D) Hotspot 4, which is present between traN and s063. Line 1: SXT, ICEVchInd4. Line 2: ICEVchInd5, ICEVchBan5; Line 3: ICESpuPO1, ICEPmiUSA; Line 4: R391, ICEVchMoz10, ICEVchBan9, ICEVflInd. Line 5: ICEPdaSpa1; Line 6: ICEPalBan1; Line 7: ICEVchMex1. (E) Hotspot 5, which is present between s026 and traI. Line 1: SXT, ICEVchInd4, ICEPdaSpa1, R391, ICEVchMoz10, ICEVchBan9; Line 2: ICEPmiUSA; Line 3: ICESpuPO1, ICEPalBan1, ICEVflInd1; Line 4: ICEVchInd5, ICEVchBan5; Line 5: ICEVchMex1.

Figure 4. Comparison of the SXT/R391 core genome with the genome of pIP1202 and defining the minimal functional SXT/R391 gene set.

(A) Alignment of the conserved core genes of SXT/R391 ICEs with the genome of the IncA/C conjugative plasmid pIP1202 from Yersinia pestis. The top line shows the same core ICE genes shown in Figure 2A. ORFs are color coded as follows: DNA processing, yellow; mating pair formation, orange; DNA recombination and repair, green; integration/excision, red; replication, purple; regulation, gray; entry exclusion, blue; homologous genes of unknown function, black; genes without corresponding counterparts in ICEs and pIP1202, white. Numbers shown in the middle represent % identity between the orthologous proteins encoded by SXT and pIP1202 [GenBank:NC_009141]. The positions of the hotspots in SXT/R391 ICEs are marked by downward pointing arrowheads. For pIP1202, the size of the sequences (which include IncA/C backbone DNA as well as variable DNA) found at these locations as well as resistance markers are indicated by upward pointing arrowheads. aphA, aadA and strAB confer resistance to aminoglycosides. sul1 and sul2 confer resistance to sulfonamides. cat, bla_SHV-1, tetAR, qacED1 and merRTPCADE confer resistance to chloramphenicol, β-lactams, tetracyclines, quaternary ammonium compounds and mercury ions, respectively. Detailed descriptions of the conserved backbone of the IncA/C conjugative plasmids have been published elsewhere ,. Regions that were deleted from SXT to investigate the function of genes of unknown function (see panel B) are indicated with straight lines. Dotted lines indicate that the deletion included DNA in the adjacent hotspots. (B) Influence of deletion of genes of unknown function on the frequency of SXT transfer. The mean values and standard deviations from three independent experiments are shown. * indicates that the frequency of transfer was below the detection level (<10⁻⁸). Deletion mutants SXTΔa, SXTΔk and SXTΔl, transferred at frequencies that were not significantly different from that of wild-type SXT (data not shown). (C) Proposed minimal set of genes necessary for a functional SXT/R391 ICE. int, integration/excision module; mob, DNA processing module; mpf, mating pair formation modules; reg, regulation module.

Figure 5. Variations in the nucleotide conservation of core ICE genes.

The nucleotide sequence of each core gene from each ICE was compared to the corresponding sequence in SXT using pairwise BLASTn analyses to determine the percent identities. The average values for all of the ICEs, excluding SXT and ICEVchInd4, are shown in the inset.

Figure 6. Phylogenetic analysis of several core ICE genes.

Nucleotide sequences of the indicated core genes were used to generate the phylogenetic trees shown. Bootstrap values are indicated at branch points. The individual scale bars represent genetic distances and reflect the number of substitutions per residue.