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Genomic islands targeting dusA in Vibrio species are distantly related to Salmonella Genomic Island 1 and mobilizable by IncC conjugative plasmids

  • Romain Durand,

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Current address: Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec, Canada

    Affiliation Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada

  • Florence Deschênes,

    Roles Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review & editing

    Affiliation Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada

  • Vincent Burrus

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    vincent.burrus@usherbrooke.ca

    Affiliation Département de biologie, Université de Sherbrooke, Sherbrooke, Québec, Canada

Abstract

Salmonella Genomic Island 1 (SGI1) and its variants are significant contributors to the spread of antibiotic resistance among Gammaproteobacteria. All known SGI1 variants integrate at the 3’ end of trmE, a gene coding for a tRNA modification enzyme. SGI1 variants are mobilized specifically by conjugative plasmids of the incompatibility groups A and C (IncA and IncC). Using a comparative genomics approach based on genes conserved among members of the SGI1 group, we identified diverse integrative elements distantly related to SGI1 in several species of Vibrio, Aeromonas, Salmonella, Pokkaliibacter, and Escherichia. Unlike SGI1, these elements target two alternative chromosomal loci, the 5’ end of dusA and the 3’ end of yicC. Although they share many features with SGI1, they lack antibiotic resistance genes and carry alternative integration/excision modules. Functional characterization of IMEVchUSA3, a dusA-specific integrative element, revealed promoters that respond to AcaCD, the master activator of IncC plasmid transfer genes. Quantitative PCR and mating assays confirmed that IMEVchUSA3 excises from the chromosome and is mobilized by an IncC helper plasmid from Vibrio cholerae to Escherichia coli. IMEVchUSA3 encodes the AcaC homolog SgaC that associates with AcaD to form a hybrid activator complex AcaD/SgaC essential for its excision and mobilization. We identified the dusA-specific recombination directionality factor RdfN required for the integrase-mediated excision of dusA-specific elements from the chromosome. Like xis in SGI1, rdfN is under the control of an AcaCD-responsive promoter. Although the integration of IMEVchUSA3 disrupts dusA, it provides a new promoter sequence and restores the reading frame of dusA for proper expression of the tRNA-dihydrouridine synthase A. Phylogenetic analysis of the conserved proteins encoded by SGI1-like elements targeting dusA, yicC, and trmE gives a fresh perspective on the possible origin of SGI1 and its variants.

Author summary

We identified integrative elements distantly related to Salmonella Genomic Island 1 (SGI1), a key vector of antibiotic resistance genes in Gammaproteobacteria. SGI1 and its variants reside at the 3’ end of trmE, share a large, highly conserved core of genes, and carry a complex integron that confers multidrug resistance phenotypes to their hosts. Unlike members of the SGI1 group, these novel genomic islands target the 5’ end dusA or the 3’ end of yicC, lack multidrug resistance genes, and seem much more diverse. We showed here that, like SGI1, these elements are mobilized by conjugative plasmids of the IncC group. Based on comparative genomics and functional analyses, we propose a hypothetical model of the evolution of SGI1 and its siblings from the progenitor of IncA and IncC conjugative plasmids via an intermediate dusA-specific integrative element through gene losses and gain of alternative integration/excision modules.

Introduction

Integrative and mobilizable elements (IMEs) are discrete, mobile chromosomal regions that can excise from the chromosome and borrow the mating apparatus of helper conjugative elements to transfer to a new bacterial host [1,2]. IMEs are usually composed of two main functional modules. The site-specific recombination module contains genes and cis-acting sequences that mediate the integration of the IMEs into and their excision from the chromosome. The mobilization module includes the cis-acting origin of transfer (oriT) and usually encodes mobilization proteins required to initiate the conjugative transfer at oriT [1]. In its simplest form, the mobilization module only consists of an oriT locus mimicking the oriT of the helper element [35]. The excision of IMEs is elicited by conjugative plasmids or integrative and conjugative elements (ICEs). These helper elements encode the type IV secretion system (T4SS) that translocates the IME DNA into the recipient cell [1].

Several distinct families of IMEs have been described to date. Most encode beneficial traits for their host, such as resistance to antibiotics and heavy metals or bacteriocin synthesis [1,6]. Salmonella Genomic Island 1 (SGI1) is certainly one of the most studied IMEs. Though first described 20 years ago, SGI1 and its siblings have only recently gained a lot of attention due to their prevalence and prominent role in the spread of multidrug resistance [7,8]. The canonical 43-kb SGI1 resides at the 3’ end of trmE (also known as mnmE or thdF) in Salmonella enterica serovar Typhimurium DT104 [9]. trmE encodes the 5-carboxymethylaminomethyluridine-tRNA synthase GTPase subunit. SGI1 variants have been reported in a wide array of Gammaproteobacteria, including Proteus mirabilis (PGI1), Acinetobacter baumannii (AGI1), Morganella, Providencia, Enterobacter, Escherichia coli, Vibrio cholerae (GI-15), and Klebsiella pneumoniae [7,10,11]. Most variants carry a class I integron structurally similar to the In104 integron of SGI1. In104 confers resistance to ampicillin, chloramphenicol/florfenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracycline [8,12]. SGI1 and its variants are an epidemiological threat exacerbated by their specific mobilization by conjugative plasmids of the incompatibility groups A (IncA) and C (IncC) [13,14]. IncC plasmids contribute to the global circulation of multidrug resistance genes, including NDM metallo-β-lactamase and carbapenemase genes, among a broad range of Gammaproteobacteria [15,16]. The transcriptional activator AcaCD encoded by IncC plasmids triggers the excision and mobilization of SGI1 [17,18].

SGI1 and most variants share a conserved core set of 28 genes, representing 27.4 kb, disrupted by insertion sequences and the class 1 integron inserted at diverse positions (Fig 1, top) [7,9,12]. Thus far, the function of a few conserved genes has been characterized. Together with the cis-acting recombination site attP, the genes int and xis form the recombination module of SGI1 [13]. int encodes the site-specific tyrosine recombinase (integrase) that targets the 3’ end of trmE. xis encodes the recombination directionality factor (RDF or excisionase) that enhances the excision reaction catalyzed by Int. The mobilization module includes the mobilization genes mpsAB and the oriT located upstream of mpsA [19]. mpsA encodes an atypical relaxase distantly related to tyrosine recombinases. Unlike most characterized IMEs, SGI1 carries a replicon composed of an iteron-based origin of replication (oriV) and the replication initiator gene rep [20,21]. SgaCD, a transcriptional activator complex expressed by SGI1 in response to a coresident IncC plasmid, controls rep expression [21,22]. The excised replicative form of SGI1 destabilizes the helper plasmid by an unknown process, and is further stabilized by its sgiAT addiction module [20,2224]. Finally, SGI1 encodes three mating pore subunits, TraNS, TraHS, and TraGS, that actively replace their counterparts in the T4SS encoded by the IncC plasmid [25]. The substitution of TraG allows SGI1 to bypass the IncC-encoded entry exclusion mechanism and transfer between cells carrying conjugative plasmids belonging to the same entry exclusion group [26].

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Fig 1. Schematic representations of SGI1-related IEs.

The position and orientation of open reading frames (ORFs) are indicated by arrowed boxes. Colors depict the function deduced from functional analyses and BLAST comparisons. Potential AcaCD binding sites are represented by green angled arrows. Each island is flanked by the attL and attR (vertical grey lines) attachment sites when integrated into the 3’ end of trmE (light blue), the 5’ end of dusA (light green), or the 3’ end of yicC (pink). The annotation of attL and attR relative to int is based on SGI1 (trmE) [9], IEAbaD1279779 of Acinetobacter baumannii D1279779 (dusA) [30] and MGIVflInd1 (yicC) [3]. Details regarding ORFs are shown in S1 Dataset.

https://doi.org/10.1371/journal.pgen.1009669.g001

Given the high similarity between SGI1 variants integrated at trmE, we undertook a search for distant SGI1-like IMEs in bacterial genomes using MpsA, TraGS, SgaC, and TraNS as baits. Here, we report the existence of distantly related IMEs integrated at the 5’ end of dusA in several species of Vibrionaceae and the 3’ end of yicC in several species of Gammaproteobacteria. We have examined the interactions between an IncC plasmid and IMEVchUSA3, a dusA-specific representative IME from an environmental V. cholerae strain. The genetic determinants required for the excision of IMEVchUSA3 and its mobilization by IncC plasmids were characterized. Finally, we took a fresh look at the emergence and evolution of SGI1 and its siblings by conducting phylogenetic analyses and proposed a hypothetical evolutionary pathway of putative IMEs resembling SGI1.

Results

Novel integrative elements (IEs) distantly related to SGI1 are inserted in dusA and yicC in various Gammaproteobacteria

To find novel SGI1-like elements, we searched the Refseq database using blastp and the primary sequences of MpsA, TraGS, SgaC, and TraNS. Considering the substitution of integration modules can change the integration site [2729], the integrase InttrmE was excluded from the analysis. We identified 24 distinct integrative elements encoding homologs of the four bait proteins in 36 different bacterial strains (Fig 1, Tables 1 and S1). 21 of these IEs are integrated into the 5’ end of dusA (tRNA-dihydrouridine synthase A) in diverse Vibrio species from various origins. The remaining three are located at the 3’ end of yicC (unknown function) in E. coli, Aeromonas veronii, P. mirabilis, S. enterica serovar Kentucky, and Pokkaliibacter plantistimulans. The size of the IEs varies from 22.8 kb to 37.1 kb. The conserved genes mpsA (together with mpsB), traG, traN, and sgaC remain in a syntenic order, though sporadically separated by variable DNA (Fig 1).

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Table 1. Main features of the IEs described in this study.

https://doi.org/10.1371/journal.pgen.1009669.t001

Consistent with the change of integration site, the respective int genes of SGI1 and the dusA- and yicC-specific IEs do not share any sequence similarity. Furthermore, unlike SGI1, these novel IEs lack xis downstream of int (Fig 1). Instead, yicC-specific IEs carry two small open reading frames (ORF) upstream of the attR site. The putative translation product of the second one shares 35% identity over 65% coverage with the excisionase RdfM of MGIVflInd1 [31]. Although dusA-specific IEs lack xis and rdfM, all carry an ORF predicted to encode a 76-aminoacyl residue protein containing the pyocin activator protein PrtN domain (Pfam PF11112). Based on its size, position, predicted DNA-binding function, conservation, and evidence presented below, we named this ORF rdfN.

Phylogenetic analysis of IntyicC proteins of yicC-specific SGI1-like IEs form a cluster distinct from the integrases of IMEs mobilizable by IncC plasmids through a MobI protein (Pfam PF19456), such as MGIVmi1, and IMEs that mimic the oriT of SXT/R391 ICEs, such as MGIVflInd1 [3,17,32] (Fig 2A).

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Fig 2. Integrases encoded by the yicC- and dusA-specific IEs.

Maximum likelihood phylogenetic analyses of IntyicC (A) and IntdusA (B). The trees are drawn to scale, with branch lengths measured in the number of substitutions per site over 400 and 359 amino acid positions for IntyicC, and IntdusA, respectively. The helper elements and mechanism of mobilization are indicated for each lineage according to the keys shown in the legend box of panel A. The inset of panel B shows logo sequences of the repeats in attL and attR attachment sites. The arrows indicate the island termini experimentally determined for IEAbaD1279779 by Farrugia et al. [30]. (C) Heatmap showing blastp identity percentages of pairwise comparison of IntdusA representative proteins. Proteins accession numbers are provided in S2 Dataset, except for IEAbaD1279779 (WP_000534871.1), IEPprPf-5 of Pseudomonas protegens Pf-5 (WP_011060295.1), and IEs of Burkholderia gladioli BSR3 (WP_013697845.1), Bradyrhizobium sp. BTAi1 (WP_012043559.1), Agrobacterium sp. H13-3 (WP_013636109.1), and Neisseria gonorrhoeae FA 1090 (EFF39980.1).

https://doi.org/10.1371/journal.pgen.1009669.g002

Phylogenetic analysis of IntdusA proteins confirmed that the integrases of these IEs form a monophyletic group exclusive to the Vibrionaceae and distinct from those encoded by other dusA-specific IEs found in other taxa, including GIAcaBra1 from Aeromonas caviae that is likely mobilizable by IncC plasmids via a MobI protein [32] (Fig 2B). IntdusA proteins of the IEs identified here share at least 75% identity, while identities drop below 60% with the non-Vibrio IntdusA proteins (Fig 2C). Sequence logos built using alignments of the attL and attR chromosomal junctions revealed a 21-bp imperfect repeat at the extremities of each IE (Fig 2B). This repeat is similar to the one reported for dusA-specific IEs found in a broader range of species [30].

Three types of dusA-integrated SGI1-related elements

Blastn and blastp analyses using SGI1ΔIn104 as the reference confirmed that the identified dusA-specific IEs share limited sequence similarities with SGI1 (S1A Fig). Besides the genes encoding MpsA, TraG, SgaC, and TraN, all carry the auxiliary mobilization factor gene mpsB and the oriT sequence (Fig 1). Secondary structure prediction of the aligned oriT sequences located upstream of mpsA using RNAalifold revealed that despite the sequence divergence, the structure of oriT with three stem-loops was strictly conserved (S2B Fig). In contrast, sgaD is not strictly conserved and highly divergent from sgaD of SGI1 when present (Figs 1 and S1A).

Comparison using IEVchUSA2 as the reference suggests that dusA-specific IEs cluster into three distinct types as confirmed by the phylogenetic analysis of concatenated MpsA-TraG-SgaC-TraN (Figs 3A, S1B, and S3). Type 1 dusA-specific IEs such as IMEVchUSA3 are mainly found in V. cholerae and lack both traH and sgaD (Figs 1 and 3A). Type 2 IEs such as IEVchUSA2 lack sgaD but carry traH. This lineage only includes two dusA-specific IEs of V. cholerae but also closely related yicC-specific IEs such as IEEcoMOD1 and the trmE-specific GIVchO27-1. Finally, type 3 IEs such as GIVchUSA5 are the most distant from the two other types and SGI1. Type 3 IEs carry both traH and sgaD and reside in diverse Vibrio species. With the exception of a few outliers encoded by IEs such as IEVchN2817, IEVchN2708 or IEPplInd1, the proteins MpsA, TraG, SgaC and TraN encoded by members of the same type typically share more than 95% identity (Figs 3B and S3). MpsA remains the least divergent protein between the three types, sharing at least 65% identity between type 1 and type 3, and from 64% to 93% with SGI1. In contrast, TraG and TraN are the most divergent between types, ranging from 46% to 59% for TraG and from 46% to 76% for TraN.

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Fig 3. Conserved genes support three main lineages of dusA-specific SGI1-like IEs.

(A) Maximum likelihood phylogenetic analysis of concatenated MpsA-TraG-SgaC-TraN. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site over 2,637 amino acid positions. Taxa corresponding to IEs targeting trmE and yicC are indicated by a light blue circle and a red circle, respectively. All other taxa correspond to dusA-specific IEs. Phylogenetic relationships of MpsA, TraG, SgaC and TraN proteins are shown separately in S2 Fig. (B) Heatmaps showing blastp identity percentages of pairwise protein comparisons for representatives of MpsA, TraG, SgaC, and TraN. Proteins accession numbers and clusters are provided in S1 Table and S2 Dataset.

https://doi.org/10.1371/journal.pgen.1009669.g003

Worthy of note, these three distinct lineages of dusA-specific IEs are supported by the phylogeny of the oriT sequences (S2A Fig). Again, oriT loci of type 3 IEs strongly diverge from those of types 1 and 2, as well as from the oriT loci of the highly homogenous SGI1 group.

Variable features found in the dusA- and yicC-specific IEs

Most variable genes in the identified IEs encode proteins of unknown function. A search for antibiotic resistance determinants using the Resistance Gene Identifier server failed to reveal any known resistance gene. Several IEs encode putative functions altering host processes and virulence, including the transport of ions and small molecules (ktrAB, trkH, and kdpD for potassium uptake and rcnAR for nickel/cobalt efflux in IEVchHai10, sulfite export in IEVchN2817 and IEVchSwe1), c-di-GMP degradation (IEVchBan1), and fimbriae (IEVchBan1) (S1 Dataset).

None of the reported IEs carries the same replication module (S004-rep-oriV) as canonical SGI1. Instead, five dusA-specific IEs belonging to the type 3 lineage (IEVchUSA5, IEVchBra2, IEVpaChn1, IEVpaChn2, and IEVpaBan1a) encode a putative replication initiator protein with the IncFII_repA domain (Pfam PF02387) (Fig 1, S1 Dataset). IEVvuUSA1 encodes a putative helicase with an UvrD_C_2 domain (Pfam PF13538), whereas, like SGI1, IEVchHai10, IEVchUSA2 and IEVchSwe1 encode a putative ATP-dependent helicase (PcrA) and a putative ATP-dependent endonuclease (YbjD). In addition, IEVchN2786, IESenUSA1 and IEEcoMOD1 encode a predicted DEAD/DEAH box helicase (Pfam PF00270 and PF00271). The three yicC-specific IEs encode a homolog of TrfA (Pfam PF07042), the replication initiator protein of broad-host-range IncP plasmids [33]. No replicative functions could be ascribed with confidence to any gene carried by the other dusA-specific IEs. Several IEs also encode toxin-antitoxin systems, such as sgiAT and higAB, which likely enhance their stability (Fig 1). In the type 3 IEs IEVchBra2, IEVchN2708, IEVpaChn1 and IEVpaBan1a, sgiAT is associated with a gene coding for a putative abortive infection bacteriophage resistance factor (Abi_2, Pfam PF07751). Likewise, IEVvuUSA1 carries a gene coding for a different putative abortive infection bacteriophage resistance factor (AbiEii toxin, Pfam PF13304).

Finally, IEVpaBan1a is integrated at dusA adjacent to a distinct IE, IEVpaBan1b, in a tandem fashion. GIVpaBan1b codes for two predicted integrases sharing 44% and 27% identity with IntdusA of IEVpaBan1a. GIVpaBan1b encodes a putative type I restriction-modification system, a MobA-like relaxase (MOBP1), the mobilization auxiliary factor MobC, and an RdfN homolog (Fig 1).

Non-canonical SGI1-like IEs carry AcaCD-responsive genes

Considering the divergence of the 24 new IEs from prototypical SGI1, we wondered whether an IncC plasmid could mobilize them like SGI1. The hallmark of IncC-dependent mobilization is the presence of AcaCD-responsive promoters in IncC-mobilizable IEs. Hence, we searched for putative AcaCD-binding sites in the sequences of trmE-specific IEs (prototypical SGI1 was used as the positive control) and the yicC- and dusA-specific IEs. In these IEs, an AcaCD-binding motif was predicted upstream of traN, traHG (or traG), S018, and xis (or rdfM or rdfN) (Figs 1 and S4). Moreover, an AcaCD-binding motif was also predicted upstream of trfA in the yicC-specific IEs.

We cloned the promoter sequences of int, traN, traG, S018, and rdfN of IMEVchUSA3 upstream of a promoterless lacZ reporter gene and monitored the β-galactosidase activity with or without AcaCD. The promoter Pint was active regardless of the presence of AcaCD (Fig 4A). In contrast, the four other promoters exhibited weak activity in the absence of AcaCD. Upon induction of acaDC expression, PtraN and PS018 remained unresponsive, while the activities of PtraG and PrdfN increased 40 and 400 times, respectively (Fig 4B). The inertia of PtraN and PS018 toward AcaCD could result from single nucleotide substitutions in the AcaCD binding site previously shown to be essential for recruiting the activator [22]: CCSAAAWW instead of CCSCAAWW in PtraN and CCCCAAAA instead of CCCAAAAA in PS018 (S4 Fig).

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Fig 4. β-galactosidase activities of the promoters Pint, PtraN, PtraG, PS018 and PrdfN of IMEVchUSA3 transcriptionally fused to lacZ.

(A) Colonies were grown on LB agar with or without arabinose to induce acaDC expression from pBAD-acaDC. (B) Induction levels of the same promoters in response to AcaCD. β-galactosidase assays were carried out using the strains of panel A. Ratios between the enzymatic activities in Miller units for the arabinose-induced versus non-induced strains containing pBAD-acaDC are shown. The bars represent the mean and standard error of the mean of three independent experiments.

https://doi.org/10.1371/journal.pgen.1009669.g004

Hence, despite their divergence and different integration sites, these IEs share with SGI1 a common activation mechanism elicited by the presence of an IncC plasmid.

IncC plasmids induce the excision and mobilization of IMEVchUSA3

Next, we tested whether a coresident IncC plasmid could trigger the excision of IMEVchUSA3 from dusA in its original host, V. cholerae OY6PG08. The derepressed IncC plasmid pVCR94Kn Δacr2 [34] was introduced into OY6PG08 by conjugation from E. coli KH40. The Δacr2 mutation improves the efficiency of interspecific transfer of the plasmid [35]. OY6PG08 KnR transconjugants were tested by PCR to amplify the attL and attR chromosomal junctions, as well as the attB and attP sites resulting from the excision of IMEVchUSA3 (S5A Fig). IMEVchUSA3 was rarely retained in the transconjugants compared to the control IncC-free OY6PG08 clones, suggesting it was unstable and rapidly lost in IncC+ cells (S5B and S5C Fig).

To test the interspecific mobilization of IMEVchUSA3 from V. cholerae OY6PG08, we inserted a selection marker upstream of traG and used pVCR94Kn Δacr2 as the helper plasmid. IMEVchUSA3Cm transferred to E. coli CAG18439 at a frequency of 7.01 × 10−5 transconjugant/donor CFUs. Amplification of attL and attR using E. coli-specific primers confirmed that IMEVchUSA3 integrates at dusA in E. coli (S5D Fig).

Excision of dusA-specific IEs depends on rdfN

To further characterize the biology of IMEVchUSA3, we measured its excision rate and copy number by qPCR, with and without coresident pVCR94Sp. We also monitored its intraspecific transfer (E. coli to E. coli) in the same context. Spontaneous excision of the IE rarely occurred (<0.001% of the cells) (Fig 5A). In contrast, in the presence of the helper plasmid, the free attB site was detected in more than 67% of the cells confirming that the IncC plasmid elicits the excision of IMEVchUSA3Kn. Likewise, the presence of the plasmid resulted in a ~3-fold increase of the copy number of IMEVchUSA3Kn (Fig 5B), suggesting that the excised form undergoes replication. The frequency of transfer of IMEVchUSA3Kn was comparable to that of the helper plasmid (~3.5×10−2 transconjugants/donor), while the frequency of cotransfer was more than two logs lower (Fig 5C).

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Fig 5. Effect of acaDC and rdfN on the IncC-dependent excision and mobilization of IMEVchUSA3.

(A) IMEVchUSA3Kn excision rate corresponds to the attB/chromosome ratio. (B) IMEVchUSA3Kn copy number corresponds to the higA/chromosome ratio. For panels A and B, all ratios were normalized using the control set to 1 and displayed in white. (C) Impact of acaC, acaDC, sgaC and rdfN deletions on the mobilization of IMEVchUSA3. Conjugation assays were performed with CAG18439 (Tc) containing the specified elements as donor strains and VB112 (Rf) as the recipient strain. The bars represent the mean and standard error of the mean obtained from a biological triplicate. ¤ indicates that the excision rate or transfer frequency was below the detection limit. Statistical analyses were performed (on the logarithm of the values for panels A and C) using a one-way ANOVA with Dunnett’s multiple comparison test. For panels A and B, statistical significance indicates comparisons to the normalization control. Statistical significance is indicated as follows: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant. (D) Schematic representation of mini-IE inserted at the 5’ end of dusA. (E) RdfN acts as a recombination directionality factor. Detection of attB, attP, attL and attR sites by PCR in colonies of E. coli EC100 dusA::mini-IE in the presence or absence of rdfN. L, 1Kb Plus DNA ladder (Transgen Biotech).

https://doi.org/10.1371/journal.pgen.1009669.g005

Thus far, the factors required to catalyze the excision of dusA-specific IEs have not been examined [30]. Whereas all dusA-specific IEs lack xis downstream of int, they carry a small ORF, here named rdfN, coding for a putative PrtN homolog (Fig 1) [30]. The deletion of rdfN abolished the excision and replication of IMEVchUSA3Kn. Complementation by ectopic expression of rdfN from the arabinose-inducible promoter PBAD restored the wild-type excision level but not the replication (Fig 5A and 5B). Likewise, deletion of rdfN abolished the mobilization of IMEVchUSA3Kn but had no impact on the transfer of the helper plasmid (Fig 5C), confirming the specific role of rdfN in the IE’s mobility.

To confirm that rdfN encodes the sole and only RDF of IMEVchUSA3, we constructed mini-IE, a minimal version of IMEVchUSA3 that only contains int and a spectinomycin-resistance marker. mini-IE is flanked by attL and attR and is integrated at dusA in E. coli EC100 (Fig 5D). Using mini-IE, attB and attP were detected only upon ectopic expression of rdfN from pBAD-rdfN, confirming that no other IMEVchUSA3-encoded protein besides Int and RdfN is required for the excision of the element (Fig 5E). rdfN is the essential RDF gene that favors the excision of IMEVchUSA3 and, most likely, all dusA-specific IEs.

A SgaC/AcaD hybrid complex activates the excision and mobilization of IMEVchUSA3

Next, we investigated the role of the transcriptional activator genes acaC and sgaC in the mobilization of IMEVchUSA3. Deletion of acaDC abolished the excision and replication of IMEVchUSA3Kn, confirming that its excision relies on rdfN, whose expression is activated by AcaCD (Figs 4, 5A, and 5B). The mutation also confirmed that SgaC provided by IMEVchUSA3Kn is insufficient by itself to elicit rdfN expression. The excision rate remained extremely low in cells that lack the helper plasmid or cells that carry pVCR94Sp ΔacaDC. However, IMEVchUSA3Kn allowed the low-frequency transfer of pVCR94Sp ΔacaDC [17] (Fig 5C). Hence SgaC alone can activate to some degree the expression of the transfer genes of the helper plasmid. In contrast, deletion of acaC had no significant impact on the excision, replication, and mobilization of IMEVchUSA3Kn, or on the transfer of the helper plasmid (Fig 5A, 5B, and 5C). The primary sequences of AcaC and SgaC from IMEVchUSA3 share 85% identity over 94% coverage, whereas AcaC and SgaC from SGI1 share only 75% identity over 92% coverage. Hence AcaD produced by the plasmid and SgaC produced by the IME likely generate a functional chimeric transcriptional complex that acts as a potent activator of rdfN and the transfer genes.

The transfer of IMEVchUSA3Kn ΔsgaC decreased nearly 3 logs compared to the wild-type IE, despite the presence of acaDC on the helper plasmid (Fig 5C). Moreover, deletion of both acaC and sgaC nearly abolished all transfer. Ectopic expression of sgaC alone from pBAD-sgaC complemented these deletions to wild-type levels (Fig 5C). These observations confirm that sgaC, not acaC, combined with acaD produces a hybrid activator complex that is essential for the excision and mobilization of IMEVchUSA3.

IMEVchUSA3 provides a new promoter and N-terminus for dusA expression

Since dusA-specific IEs insert within the 5’ end of dusA, we wondered whether the gene remains expressed after the integration event. Sequence analysis of the attR junction of E. coli K12 transconjugants revealed that IMEVchUSA3 provides a new 5’ coding sequence that diverges significantly from the native E. coli dusA gene (Fig 6A). This alteration of the 5’ end of dusA results in a novel N-terminus of identical length sharing 61% identity over the 35 initial amino acid residues with native DusA. To test the expression of dusA, we constructed a translational lacZ fusion to its fortieth codon downstream of the attR junction in E. coli CAG18439 and BW25113 (Fig 6B). β-galactosidase assays revealed that dusA remains expressed after integration in both strains, confirming that IMEVchUSA3 provides a new promoter (Fig 6C). However, we observed a statistically significant reduction of dusA expression resulting from the integration of the IE in both strains, suggesting that the transcription or translation signals brought by the IE are weaker than the original ones upstream of E. coli dusA.

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Fig 6. IMEVchUSA3 drives the expression of dusA.

(A) Comparison of the coding sequences of the 5’ end of dusA in E. coli K12 MG1655 before (attB site) and after (attR junction) the integration of IMEVchUSA3. The core sequence of the attB and attR recombination sites is indicated with red shading. The ATG start codon of dusA is shown in bold. The sequence shown in blue is internal to IMEVchUSA3. Amino acid residues shown in red differ from the native N-terminus of DusA. This sequence was obtained by sequencing the attR junction of an E. coli CAG18439 dusA::IMEVchUSA3Kn transconjugant colony. (B) Schematic representation of the dusA’-lacZ translational fusion for the detection of dusA expression. (C) β-galactosidase activity of the dusA’-‘lacZ fusion before (-) and after (+) insertion of IMEVchUSA3Kn in E. coli CAG18439 (FD034) and BW25113 (FD036). The bars represent the mean and standard error of the mean of three independent experiments. Statistical analyses were performed using an unpaired t test to compare the expression before and after integration of IMEVchUSA3Kn for each strain. Statistical significance is indicated as follows: **, P < 0.01; *, P < 0.05.

https://doi.org/10.1371/journal.pgen.1009669.g006

Discussion

SGI1-like elements integrated at the 3’ end of trmE are widespread in a broad range of Enterobacteriaceae and sporadically found in a few Vibrio species [7]. The integrase of SGI1 and its variants occasionally targets the intergenic region between sodB and purR genes, a secondary attachment site [36]. Here, we report the identification of distant SGI1-like elements that specifically target the 5’ end of dusA in multiple Vibrio species and the 3’ end of yicC in Enterobacteriaceae and Balneatrichaceae. Farrugia et al. [30] already described IEs integrated at the 5’ end of dusA, mostly prophages or phage remnants found exclusively in Alpha-, Beta- and Gammaproteobacteria. These authors identified IEVchBan1 and IEVchBra2 in V. cholerae, and several other IEs predicted to encode conjugative functions in Bradyrhizobium, Caulobacter, Mesorhizobium, Paracoccus, Pseudomonas, and Rhodomicrobium [30]. Our group recently reported a dusA-specific IE in Aeromonas caviae 8LM potentially mobilizable by IncC plasmids [32]. GIAca8LM lacks tra genes but encodes a mobilization protein (MobI) under the control of an AcaCD-responsive promoter. Together, these reports confirm that dusA is an insertion hotspot for distinct families of mobile elements across at least three Proteobacteria phyla.

Thus far, only the dusA-specific IEs in A. baumannii D1279779 and P. protegens Pf-5 were shown to excise from the chromosome, albeit at a low level [30]. Neither IE has been tested for intercellular mobility. Here, we characterized IMEVchUSA3, a representative member of a subgroup of dusA-specific IEs circulating in Vibrio species and distantly related to SGI1. We demonstrated that IMEVchUSA3 is mobilizable by IncC conjugative plasmids to E. coli. In the presence of an IncC plasmid, this IME excises in practically all cells of the population and becomes highly unstable (Figs 5A and S5B). We showed that its excision was under the control of AcaCD provided by the IncC plasmid and required rdfN, a gene whose expression is driven by an AcaCD-responsive promoter (Fig 4). rdfN encodes a novel RDF distantly related to the pyocin activator protein PrtN of Pseudomonas. rdfN seems to be ubiquitous, yet highly divergent, in dusA-specific IEs reported by Farrugia et al. [30]. For instance, RdfN (PrtN) encoded by the IE of P. protegens Pf-5 shares only 29% identity with RdfN of IMEVchUSA3, and their promoters are unrelated. Hence, the expression of rdfN homologs encoded by different families of dusA-specific IEs is likely controlled by different factors. Only the IEs that have evolved AcaCD-responsive promoters for their RDF gene are expected to be mobilizable by IncC or related plasmids.

Excision and mobilization of IMEVchUSA3 occurred in the presence of a ΔacaC but not a ΔacaDC mutant of the helper plasmid (Fig 5), confirming that sgaC of the IME produces a functional activator subunit that can interact with AcaD provided by the plasmid. Furthermore, we showed here that, unlike acaC, sgaC plays a central role in the biology of IMEVchUSA3 as the absence of acaC had no effect on the excision or transfer of the IME, while the absence of sgaC in spite of the presence of acaC, compromised its mobilization (Fig 5A, 5B, and 5C). We recently showed that AcaD most likely stabilizes the binding of AcaC to the DNA [22]. Therefore, AcaD and SgaC from IMEVchUSA3 likely interact to form a chimerical activator complex. This interaction could compensate for the loss of sgaD in yicC- and type 1 and 2 dusA-specific IEs (Fig 1). The primary sequences of AcaC and SgaC of IMEVchUSA3 (type 1) share 85% identity. In contrast, AcaC only shares 75% identity with SgaC of SGI1 and 64% identity with SgaC of GIVchUSA5 (type 3), suggesting that retention of sgaD allowed faster divergence of SgaC from AcaC. Retention of sgaC in the IEs could result from its essential role as the elicitor of excision and replication reported for SGI1. Indeed, although AcaCD binds to the promoters Pxis and Prep of SGI1, it fails to initiate transcription at these two promoters, unlike SgaCD [22]. Nonetheless, Pxis and Prep are not conserved in the IEs described here. S004-rep is missing, whereas rdfN or rdfM replaced xis, under the control of novel AcaCD-responsive promoters (Figs 4 and S4). This observation raises intriguing questions regarding the recruitment of functional gene cassettes and their assimilation in a regulatory pathway. How did xis, rdfN, and rdfM acquire their AcaCD-responsive promoters? Is it by convergent evolution? What are the signals driving rdfN expression and IE excision in dusA-specific IEs resembling prophages?

Approximately 3 copies per cell of IMEVchUSA3 were detected in the presence of the helper IncC plasmid (Fig 5B), lower than the copy number reported for SGI1 (~8 copies/cell) [20,22]. IMEVchUSA3 lacks SGI1’s replication module (S004-rep-oriV); however, one of the multiple genes of unknown function could encode an unidentified replication initiator protein. Notably, GIVchO27-1 encodes a putative replication protein with an N-terminal replicase domain (PF03090) and a C-terminal primase domain (PriCT-1, PF08708) [20]. Multiple IEs also carry putative replicons based on repA and trfA (Fig 1), suggesting that independent replication is crucial in their lifecycle, perhaps to improve their stability in the presence of a helper plasmid [2024].

Farrugia et al. [30] hypothesized that dusA-specific IEs could restore the functioning of DusA. We demonstrated here that IMEVchUSA3 provides a new promoter allowing expression of dusA, though at a lower level than in IME-free cells, and restores the open reading frame with an altered N-terminus (Fig 6). Similarly, the ICE SXT that targets the 5’ end of the peptide chain release factor 3 (RF3) gene prfC provides a new promoter and N-terminus in both V. cholerae, its original host, and E. coli [37]. In both cases, the consequences of the alteration of the N-terminus on the activity of the protein remain unknown.

The relative positions of int and rdfN/rdfM across the attP site suggest that, to remain functional, the recombination modules must be acquired or exchanged when the IEs are in their excised circular form. The promiscuity of different families of IEs targeting yicC, dusA, and trmE and mobilizable by IncC plasmids could act as the catalyst for these recombination events. During entry into a new host cell by conjugation, IncC plasmids elicit the excision of such IEs and promote homologous recombination between short repeated sequences in response to double-stranded break induced by host defense systems (CRISPR-Cas3) [34]. Hence the diversification of IncC plasmid-mobilizable IEs could be a side effect of the DNA repair mechanism used by these plasmids.

Unlike SGI1 and its siblings, all dusA-specific SGI1-like IEs reported here lack antibiotic resistance genes. Furthermore, SGI1 variants are prevalent in several pathogenic species and relatively well-conserved, whereas their dusA-specific relatives are scarce and highly divergent. These observations suggest that despite the considerable functional resemblances between trmE- and dusA-specific SGI1-like IEs, the epidemiological success of the SGI1 lineage has directly stemmed from the acquisition of class I integrons conferring multidrug resistance by forerunner elements such as SGI0 [38]. Based on the phylogenetic relationships between the core proteins MpsA, TraG, SgaC and TraN, oriT loci, and integrase proteins (Figs 2, 3, S2A, and S3), we propose a hypothetical evolutionary pathway leading to the emergence of the different types of IEs described here (Fig 7). The diversity of dusA-specific IEs and relative homogeneity of the SGI1 group suggest that the latter originated from the progenitor of IncA and IncC plasmids via a dusA-specific IE intermediate.

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Fig 7. Proposed hypothetical evolutionary pathway of SGI1-like IEs.

The sequence of events was inferred from the phylogenetic trees presented in this study, site of integration and conservation of traH and sgaD in the IEs. The proposed pathway ignores the gene cargo and presumes that the IE lineages evolved from the progenitor of IncA and IncC plasmids. The dusA-specific recombination module was chosen as the progenitor to minimize gain/loss and recombination events. Green and red arrows indicate gene gains and losses, respectively. The orange dashed line indicates a probable recombination event from which stemmed GIVchO27-1.

https://doi.org/10.1371/journal.pgen.1009669.g007

Materials and methods

Bacterial strains and media

Bacterial strains and plasmids used in this study are described in Table 2. Strains were routinely grown in lysogeny broth at 37°C in an orbital shaker/incubator and were preserved at -75°C in LB broth containing 20% (vol/vol) glycerol. Antibiotics were used at the following concentrations: ampicillin (Ap), 100 μg/ml; chloramphenicol (Cm), 20 μg/ml; erythromycin (Em), 200 μg/ml; kanamycin (Kn), 10 μg/ml for single-copy integrants of pOPlacZ-derived constructs, 50 μg/ml otherwise; nalidixic acid (Nx), 40 μg/ml; rifampicin (Rf), 50 μg/ml; spectinomycin (Sp), 50 μg/ml; tetracycline (Tc), 12 μg/ml. Diaminopimelate (DAP) was supplemented to a final concentration of 0.3 mM when necessary.

Mating assays

Conjugation assays were performed as previously described [25]. However, mixtures of donor and recipient cells were incubated on LB agar plates at 37°C for 4 hours. Donors and recipients were selected according to their sole chromosomal markers. When required, mating experiments were performed using LB agar plates supplemented with 0.02% arabinose to induce expression of pBAD30-derived complementation vectors. Frequencies of transconjugant formation were calculated as ratios of transconjugant per donor CFUs from three independent mating experiments.

Molecular biology

Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (Qiagen), according to manufacturer’s instructions. Restriction enzymes used in this study were purchased from New England Biolabs. Q5 DNA polymerase (New England Biolabs) and EasyTaq DNA Polymerase (Civic Bioscience) were used for amplifying cloning inserts and verification, respectively. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), according to manufacturer’s instructions. E. coli was transformed by electroporation as described by Dower et al. [39] in a Bio-Rad GenePulser Xcell apparatus set at 25 μF, 200 Ω and 1.8 kV using 1-mm gap electroporation cuvettes. Sanger sequencing reactions were performed by the Plateforme de Séquençage et de Génotypage du Centre de Recherche du CHUL (Québec, QC, Canada).

Plasmids and strains constructions

Plasmids and oligonucleotides used in this study are listed in Tables 2 and S2, respectively. IMEVchUSA3Cm was constructed by inserting the pir-dependent replication RP4-mobilizable plasmid pSW23T [40] at locus CGT85_RS05425 of V. cholerae OYP6G08 (Genbank NZ_NMSY01000009) by homologous recombination. Briefly, CGT85_RS05425 was amplified using primer pair dusAigEcoRIF/dusAigEcoRIR. The amplicon was digested with EcoRI and cloned into EcoRI-digested pSW23T using T4 DNA ligase. The resulting plasmid was confirmed by restriction profiling and DNA sequencing, then introduced into the DAP-auxotrophic E. coli β2163 [40] by transformation. This strain was used as a donor in a mating assay to transfer the plasmid into V. cholerae OYP6G08, generating IMEVchUSA3Cm. Single-copy integration of the pSW23T derivative was confirmed by PCR and antibiotic resistance profiling.

IMEVchUSA3Kn was constructed from IMEVchUSA3Cm. Briefly, pVCR94Kn Δacr2 was transferred from the DAP-auxotrophic E. coli KH40 into OYP6G08 bearing IMEVchUSA3Cm. After selection on LB agar medium supplemented with chloramphenicol and kanamycin, CmR KnR V. cholerae OYP6G08 transconjugants were confirmed by growth on thiosulfate-citrate-bile salts-sucrose (TCBS) agar medium (Difco). In V. cholerae, the integration and excision of the IME were confirmed by amplification of the attL, attR, attB, and attP sites with primer pairs oRD4/ORD6, oRD1/oRD3, oRD1/oRD6, and oRD4/oRD3, respectively. IMEVchUSA3Cm was then mobilized from OYP6G08 to E. coli CAG18439. In E. coli, the integration and excision of the IME were confirmed by amplification of the attL, attR, attB and attP sites with primer pairs oRD4/ORD5, oRD2/oRD3, oRD2/oRD5 and oRD4/oRD3, respectively. IMEVchUSA3Kn was constructed by replacing pSW23T with a single kanamycin resistance marker using the one-step chromosomal gene inactivation technique with primer pair dusAscarNoFRTf/dusAscarNoFRTr and pKD13 as the template. The deletions ΔsgaC and ΔprtN in IMEVchUSA3Kn were obtained using the primer pairs oFD26r/oFD26f and DelprtNr/DelprtNf, and pKD3 and pVI36 as the templates, respectively. The ΔdapA deletion mutant of E. coli MG1655 was constructed using primer pair FwDeltaDapA-MG1655/ RvDeltaDapA-MG1655 and pKD3 as the template. The ΔlacZ mutation was introduced in E. coli CAG18439 using primer pair lacZW-B/lacZW-F and plasmid pKD4 as the template. The dusA-‘lacZ fusion was introduced in E. coli BW25113 and CAG18439 using primer pair oDF15/oDF16 and pVI42B as the template. The fortieth codon of dusA was fused to the eighth codon of lacZ downstream of the attB site. The λRed recombination system was expressed using either pSIM6, pSIM9 or pKD46 [41,42]. When appropriate, resistance cassettes were excised from the resulting constructions using the Flp-encoding plasmid pCP20 [43]. All deletions were validated by antibiotic profiling and PCR.

Fragments encompassing promoter regions upstream of int, traN, traG, s018 and rdfN were amplified using primer pairs oFD6.f/oFD6.r, oFD1.f/oFD1.r, oFD3.f/oFD3.r, oFD5.f/oFD5.r and oFD4.f/oFD4.r, respectively, and genomic DNA from E. coli CAG18439 dusA::IMEVchUSA3Kn as the template. The amplicons were digested with PstI/XhoI and cloned into PstI/XhoI-digested pOPlacZ [17]. The resulting constructs were single-copy integrated into the attBλ chromosomal site of E. coli BW25113 using pINT-ts [44]. To construct the expression vectors pBAD-rdfN and pBAD-sgaC, PCR fragments containing rdfN or sgaC were amplified from genomic DNA of E. coli CAG18439 bearing IMEVchUSA3 as the template and primer pairs prtNEcoRIf/prtNHindIIIrev and oFD38r/oFD44f, respectively. The PCR fragments were digested by either EcoRI or SacI, and HindIII and cloned into pBAD30 cut with the same enzymes.

mini-IE was constructed as follows. The 1,591-bp fragment of excised circular IMEVchUSA3Kn that contains attP-int was amplified using primer pair oVB12/oVB10 and genomic DNA from E. coli CAG18439 dusA::IMEVchUSA3Kn as the template. The 1,421-bp fragment of pVI36 that contains aadA7 was amplified using primer pair oVB11/oVB13. Both fragments were joined using the PCR-based overlap extension method [45]. After the final PCR amplification using oVB12/oVB13, the amplicon was purified, digested with SacI, and ligated. The ligation mixture was then transformed into E. coli EC100. Transformant colonies were selected on LB agar supplemented with spectinomycin. The constitutive expression of int and the absence of replicon prompted the spontaneous integration of mini-IE at the 5’ end of dusA in EC100.

All final constructs were verified by PCR and DNA sequencing by the Plateforme de Séquençage et de Génotypage du Centre de Recherche du CHUL (Québec, QC, Canada).

qPCR assays

qPCR assays for quantification of excision and copy number of IMEVchUSA3Kn were carried out as described previously [22] with the following modification. attBdusA (241 bp) and higA (229 bp) of IMEVchUSA3Kn were quantified using primer pairs attBdusAqPCRfwd/ attBdusAqPCRrev and higAqPCRfwd/ higAqPCRrev, respectively (S2 Table). The excision rate and copy number of IMEVchUSA3Kn were calculated as the ratio of free attBdusA site per chromosome and as the ratio of higA per chromosome, respectively. The data were analyzed and normalized using all three chromosomal genes dnaB, hicB and trmE as references and the qBase framework as described previously [22,46].

β-galactosidase assays

The assays were carried out on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or in LB broth using o-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate as described previously [32]. acaDC expression from pBAD-acaDC was induced by adding 0.2% arabinose to a refreshed culture grown to an OD600 of 0.2, followed by a 2-h incubation at 37°C with shaking prior to cell sampling.

Comparative analyses

Sequences were obtained using blastp against the Genbank Refseq database with the primary sequences of key proteins MpsA, TraGS, SgaC, TraNS of SGI1 (Genbank AAK02039.1, AAK02037.1, AAK02036.1, AAK02035.1, respectively), and IntdusA of IEVchBra2 (Genbank EEO15317.1) and IntyicC of IEEcoMOD1 (Genbank WP_069140142.1). Hits were exported, then sorted by accession number to identify gene clusters that likely belong to complete IEs. Sequences of IEs were manually extracted and the extremities were identified by searching for the direct repeats contained in attL and attR sites. When an IE sequence spanned across two contigs (e.g., IEVchHai10 and IEPplInd1), the sequence was manually assembled. IE sequences were clustered using cd-hit-est with a 0.95 nucleotide sequence identity cut-off [47]. Some of the annotated sequences were manually curated to correct missing small open reading frames such as mpsB, and inconsistent start codons. Pairwise comparisons of Int, MpsA, TraG, SgaC and TraN proteins were generated with blastp using sets of representative proteins selected after clustering using cd-hit with a 0.95 sequence identity cut-off (Int, MpsA, TraG, SgaC) or a 0.90 sequence identity cut-off (TraN) [47]. Heatmaps showing the blastp identity scores were drawn using the Python library seaborn v0.11.1 [48]. Circular blast representations (blast atlases) were generated with the Blast Ring Image Generator (BRIG) 0.95 [49], with blastn or blastp, against SGI1ΔIn104 and IEVchUSA2, with an upper identity threshold of 80% and a lower identity threshold of 60%. Antibiotic resistance gene prediction was conducted using the Resistance Gene Identifier (RGI) software and CARD 3.1.3 database [50]. AcaCD binding motifs were identified using FIMO and MAST [51] with the AcaCD motif matrix (S1 Matrix) described previously [17]. Logos for attL and attR repeats were generated with MAST [51] using alignments of sequences flanking the IEdusA elements identified in this work.

Phylogenetic analyses

Evolutionary analyses were conducted in MEGA X [52] and inferred by using the maximum likelihood method based on the JTT (MpsA or SgaC proteins), LG (IntdusA, IntyicC, TraG or RepAIncFII proteins) or WAG (TraN) matrix-based models [5355]. Protein sequences were aligned with Muscle [56]. Aligned sequences were trimmed using trimal v1.2 using the automated heuristic approach [57]. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with the superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories) for IntdusA (parameter = 3.5633), IntyicC (parameter = 2.6652), SgaC (parameter = 1.4064), TraG (parameter = 1.9005) and TraN (parameter = 1.6476) proteins. For IntdusA, MpsA and TraG, the rate variation model allowed for some sites to be evolutionarily invariable ([+I], 7.81% sites for IntdusA, 44.62% sites for MpsA and 5.22% sites for TraGS). The trees are all drawn to scale, with branch lengths measured in the number of substitutions per site. In all trees, bootstrap supports are shown as percentages at the branching points only when > 80%.

oriT sequences were obtained manually using the previously identified oriT of SGI1 as the reference [19], then clustered using cd-hit-est with a 1.0 nucleotide sequence identity cut-off. Sequences were then aligned using Muscle and a NeighborNet phylogenetic network was built using SplitsTree4 [58] with default parameters (Uncorrected_P method for distances and EqualAngle drawing method). The secondary structures of the aligned oriT sequences were predicted using RNAalifold v2.4.17 from the ViennaRNA package [59]. Default options were used (including no RIBOSUM scoring), except for the following: no substituting "T" for "U" (—noconv), no lonely pairs (—noLP), no GU pairs (—noGU) and DNA parameters (-P DNA). The predicted Vienna output and the annotated alignment were merged into a predicted secondary structure of SGI1 oriT color-coded to display the inter-island diversity.

Statistical analyses and figures preparation

Numerical data presented in graphs are available in S3 Dataset. Prism 8 (GraphPad Software) was used to plot graphics and to carry out statistical analyses. All figures were prepared using Inkscape 1.0 (https://inkscape.org/).

Supporting information

S1 Fig. Comparative sequence analysis of SGI1-like dusA-specific IEs.

Blastn and blastp atlases using either SGI1ΔIn104 (A) or IEVchUSA2 (B) as the reference. Coding sequences appear on the outermost circle in blue for the positive strand and red for the negative strand, with the oriT depicted as a grey arc. All other sequences are represented only according to their homology with the reference, with full opacity corresponding to 100% identity and gaps indicating identity below 60%. The order of the IEs in the atlases is indicated according to the color keys shown in the inset of panel B.

https://doi.org/10.1371/journal.pgen.1009669.s001

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S2 Fig. NeighborNet phylogenetic network (A) and predicted secondary structure of 39 oriT loci (B) of SGI1-like IEs.

Each IE’s integration site and type are annotated. The sequence of canonical SGI1 (Genbank AF261825.2) was used as a reference to show the predicted secondary structure of all oriT sequences. Pairs can be perfectly conserved, imperfectly conserved (1/39 not conserved), not conserved (> 1/39), or an A-T or G-C pair only. In the latter case, the sequence is not conserved, but the predicted local secondary structure is.

https://doi.org/10.1371/journal.pgen.1009669.s002

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S3 Fig. Maximum likelihood phylogenetic analysis of key proteins of SGI1-related IEs.

The trees for MpsA (A), TraG (B), SgaC (C) and TraN (D) proteins are drawn to scale, with branch lengths measured in the number of substitutions per site over 321, 1,145, 188, and 968 amino acid positions, respectively. For clarity, the lengths of the branches linking the two groups in panels A and C were artificially divided by 8 and 4, respectively. Taxa corresponding to IEs targeting trmE and yicC are shown by a light blue circle and a red circle, respectively. All other taxa correspond to dusA-specific IEs. Proteins accession numbers are provided in S1 Table and S2 Dataset.

https://doi.org/10.1371/journal.pgen.1009669.s003

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S4 Fig. Alignment of AcaCD-responsive promoters predicted in IEs targeting dusA, yicC and trmE.

Promoter sequences are grouped based on the function of the expressed genes as follows: (A) RDFs; (B) mating pair stabilization; (C) mating pair formation and stabilization; (D) unknown. AcaCD binding sites are shown in green. Logo sequences and p-values were generated by MAST [51]. Known transcription start sites are shown in blue [17,22]. Predicted Shine-Dalgarno sequences are shown in pink. The initiation start codon is shown in bold letters.

https://doi.org/10.1371/journal.pgen.1009669.s004

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S5 Fig. Excision of IMEVchUSA3 is enhanced in IncC+ cells.

(A) Model of excision of IMEVchUSA3. (B and C) Detection of attB, attP, attL and attR sites by PCR in colonies of V. cholerae OYO6G08 bearing (lanes 9 to 16) or lacking (lanes 1 to 8) pVCR94Kn Δacr2. Control lanes: L, 1Kb Plus DNA ladder (Transgen Biotech); +, V. cholerae N16961 genomic DNA. (D) Detection of attB, attP, attL and attR sites by PCR in transconjugant colonies of E. coli CAG18439 (lanes 1 to 4). L, 100bp Plus II DNA Ladder (Transgen Biotech)

https://doi.org/10.1371/journal.pgen.1009669.s005

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S1 Table. Features of the identified IEs and associated strains.

https://doi.org/10.1371/journal.pgen.1009669.s006

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S2 Table. Oligonucleotides used in this study.

https://doi.org/10.1371/journal.pgen.1009669.s007

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S1 Dataset. Features of ORFs in the identified IEs.

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S2 Dataset. Clusters generated by cd-hit for Int, MpsA, TraG, SgaC, and TraN.

https://doi.org/10.1371/journal.pgen.1009669.s009

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S3 Dataset. Numerical data presented in Figs 46.

https://doi.org/10.1371/journal.pgen.1009669.s010

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S1 Matrix. AcaCD motif matrix to identify AcaCD binding sites.

https://doi.org/10.1371/journal.pgen.1009669.s011

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Acknowledgments

We are grateful to Yann Boucher for the kind gift of Vibrio cholerae OYP6G08 and Kévin T. Huguet for technical assistance. We thank Nicolas Rivard and David Roy for their insightful comments on the manuscript.

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