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Open Access

Peer-reviewed

Research Article

Salmonella genomic island 1 (SGI1) reshapes the mating apparatus of IncC conjugative plasmids to promote self-propagation

Abstract

IncC conjugative plasmids and Salmonella genomic island 1 (SGI1) and relatives are frequently associated with multidrug resistance of clinical isolates of pathogenic Enterobacteriaceae. SGI1 is specifically mobilized in trans by IncA and IncC plasmids (commonly referred to as A/C plasmids) following its excision from the chromosome, an event triggered by the transcriptional activator complex AcaCD encoded by these helper plasmids. Although SGI1 is not self-transmissible, it carries three genes, traNS, traHS and traGS, coding for distant homologs of the predicted mating pore subunits TraNC, TraHC and TraGC, respectively, encoded by A/C plasmids. Here we investigated the regulation of traNS and traHGS and the role of these three genes in the transmissibility of SGI1. Transcriptional fusion of the promoter sequences of traNS and traHGS to the reporter gene lacZ confirmed that expression of these genes is inducible by AcaCD. Mating experiments using combinations of deletion mutants of SGI1 and the helper IncC plasmid pVCR94 revealed complex interactions between these two mobile genetic elements. Whereas traNC and traHGC are essential for IncC plasmid transfer, SGI1 could rescue null mutants of each individual gene revealing that TraNS, TraHS and TraGS are functional proteins. Complementation assays of individual traC and traS mutants showed that not only do TraNS/HS/GS replace TraNC/HC/GC in the mating pore encoded by IncC plasmids but also that traGS and traHS are both required for SGI1 optimal transfer. In fact, remodeling of the IncC-encoded mating pore by SGI1 was found to be essential to enhance transfer rate of SGI1 over the helper plasmid. Furthermore, traGS was found to be crucial to allow DNA transfer between cells bearing IncC helper plasmids, thereby suggesting that by remodeling the mating pore SGI1 disables an IncC-encoded entry exclusion mechanism. Hence traS genes facilitate the invasion by SGI1 of cell populations bearing IncC plasmids.

Author summary

Acquisition and dissemination of multidrug resistance genes among Enterobacteriaceae is in part driven by IncA and IncC (A/C) conjugative plasmids and Salmonella genomic island 1 (SGI1). Although unrelated, SGI1 relies on the self-transmissible A/C plasmids to disseminate within bacterial populations. The mechanisms allowing SGI1 to hijack the mating apparatus synthesized by A/C plasmids have not been previously established. Here, we show that IncC plasmids trigger the expression of three SGI1-borne genes that code for functional mating pore subunits distantly related to those encoded by the IncC helper plasmids. Our results indicate that these subunits alter the mating pore encoded by IncC plasmids to ensure optimal transfer of SGI1 and promote SGI1 dissemination in cell populations harboring IncC plasmids. Apart from SGI1 and relatives, documented mobilizable genomic islands are not known to code for mating pore components, possibly because of redundancy with those encoded by helper conjugative elements. Instead they usually code for mobilization proteins such as a relaxase and auxiliary factors involved in DNA recognition, processing and docking to the mating pore encoded by their helper conjugative element. From an ecological and epidemiological perspective, the strategy used by SGI1 likely confers a strong competitive advantage to SGI1 over IncC plasmids in clinical settings and could account for the high prevalence of SGI1 and relatives in multidrug-resistant Salmonella enterica and Proteus mirabilis.

Citation: Carraro N, Durand R, Rivard N, Anquetil C, Barrette C, Humbert M, et al. (2017) Salmonella genomic island 1 (SGI1) reshapes the mating apparatus of IncC conjugative plasmids to promote self-propagation. PLoS Genet 13(3): e1006705. https://doi.org/10.1371/journal.pgen.1006705

Editor: Danielle A. Garsin, The University of Texas Health Science Center at Houston, UNITED STATES

Received: August 4, 2016; Accepted: March 20, 2017; Published: March 29, 2017

Copyright: © 2017 Carraro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by Discovery Grants [326810-2011 and 2016-04365] from the Natural Sciences and Engineering Council of Canada (NSERC) to VB. VB holds a Canada Research Chair in bacterial molecular genetics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Conjugation is a nearly ubiquitous mechanism of horizontal gene transfer in bacteria, allowing the exchange of the largest number of genes per transfer event, often across taxonomical barriers [1–3]. In Gram-negative and most Gram-positive bacteria, conjugation is mediated by a complex nano-machine called type IV secretion system (T4SS). Conjugative T4SSs are multiprotein complexes that span the cell envelope and translocate DNA substrates from a donor to a recipient cell [4]. Conjugative plasmids and integrative and conjugative elements (ICEs) code for T4SS to promote their own dissemination by conjugation. These self-transmissible mobile genetic elements often bear a gene cargo of metal and antibiotic resistance genes, virulence determinants and other traits with potential selective advantages for the bacterial host [2,5–7].

Conjugative plasmids of the incompatibility group C (IncC) have been found in a broad range of Enterobacteriaceae and in Vibrio cholerae in which they can replicate and efficiently transfer [8–11]. IncC plasmids are closely related to IncA plasmids, and together are collectively referred to as A/C plasmids [8]. IncC plasmids are often recovered from clinical isolates of major pathogenic bacteria to which they confer resistance against multiple drugs, including last-resort antibiotics such as carbapenems [8,12,13]. IncC plasmids share a common scaffold of genes necessary for their replication, stability, conjugative transfer and regulation (Fig 1) [8,14]. Expression of the conjugative transfer genes of IncC plasmids is controlled by repressors Acr1 and Acr2 [15]. These two transcriptional repressors control the transcription of an operon containing acaC and acaD that code for the two subunits of the master activator AcaCD [15,16]. In IncC plasmids, AcaCD specifically binds to and activates 18 promoters that drive the expression of multiple genes and operons, most of which are of unknown function [15]. A third of these promoters drive the expression of tra genes coding for F-type T4SS assembly (traLEKB, traVA, dsbC/traC/trhF/traWU, traFHG), mating pair stabilization (traN), and the relaxase and type IV coupling protein (traIDJ) necessary for conjugative transfer (Fig 1) [17,18].

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Fig 1. Linear schematic representation of the core sequence of IncC plasmids and of Salmonella genomic island 1 (SGI1).

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. AcaCD binding sites are represented by green angled arrows. SGI1 is flanked by the attL (vertical blue line on the left) and attR (vertical blue line on the right) attachment sites when integrated into the 3’ end of the trmE gene in the chromosome of Salmonella enterica serovar Typhimurium DT104. ACSSuT, resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole and tetracycline.

https://doi.org/10.1371/journal.pgen.1006705.g001

AcaCD also activates the expression of genes carried by unrelated mobilizable genomic islands (MGIs), thereby triggering their excision from the chromosome and their IncC-dependent dissemination into new bacterial hosts. Such MGIs include MGIVmi1 from Vibrio mimicus, MGIVchHai6 from Vibrio cholerae and Salmonella genomic island 1 (SGI1) from Salmonella enterica serovar Typhimurium DT104 [15,19–22]. SGI1 and its multiple variants are frequently found in S. enterica and Proteus mirabilis clinical isolates [23–25]. SGI1 and relatives all share a common 26-kb core region disrupted by a complex class 1 integron conferring multidrug resistance (Fig 1) [23,26,27].

SGI1 is thought to hijack the conjugative apparatus encoded by IncA and IncC plasmids to transfer to a new host cell by a mechanism that remains unknown as no origin of transfer (oriT) or mobilization protein such as a relaxase has been identified in SGI1 to date [28]. Remarkably, SGI1 was reported to be mobilized at a much higher rate than MGIVmi1 and MGIVchHai6 (~3 logs higher) when mobilized by the same IncC plasmid, even outperforming the helper plasmid by 10 fold [15,20]. Unlike MGIVmi1 and MGIVchHai6, the core region conserved in SGI1-related elements codes for two putative T4SS subunits, TraGS and TraHS, as well as a putative mating pair stabilization protein, TraNS, that are distantly related to the counterparts TraGC (Vcrx144), TraHC (Vcrx143) and TraNC (Vcrx084) encoded by IncC plasmids (Fig 1, Table 1) [18,29–33]. While these observations suggest that the putative T4SS subunits encoded by SGI1 could be involved in SGI1 spread, they have been shown to be dispensable for its mobilization [28]. Like xis, a gene coding for the recombination directionality factor Xis that facilitates the excision of SGI1 from the chromosome, expression of the three tra genes of SGI1 has recently been reported to be under the control of AcaCD (Fig 1) [15,34,35]. This raises the question of the functional role of the putative tra genes of SGI1.

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Table 1. Protein homologs encoded by IncC plasmids and SGI1.

https://doi.org/10.1371/journal.pgen.1006705.t001

In this study, we investigated whether SGI1 could alter the mating pore encoded by IncC plasmids to enhance its own transfer. First, we confirmed that the genes traNS, and traHGS of SGI1 are under AcaCD control and that this cluster of tra genes is important for SGI1 mobilization. Using combinations of deletion mutants and complementation assays, we explored the role of each TraC/TraS subunit on the formation of the mating pore and its proficiency to mediate transfer of SGI1 and/or the helper IncC plasmid. Finally, we demonstrated that substitution of the TraG subunit enables SGI1 to escape an entry exclusion mechanism encoded by IncC plasmids.

Results

SGI1 traS genes are induced by the master activator of transfer of IncC plasmids

Expression of traNC and traHGC of IncC plasmids is activated by AcaCD, which recognizes a specific AcaCD box upstream of the -35 sequence in the promoters PtraNc and PtraF, respectively (Fig 1) [15]. Two AcaCD boxes were also predicted in intergenic sequences upstream of traNS and traHGS. To test whether expression of SGI1 tra genes is AcaCD-dependent, promoters of traNS (PtraNs) and traHGS (PtraHs) were cloned upstream of a promoterless lacZ gene and their activity was measured by β-galactosidase assays. Controls included the constitutive promoter Pint and the AcaCD-inducible promoter Pxis that drive the expression of the integrase- and Xis-coding genes of SGI1, respectively.

Activity of PtraNs was below the level of detection in absence of AcaCD, whereas in the same condition, PtraHs exhibited a weak yet detectable constitutive activity that remained weaker than the constitutive expression of Pint (Fig 2A). AcaCD increased the activity of PtraNs and PtraHs by 6 and 10 fold, respectively, as observed for Pxis (Fig 2A and 2B). Therefore, like their IncC counterparts, expression of SGI1-borne traS genes is directly stimulated by AcaCD, suggesting that TraNS, TraHS and TraGS of SGI1 are produced alongside with TraNC, TraHC and TraGC of IncC plasmids. Thus, the SGI1-coded TraS subunits might complement or even compete with those encoded by IncC plasmids and lead to the synthesis of a hybrid T4SS with altered properties.

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Fig 2. Regulation of expression of int, xis, traNS and traHGS of SGI1.

(A) Activity of Pint, Pxis, PtraNs and PtraHs was monitored from single-copy, chromosomally integrated lacZ transcriptional fusions in E. coli BW25113 Nx. Colorimetric assays of β-galactosidase activity were carried out on LB medium supplemented with (+) or without (-) arabinose to express acaCD from PBAD on pacaCD. (B) Induction of Pint, Pxis, PtraNs and PtraHs in response to AcaCD. β-galactosidase assays were carried out using the same strains as in panel A. Ratios between normalized OD420 values in the arabinose-induced over non-induced pacaCD, and non-induced pacaCD over arabinose-induced empty pBAD30 vector are shown. The bars represent the mean and standard deviation values obtained from at least three independent experiments. Statistical analyses were performed using the one-way ANOVA followed by Sidak’s post-test to compare each induction ratio to its corresponding control. Statistical significance is indicated as followed: ****, P < 0.0001; ***, P < 0.001; ns, not significant.

https://doi.org/10.1371/journal.pgen.1006705.g002

TraNC, TraHC and TraGC are essential for IncC plasmid transfer

While predicted to be part of the mating apparatus of IncC plasmids, whether TraNC, TraHC or TraGC are necessary for conjugative transfer has not yet been established [8]. To investigate this, we carried out mating experiments using a set of deletion mutants of pVCR94ΔX2 (Su Sp) as well as complementation assays aimed at evaluating their importance for conjugative transfer. Individual deletion of traNC, traHC or traGC completely abolished conjugative transfer of pVCR94ΔX2 (Fig 3). Trans-complementation of each deletion mutant by expressing the missing gene from the medium-copy plasmid pBAD30 under the control of the arabinose-inducible PBAD promoter restored pVCR94ΔX2 transfer to wild-type level, thereby confirming that the mutations were non-polar (Fig 3). Therefore, the predicted T4SS subunits TraNC, TraGC and TraHC are essential for conjugative transfer of IncC plasmids.

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Fig 3. Role of TraN/G/HC on conjugative transfer of IncC plasmids.

Effect of traNC, traGC and traHC on conjugative transfer of pVCR94ΔX2. Conjugation assays were carried out using E. coli BW25113 Nx containing the indicated elements as donor strains and E. coli CAG18439 (Tc) as the recipient strain. Wild-type (WT) or derivative mutants of pVCR94ΔX2 as well as the gene expressed from pBAD30 are indicated below each graph; (-) indicates that the plasmid is not present in the donor cell. For clarity, gene names traXY were shortened XY. Transfer frequencies are expressed as the number of transconjugants per NxR KnR SpR donor CFUs and the ratio of transfer frequencies relative to WT is indicated at the base of each bar. The bars represent the mean and standard deviation values obtained from at least three independent experiments. “x” indicates that the frequency of transfer was below the detection limit (<10−7). Statistical analyses were carried out on the logarithm of the values using the one-way ANOVA with Tukey’s multiple comparison test. Statistical significance is indicated as followed: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

https://doi.org/10.1371/journal.pgen.1006705.g003

The traS gene cluster is important for SGI1 mobilization

To facilitate further investigations on SGI1, we substituted its multidrug resistance locus In104 with a kanamycin-resistance (Kn) cassette, while preserving the core genes conserved in all SGI1-like elements (Fig 1). Resulting SGI1ΔIn104 (Kn) was then used with pVCR94ΔX2 to investigate the role of traNS and traHGS, and of their respective IncC-borne homologs in the dissemination of SGI1 and IncC plasmids.

As shown in Fig 2, expression of traNS, traHS and traGS in SGI1 is triggered by AcaCD of IncC plasmids. Nevertheless, whether these genes code for proteins able to contribute to the formation of a functional T4SS was unclear. Kiss et al. [28] reported that mobilization of a natural SGI1 variant lacking ~10 kb (from 3,610 to 13,537 bp) including open reading frames from traNS to traHS was not significantly impacted by the deletion. This suggests the region encompassing traNS to traHS does not contain indispensable functions for SGI1 mobilization. To verify this, we constructed a ΔtraS mutant of SGI1ΔIn104 lacking the whole traNS to traHS region and compared the mobilization by pVCR94ΔX2 of SGI1ΔIn104 and its ΔtraS mutant. Unlike previously reported [28], we observed that ΔtraS led to ~4,000-fold decrease of transfer (2.27 ± 1.42 for SGI1ΔIn104 vs 5.46×10−4 ± 1.20×10−4 for its ΔtraS mutant), thereby suggesting that genes included in the traS gene cluster are important for SGI1 mobilization.

SGI1 complements a defective IncC-encoded T4SS with functional subunits

We took advantage of the non-transmissible traNC, traHC and traGC null mutants of pVCR94ΔX2 that likely produce a non-functional mating apparatus to test whether SGI1ΔIn104 could rescue such mutants. If the putative T4SS subunits encoded by SGI1ΔIn104 can replace the ones missing in the T4SS encoded by pVCR94ΔX2, transmissibility of the plasmid should be restored. Remarkably, SGI1ΔIn104 could restore conjugative transfer of each individual mutant to levels that were comparable to wild-type pVCR94ΔX2, while also allowing its own transfer (second pair of bars in Fig 4A, 4B and 4C). These results indicate that SGI1 produces functional mating pore components that can replace the missing corresponding parts in the IncC T4SS.

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Fig 4. Role of traC and traS genes on conjugative transfer of IncC plasmids and SGI1.

(A) Effect of traGC and traGS on conjugative transfer of pVCR94ΔX2 (light gray bars) and SGI1ΔIn104 (dark gray bars). (B) Effect of traHC and traHS. (C) Effect of traNC and traNS. (D) Effect of combinatory deletions. For details, refer to legend of Fig 3. Two one-way ANOVA with Tukey’s multiple comparison test were carried out separately for each element. The unpaired t-test (two-tailed) was performed to compare the bars of different elements. WT frequencies of transfer for both elements come from a single set of experimental replicates but are displayed throughout panels A to D as a reference in each statistical analysis.

https://doi.org/10.1371/journal.pgen.1006705.g004

To confirm this hypothesis, trans-complementation of the traNC, traGC and traHC null mutants of pVCR94ΔX2 was carried out by providing donor cells with traNS, traGS and traHS, respectively, expressed from pBAD30. Expression of traNS restored conjugative transfer of pVCR94ΔX2 ΔtraNC to wild-type level (Fig 3). Surprisingly, despite its strong divergence from TraGC (Table 1), TraGS restored conjugative transfer of pVCR94ΔX2 ΔtraGC with only a mere 5-fold reduction compared to wild-type (Fig 3). In contrast, although TraHC and TraHS share 64% identity, expression of traHS failed to restore conjugative transfer of pVCR94ΔX2 ΔtraHC (Fig 3). This latter observation was puzzling because SGI1ΔIn104 could complement the traHC null mutant of pVCR94ΔX2.

TraGS is required for optimal SGI1 transfer

As shown above, TraGS is a functional substitute for TraGC as it restored transfer of pVCR94ΔX2 ΔtraGC (Fig 3). In contrast, when the reciprocal experiment was carried out, we found that TraGC is a poor substitute for TraGS for mediating transfer of SGI1ΔIn104. While SGI1ΔIn104 transfer was unaffected by the ΔtraGC mutation in pVCR94ΔX2 (Fig 4A), SGI1ΔIn104 ΔtraGS transfer was strongly reduced despite the presence of wild-type pVCR94ΔX2. Taken together, these results suggest that TraGS is required for optimal transfer of SGI1 whereas efficient transmissibility of IncC plasmids can be mediated by TraGC and/or TraGS (Figs 3 and 4A).

Combination of both traGC and traGS mutations completely abolished transfer of both elements, confirming the key role of this T4SS component (Fig 4A and S1A Fig). In this context, overexpression of either traGC or traGS nearly restored full transfer of pVCR94ΔX2 ΔtraGC. However, traGC was inefficient at complementing SGI1ΔIn104 ΔtraGS and resulted in a 250-fold decrease of transfer compared to SGI1ΔIn104 (Fig 4A). In contrast, providing traGS in trans fully restored transfer of SGI1ΔIn104 ΔtraGS. Therefore, traGS seems to be a key factor for enhancing the transmissibility of SGI1 relatively to IncC plasmids.

TraHS is specifically required with TraGS for SGI1 optimal transfer

Overexpression of traHS was unable to complement a traHC null mutant of pVCR94ΔX2, whereas wild-type SGI1ΔIn104 rescued pVCR94ΔX2 ΔtraHC (Figs 3 and 4B). As traHS and traGS seem to be part of the same operon in SGI1, like their homologs traHC and traGC in IncC plasmids (Fig 1), we suspected that the products of these genes might be interacting partners in the T4SS [14,15]. Substitution of a cognate partner within a pair of interacting proteins by a homologous protein encoded by another element is likely to impair these interactions and affect the functionality of the resulting hybrid mating pore. To test this hypothesis, complementation assays were performed using ptraHGS to coexpress both traHS and traGS in cells bearing either pVCR94ΔX2 ΔtraHC or pVCR94ΔX2 ΔtraHGC (Fig 3). In both cases, conjugative transfer of pVCR94ΔX2 was partially restored, thereby confirming that TraGS and TraHS work together within the mating pore. Together with the lack of complementation of pVCR94ΔX2 ΔtraHC by ptraHS, this latter observation likely reflects the inability of the SGI1-encoded TraHS subunit to interact with the IncC plasmid-encoded TraGC subunit to form a functional T4SS.

Although SGI1ΔIn104 ΔtraHS combined with pVCR94ΔX2 did not prevent the formation of a functional mating pore since pVCR94ΔX2 transferred at wild-type level, we observed that transfer of SGI1ΔIn104 ΔtraHS was reduced by 100 fold (Fig 4B). Because TraGS is required for optimal transfer of SGI1ΔIn104 (Fig 4A), this observation suggests that although TraHS does not work in association with TraGC, TraHC associated with TraGS could form a functional mating pore.

To investigate further these interactions, we combined pVCR94ΔX2 and SGI1ΔIn104 mutants, as well as expression of TraC/TraS subunits from pBAD30 vectors in conjugative transfer experiments. Neither element transferred when the traHC/traHS combination of mutants was used (Fig 4B and S1B Fig). However, complementation by providing either traHC or traHS in trans partially restored transfer of both elements. When pVCR94ΔX2 ΔtraHC was combined with SGI1ΔIn104 ΔtraGS, no transfer was detected for either element (Fig 4D), thereby confirming that TraGC and TraHS are incompatible and unable to form a functional T4SS. In contrast, the reciprocal association of pVCR94ΔX2 ΔtraGC with SGI1ΔIn104 ΔtraHS allowed transfer of both elements to near wild-type levels, thereby confirming that association of TraHC with TraGS can sustain formation of a functional and efficient hybrid T4SS (Fig 4B).

Altogether, these results revealed that while SGI1 can use a mating pore entirely encoded by IncC plasmids, the expression and association of TraHS with TraGS are necessary for its optimal transfer, which largely surpasses the transfer rate of the helper plasmid.

TraNC and TraNS proteins are exchangeable

Additional experiments were performed to assess the impact of traNC and traNS on the transfer ability of pVCR94ΔX2 and SGI1. Transfer assays using a donor bearing pVCR94ΔX2 and SGI1ΔIn104 ΔtraNS revealed that traNS was dispensable for SGI1 transfer if the IncC helper plasmid provided TraNC (Fig 4C). Combination of ΔtraNC and ΔtraNS mutations abolished pVCR94ΔX2 transfer, although it allowed residual transfer of SGI1ΔIn104 ΔtraNS (>4 logs below SGI1ΔIn104 level). Complementation of both mutations using either ptraNC or ptraNS restored the transmissibility of both elements (Fig 4C and S1C Fig).

TraNS enhances SGI1 transfer through the hybrid T4SS

Attempts to rescue pVCR94ΔX2 ΔtraHGC with ptraHGS only partially restored transfer of the mutant plasmid (250-fold reduction of transfer compared to wild-type) (Fig 3), whereas wild-type SGI1ΔIn104 restored transfer of pVCR94ΔX2 ΔtraHGC to near wild-type level (Fig 4D). This observation suggests that TraNS of SGI1 interacts specifically with the TraHGS-containing T4SS. This prompted us to test whether traNS could act cooperatively with traHGS to enhance the transmissibility of SGI1ΔIn104. We found that although SGI1ΔIn104 ΔtraNS transfer is not affected by the mutation in the context of wild-type pVCR94ΔX2, the concomitant absence of traHGC in pVCR94ΔX2 resulted in a 330-fold decrease of SGI1ΔIn104 ΔtraNS, despite the presence of SGI1ΔIn104-borne traHGS and pVCR94ΔX2-borne traNC (Fig 4D). In contrast, transfer of pVCR94ΔX2 was not affected in this context. These results showed that all three SGI1 Tra subunits seem to work together to promote its optimal transfer.

IncC plasmids exert entry exclusion

In F-type T4SSs, TraG is known to be a determinant of entry exclusion in the donor cell [36]. Entry exclusion is a process by which DNA transport from the donor cell is blocked by a recipient cell that contains a plasmid belonging to the same exclusion group. IncA and IncC plasmids seem to have been combined into A/C based on entry exclusion rather than incompatibility [37]. However, whether A/C plasmids exert entry exclusion had yet to be demonstrated. To test this, we monitored the mobilization of pSU4628, a derivative of the broad-host-range mobilizable plasmid CloDF13 (Table 2). Although pSU4628 lacks T4SS genes, it codes for a mobilization protein that recognizes its cognate oriT and enables its translocation through P- and F-type T4SSs [38]. As a control, we first tested whether pSU4628 could be efficiently mobilized from E. coli SM10λpir which bears conjugative plasmid RP4 (P-type) to E. coli bearing or lacking pVCR94ΔX4. pSU4628 transferred at high frequency regardless of the presence of the IncC plasmid in the recipient (Fig 5A), thereby indicating that IncC plasmids do not exclude entry of DNA mediated by IncP RP4. In contrast, when pVCR94ΔX2 was used to mobilize pSU4628 to the same recipient strains, a 160-fold reduction of transfer (Exclusion index (EI)) was observed if pVCR94ΔX4 was present in the recipient, thereby confirming that IncC plasmids exert entry exclusion.

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Fig 5. Suppression by traGS of IncC entry exclusion.

(A) IncC entry exclusion inhibits mobilization of pSU4628. E. coli SM10λpir (KnR) bearing pSU4628 (ApR) (hatched bars) or E. coli BW25113 Nx bearing pSU4628 and pVCR94ΔX2 (SpR) in the absence or presence of SGI1ΔIn104 (KnR) or its mutants were used as donors. E. coli CAG18439 (TcR) bearing or lacking pVCR94ΔX4 (CmR) was used as the recipient. Transfer frequencies are expressed as the number of TcR ApR transconjugants per KnR ApR donor CFUs (hatched bars) or per NxR (KnR) SpR ApR donor CFUs. Exclusion index (EI) is indicated at the bottom of each gray bar. (B) Effect of traGS on mobilization of SGI1ΔIn104 when the helper IncC plasmid is in the recipient. E. coli BW25113 Nx bearing SGI1ΔIn104 (WT or ΔtraGS, KnR) and E. coli CAG18439 (TcR) bearing pVCR94ΔX4 (CmR) were crossed. traGS and traGC complementation assays were carried out with ptraGS or ptraGC, respectively. Transfer frequencies are expressed as the number of NxR CmR transconjugants per TcR CmR donor CFUs for pVCR94ΔX4 and TcR KnR transconjugants per NxR CmR donor CFUs for SGI1ΔIn104. The bars represent the mean and standard deviation values obtained from at least three independent experiments. “x” indicates that the frequency of transfer was below the detection limit (<10−7). Statistical analyses were carried out on the logarithm of the values using the one-way ANOVA with Sidak’s post-test (A) to compare each bar to its corresponding control using an empty recipient, and with Tukey’s multiple comparison test (B) for each element. Statistical significance is indicated as followed: ****, P < 0.0001; *, P < 0.05; ns, not significant.

https://doi.org/10.1371/journal.pgen.1006705.g005

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Table 2. Strains and plasmids used in this study.

https://doi.org/10.1371/journal.pgen.1006705.t002

TraGS disables entry exclusion between cells bearing IncC plasmids

Genes mediating entry exclusion of A/C plasmids have not yet been characterized; however, by analogy with other F-type T4SSs [36], TraGC is likely the determinant of entry exclusion in donor cells. Since SGI1 codes for its own TraGS subunit, we hypothesized that SGI1 would escape IncC entry exclusion, thereby facilitating DNA exchange between cells bearing IncC plasmids. To test this, we monitored pSU4628 mobilization from a donor bearing both pVCR94ΔX2 and SGI1ΔIn104 to recipient cells bearing or lacking pVCR94ΔX4. pSU4628 mobilization by pVCR94ΔX2 was enhanced by SGI1ΔIn104 at a rate comparable to mobilization by RP4 regardless of the presence of pVCR94ΔX4 in the recipient (EI = 1.6) (Fig 5A), thereby confirming that SGI1 disables IncC entry exclusion. Deletion of traHS had no significant impact (EI = 1.3) suggesting it plays no role in disabling IncC entry exclusion. In contrast, when SGI1ΔIn104 ΔtraGS was used, mobilization of pSU4628 was much reduced and the presence of the IncC plasmid in the recipient resulted in exclusion (EI = 27) (Fig 5A). Mobilization of pSU4628 in the presence of SGI1ΔIn104 ΔtraHGS was comparable with donors lacking SGI1ΔIn104 (EI = 19).

Recently, Sibor et al. [24] showed SGI1 mobilization when the IncC helper plasmid resides in the recipient strain. Since SGI1 is not self-transmissible, this observation suggests that prior to SGI1 mobilization, the donor strain acquires the IncC plasmid, which can then mobilize SGI1 toward the recipient. Such a two-step transfer of SGI1 can only occur if SGI1 disables IncC entry exclusion. We verified this using as a donor E. coli BW25113 Nx containing either SGI1ΔIn104 (Kn) or its ΔtraGS mutant, and as a recipient E. coli CAG18439 bearing pVCR94ΔX4. Selection of intermediate BW25113 Nx transconjugants bearing pVCR94ΔX4 showed that the helper plasmid transferred efficiently regardless of the absence or presence of traGS (Fig 5B). Furthermore, while SGI1ΔIn104 transferred at high frequency to CAG18439 with pVCR94ΔX4, we failed to detect transfer of SGI1ΔIn104 ΔtraGS (Fig 5B). Mobilization was restored to wild-type level when the ΔtraGS mutant was complemented with traGS, whereas overexpression of traGC partially rescued transfer to levels 4 logs below the wild-type. This confirms that SGI1 fails to transfer to cells harboring an IncC helper if it must rely on a TraGC-based T4SS. TraGS-based T4SS is critical for SGI1 propagation across bacterial population bearing IncC plasmids.

Discussion

Most known MGIs are opportunistic passengers riding the T4SS encoded by their conjugative helper element [39]. IncC conjugative plasmids have hitherto been shown to mobilize in trans three different MGIs: MGIVmi1 from V. mimicus, MGIVchHai6 from V. cholerae and SGI1 from S. enterica. Mobilization of both MGIVmi1 and MGIVchHai6 would rely on the auxiliary mobilization protein MobI, which would play the role of adaptor between the oriT of the MGIs and the relaxase of IncC plasmids [15,19,20]. However, this mechanism of mobilization is suboptimal with transfer rates 150 to 200 times lower than the IncC helper plasmid. In strong contrast, SGI1 was reported to transfer more than 10 times better than the same IncC helper plasmid [15,40]. Hence, SGI1 is not merely a free rider of the T4SS encoded by IncC plasmids, but rather tweaks the engine to its own benefit.

This study confirmed the key role of TraNC, TraGC and TraHC in the formation of the T4SS of IncC plasmids. Expression of each protein restored the transfer of the corresponding mutant, although it did not enhance its transfer rate above wild-type level. Therefore, unlike the master regulator AcaCD [15], individual production of TraNC, TraGC or TraHC is not a limiting step for plasmid transfer. Moreover, our study of the three putative tra genes of SGI1 confirmed that not only is expression of these genes AcaCD-dependent as recently shown by Murányi et al. [35], but each one also codes for a fully functional T4SS subunit; together the Tra subunits of SGI1 could complement individual traGC, traHC and traNC deletion mutants of pVCR94ΔX2. Heterologous complementation of T4SS functions by subunits encoded by different plasmids has already been reported for P-type T4SSs. For instance, the peptide hydrolase TraL from the IncN plasmid pKM101 can replace VirB1 of the VirB T4SS of Agrobacterium tumefaciens despite low sequence identity (31%) [41]. Likewise, their TraC and VirB5 components can also be exchanged [42]. Furthermore, the VirB10 homolog TrwE of IncW plasmid R388 can be partially substituted for conjugation by TrwE of Bartonella tribocorum, a component of a T4SS involved in pathogenicity [43]. However, such exchanges of T4SS subunits between mobile genetic elements is uncommon and usually prevented in natural systems. For instance, interference between multiple functionally divergent T4SSs that co-occur in Bartonella is avoided by tight spatiotemporal regulation of expression or rapid diversification of the T4SS components [44].

Optimal SGI1 transfer depends on which of the subunits are composing the mating pore. Fig 6 illustrates the possible combinations of T4SS subunits and their outcome on SGI1 transfer efficiency inferred from our results (Figs 3 and 4). Our findings challenge a previous report by Kiss et al. [28] suggesting that SGI1 traS genes are not involved in SGI1 mobilization. In fact, our results indicate that traGS (collaboratively with traHS and traNS) enhances the transfer rate of SGI1ΔIn104 over the helper IncC plasmid (Fig 4). Moreover, traGC substitution by traGS enables SGI1 to invade cell populations bearing IncC plasmids likely by evading IncC entry exclusion (Fig 5B). Since SGI1 has also been shown to destabilize IncC plasmids [40], this mechanism sheds a new light on the ecological and epidemiological significance of SGI1 and relatives in the propagation of multidrug resistance. In fact, we predict that combination of entry exclusion escaping and IncC plasmid destabilization would result in displacement of IncC plasmids by SGI1 in enterobacterial cell populations bearing IncC plasmids upon contact with a small subpopulation of cell bearing only SGI1.

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Fig 6. Schematic representation of mating pore configurations and impact on IncC plasmids and SGI1 transfer efficiency.

Each mating pore configuration shows only a single subunit of TraNC/S, TraGC/S, TraHC/S, which are represented as described in the figure, and color-coded in purple for IncC plasmid-encoded subunits and blue for SGI1-encoded subunits. The rest of the mating apparatus is not shown and is assumed to be provided by the IncC plasmid. Efficiency of transfer of both elements is indicated by a green circle (optimal or efficient transfer for both elements), an orange triangle (impaired transfer for SGI1 but not IncC plasmid) or a red cross (abolished transfer for both elements) under the corresponding mating pore configuration as inferred from the results (Figs 3 and 4).

https://doi.org/10.1371/journal.pgen.1006705.g006

Deletion of both traNC and traNS revealed that, despite the lack of putative adhesin that seems to be required for transfer of IncC plasmids, SGI1 can still transfer at low frequency. The TraNC/TraNS adhesins, which are thought to stabilize the mating cell pair [18,30,31], are likely required for the transfer of a large DNA molecule such as pVCR94ΔX2 (~120 kb), while the smaller size of SGI1 (~26 kb for SGI1ΔIn104) would render it less vulnerable to premature separation of the mating partners due to the shorter transfer time required to transfer the whole element. In addition, traNC and traNS could be easily exchanged without drastic impairment of SGI1ΔIn104 or pVCR94ΔX2 transfer. This result is not surprising considering that TraNC and TraNS are the least divergent proteins of the three orthologous pairs (78% identity) (Fig 1, Table 1). An H (TraHC or TraHS) and a G (TraGC or TraGS) subunit are both required for assembly of a functional mating apparatus. However, we showed that all combinations are not functionally equivalent, as TraHS and TraGC appeared to be incompatible (Fig 4B). Thus, SGI1-encoded subunit TraHS specifically interacts with TraGS, strongly enhancing the efficiency of SGI1 transfer. Altogether, our observations suggest that the TraHGS association allows a specific interaction with protein(s) and/or DNA of SGI1 to optimize its transfer. One candidate could be the relaxosome, i.e. the machinery that processes DNA at the SGI1-borne origin of transfer (oriT) to allow its transfer. oriT of SGI1 and components of the relaxosome that process it remain to be identified and could be partly encoded by SGI1 to confer specificity to the altered mating pore.

Chromosomally integrated SGI1 is not entirely quiescent. While expression of IncC plasmid-borne traC genes strictly depends on AcaCD activation [15], traHGS are constitutively transcribed at low level in an AcaCD-independent fashion (Fig 2). Basal expression of traHGS strongly suggests that SGI1 primes the bacterial cell to accumulate TraGS and TraHS subunits. While production of these T4SS subunits is likely vain when SGI1 is alone, upon arrival of a helper IncC plasmid, cells primed with TraGS and TraHS might be more prone to rapidly incorporate SGI1-encoded subunits in lieu of IncC-plasmid-encoded ones, thereby favoring the transfer of SGI1 over the helper plasmid. In contrast, expression of traNS is strictly dependent upon AcaCD activation (Fig 2) [35]. Tight control over traNS expression may have been selected to prevent futile expression of the cell surface-exposed adhesin, which could potentially serve as a receptor for infection by bacteriophages.

In conclusion, unlike any other known mobilizable genomic island described to date, SGI1 not only hijacks the mating pore encoded by IncC plasmids, but also customizes it by inserting its T4SS subunits. This strategy enhances the propagation of SGI1 in bacterial populations as a result of enhanced transfer rates and expansion of its host range to recipient cells bearing IncC plasmids. This study takes us one step further into the comprehension of the intimate relation that links the mobility of unrelated classes of multidrug resistance-conferring mobile genetic elements.

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 (LB-Miller, EMD) at 37°C in an orbital shaker/incubator and were preserved at -80°C in LB broth containing 15% (vol/vol) glycerol. Antibiotics were used at the following concentrations: ampicillin (Ap), 100 μg/ml; chloramphenicol (Cm), 20 μg/ml; hygromycin B (Hy), 50 μg/ml; kanamycin (Kn), 50 μg/ml or 10 μg/ml for single copy integrants of pOPlacZ; nalidixic acid (Nx), 40 μg/ml; spectinomycin (Sp), 50 μg/ml; streptomycin (Sm), 200 μg/ml; sulfamethoxazole (Su), 160 μg/ml; tetracycline (Tc), 12 μg/ml; trimethoprim (Tm), 32 μg/ml. When required, bacterial cultures were supplemented with either 0.02 or 0.2% L-arabinose.

Mating assays

Conjugation assays were performed by mixing 100 μl of donor cells and 100 μl of recipient cells (typically ~2×109 cells/ml each) that were grown overnight in LB broth at 37°C with suitable antibiotics to ensure retention of the plasmid and SGI1 derivatives. Cells were pelleted by centrifugation for 3 min at 1,200 g, washed once in 200 μl of LB broth and resuspended in 10 μl of LB broth. Mating mixtures were then deposited as drops on LB agar plates and incubated at 37°C for 6 hours. The cells were recovered from the plates in 800 μl of LB broth, vortexed and diluted via serial 10-fold dilutions before plating on LB agar plates containing suitable antibiotics. Donors were selected using a chromosomal marker, and as necessary a marker for pVCR94, SGI1ΔIn104 and/or pSU4628. To induce expression of tra genes in complementation assays, mating experiments were carried out onto LB agar plates with 0.02% arabinose. Frequency of transfer was calculated as transconjugants/donor from data obtained from at least 3 parallel mating experiments.

Molecular biology methods

Plasmid DNA was prepared using the EZ-10 Spin Column Plasmid DNA Minipreps Kit (Bio Basic) according to manufacturer’s instructions. All enzymes used in this study were purchased from New England Biolabs. PCR assays were performed with the primers described in S1 Table. PCR conditions were as follows: (i) 3 min at 94°C; (ii) 30 cycles of 30 sec at 94°C, 30 sec at the appropriate annealing temperature, and 1 minute/kb at 68°C; and (iii) 5 min at 68°C. When necessary, PCR products were purified using an EZ-10 Spin Column PCR Products Purification Kit (Bio Basic) according to manufacturer’s instructions. E. coli was transformed by electroporation as described by Dower et al. [53] in a Bio-Rad GenePulser Xcell apparatus set at 25 μF, 200 V and 1.8 kV using 1-mm gap electroporation cuvettes. Sequencing reactions were performed by the Plateforme de Séquençage et de Génotypage du Centre de Recherche du CHUL (Québec, QC, Canada).

Plasmid and strain construction

Plasmids and oligonucleotides used in this study are listed in Table 2 and S1 Table. Plasmids used for complementation assays were derived from pBAD30. traNC, traGC, and traHC were amplified using primer pairs 94traN84EcoRI.for/94traN84EcoRI.rev, 94traG144EcoRI.for/94traG144EcoRI.rev, 94traH143EcoRI.for/94traH143EcoRI.rev, and genomic DNA of E. coli BW25113 Nx containing pVCR94ΔX2 as the template. Amplicons were digested by EcoRI and cloned into EcoRI-digested pBAD30 using T4 DNA ligase, generating ptraNC, ptraGC and ptraHC. Likewise, traNS, traGS, traHS and traHGS were amplified using primer pairs SGI105traNSalI.for/SGI105traNSalI.rev, SGI111traGSalI.for/SGI111traGSalI.rev, SGI1s012EcoRI.for/SGI1s012EcoRI.rev, and SGI1s012SalI.for/SGI111traGSalI.rev, and genomic DNA of E. coli BW25113 Nx containing SGI1 as template. Amplicons were digested by SalI or EcoRI and cloned into SalI or EcoRI-digested pBAD30 using the T4 DNA ligase, generating ptraNS, ptraGS, ptraHS and ptraHGS.

PCR fragments containing the promoter region upstream of int, xis, traNS, traHGS were amplified using primer pairs SGI1promintPstI.for/SGI1promintPstI.rev, SGI1promxisPstI.for/SGI1promxisPstI.rev, SGI1promtraNPstI.for/SGI1promtraNPstI.rev, SGI1promtraHPstI.for/SGI1promtraHPstI.rev and cloned into the PstI restriction site of pOPlacZ to produce pPromint, pPromxis, pPromtraNS, pPromtraHS, respectively [15]. The resulting plasmids were verified by restriction profiling and DNA sequencing. These vectors were integrated in single copy into the chromosomal site attBλ of E. coli BW25113 Nx using pINT-Ts [52].

Deletion mutants of pVCR94ΔX2 and SGI1 were constructed using the one-step chromosomal gene inactivation technique with pSIM6 or pSIM18 (Table 2) [45]. For pVCR94ΔX2, deletions of traNC, traGC, traHC and traHGC were obtained using primer pairs, 94del84traN.for/94del84traN.rev, 94del144traG.for/94del144traG.rev, 94del143traH.for/94del143traH.rev, 94del143traH.for/94del144traG.rev, respectively, and pKD3 as the template (Table 2 and S1 Table). SGI1 derivative SGI1ΔIn104 was obtained using primer pair SGI1delVar.for/SGI1delVar.rev and pKD13 as the template. Subsequent deletions of traNS, traGS, traHS, traHGS and traNS-traHS region in SGI1ΔIn104 were obtained using primer pairs, SGI1delS005.for/SGI1del05traN.rev, SGI1delS011.for/SGI1delS011.rev, SGI1delS012.for/SGI1delS012.rev, SGI1delS012.for/SGI1delS011.rev and SGI1delS012.for/SGI1del05traN.rev, respectively, and pKD3 as the template. Substitution of the aph (Kn) resistance gene with the cat (Cm) resistance gene in pVCR94ΔX3 was carried out using the same approach with primers 94DelXnoFRTcm.for and 94DelXnoFRTcm.rev, and pKD3 as the template, yielding pVCR94ΔX4.

When possible, the antibiotic resistance cassette was removed from the resulting construction by Flp-catalyzed excision using the pCP20 vector [50]. All deletions were verified by PCR and antibiotic resistance profiling.

β-galactosidase assays

Qualitative assays on solid LB agar plate were done using 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as the substrate with or without 0.02% arabinose. Plates were observed after overnight incubation at 37°C.

Quantitative liquid assays using o-2-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate were done according to a protocol adapted from Miller [54]. After overnight incubation at 37°C in 4 ml LB broth supplemented with appropriate antibiotics, cultures were refreshed 1:100 in 4 ml LB broth supplemented with 10 μg/ml kanamycin, 25 μg/ml ampicillin and 0.2% arabinose except for the non-induced controls. Cultures were incubated for 5 hours at 37°C with shaking prior to sampling for enzymatic assays. OD measurements for enzymatic assays were performed using a Multiskan Go Microplate Spectrophotometer (Thermo Scientific). Each experiment was performed in at least three independent biological replicates. Induction ratios were calculated by dividing the “induced” values by the “non-induced” values whereas the control ratios were calculated by dividing the “non-induced” values by the control values.

Supporting information

S1 Fig. Effect of traC/S genes on cotransfer of IncC plasmids and SGI1.

Effect of traGC and traGS (A), and traHC and traHS (B) and traNC and traNS (C) and combinatory mutants (D), on cotransfer of pVCR94ΔX2 and SGI1ΔIn104. For details, refer to legend of Fig 3.

https://doi.org/10.1371/journal.pgen.1006705.s001

(PDF)

S1 Table. Primers used in this study.

https://doi.org/10.1371/journal.pgen.1006705.s002

(PDF)

Acknowledgments

We are grateful to Reynold Gerardo Farrera Calderón for his technical assistance and Alain Lavigueur for his insightful comments on the manuscript.

Author Contributions

  1. Conceptualization: NC RD VB.
  2. Formal analysis: NC RD NR MH.
  3. Funding acquisition: VB.
  4. Investigation: NC RD NR CA CB MH.
  5. Methodology: NC VB.
  6. Project administration: VB.
  7. Resources: VB.
  8. Supervision: NC VB.
  9. Validation: NC RD NR MH.
  10. Visualization: NC RD.
  11. Writing – original draft: NC.
  12. Writing – review & editing: NC RD NR VB.

References

  1. 1. Smillie C, Garcillán-Barcia MP, Francia MV, Rocha EPC, de la Cruz F. Mobility of plasmids. Microbiol Mol Biol Rev MMBR. 2010;74: 434–452. pmid:20805406
  2. 2. Norman A, Hansen LH, Sorensen SJ. Conjugative plasmids: vessels of the communal gene pool. Philos Trans R Soc B Biol Sci. 2009;364: 2275–2289.
  3. 3. Guglielmini J, Quintais L, Garcillán-Barcia MP, de la Cruz F, Rocha EPC. The Repertoire of ICE in Prokaryotes Underscores the Unity, Diversity, and Ubiquity of Conjugation. PLoS Genet. 2011;7: e1002222. pmid:21876676
  4. 4. Christie PJ. The Mosaic Type IV Secretion Systems. EcoSal Plus. 2016;7.
  5. 5. Carraro N, Burrus V. Biology of Three ICE Families: SXT/R391, ICEBs1, and ICESt1/ICESt3. Microbiol Spectr. 2014;2.
  6. 6. Johnson CM, Grossman AD. Integrative and Conjugative Elements (ICEs): What They Do and How They Work. Annu Rev Genet. 2015;49: 577–601. pmid:26473380
  7. 7. Wozniak RAF, Waldor MK. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nat Rev Microbiol. 2010;8: 552–563. pmid:20601965
  8. 8. Harmer CJ, Hall RM. The A to Z of A/C plasmids. Plasmid. 2015;80: 63–82. pmid:25910948
  9. 9. Fricke WF, Welch TJ, McDermott PF, Mammel MK, LeClerc JE, White DG, et al. Comparative genomics of the IncA/C multidrug resistance plasmid family. J Bacteriol. 2009;191: 4750–4757. pmid:19482926
  10. 10. Welch TJ, Fricke WF, McDermott PF, White DG, Rosso M-L, Rasko DA, et al. Multiple antimicrobial resistance in plague: an emerging public health risk. PLoS One. 2007;2: e309. pmid:17375195
  11. 11. Carraro N, Sauvé M, Matteau D, Lauzon G, Rodrigue S, Burrus V. Development of pVCR94ΔX from Vibrio cholerae, a prototype for studying multidrug resistant IncA/C conjugative plasmids. Front Microbiol. 2014;5: 44. pmid:24567731
  12. 12. Wailan AM, Sartor AL, Zowawi HM, Perry JD, Paterson DL, Sidjabat HE. Genetic contexts of blaNDM-1 in patients carrying multiple NDM-producing strains. Antimicrob Agents Chemother. 2015;59: 7405–7410. pmid:26392493
  13. 13. Wang R, Yu D, Zhu L, Li J, Yue J, Kan B. IncA/C plasmids harboured in serious multidrug-resistant Vibrio cholerae serogroup O139 strains in China. Int J Antimicrob Agents. 2015;45: 249–254. pmid:25532743
  14. 14. Poulin-Laprade D, Carraro N, Burrus V. The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids. Front Microbiol. 2015;6: 837. pmid:26347724
  15. 15. Carraro N, Matteau D, Luo P, Rodrigue S, Burrus V. The master activator of IncA/C conjugative plasmids stimulates genomic islands and multidrug resistance dissemination. PLoS Genet. 2014;10: e1004714. pmid:25340549
  16. 16. Lang KS, Johnson TJ. Characterization of Acr2, an H-NS-like protein encoded on A/C2-type plasmids. Plasmid. 2016; 87–88:17–27.
  17. 17. Guglielmini J, Néron B, Abby SS, Garcillán-Barcia MP, de la Cruz F, Rocha EPC. Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion. Nucleic Acids Res. 2014;42: 5715–5727. pmid:24623814
  18. 18. Lawley TD, Klimke WA, Gubbins MJ, Frost LS. F factor conjugation is a true type IV secretion system. FEMS Microbiol Lett. 2003;224: 1–15. pmid:12855161
  19. 19. Carraro N, Matteau D, Burrus V, Rodrigue S. Unraveling the regulatory network of IncA/C plasmid mobilization: when genomic islands hijack conjugative elements. Mob Genet Elem. 2015;5: 1–5.
  20. 20. Carraro N, Rivard N, Ceccarelli D, Colwell RR, Burrus V. IncA/C Conjugative Plasmids Mobilize a New Family of Multidrug Resistance Islands in Clinical Vibrio cholerae Non-O1/Non-O139 Isolates from Haiti. mBio. 2016;7: e00509–16. pmid:27435459
  21. 21. Douard G, Praud K, Cloeckaert A, Doublet B. The Salmonella genomic island 1 is specifically mobilized in trans by the IncA/C multidrug resistance plasmid family. PLoS One. 2010;5: e15302. pmid:21187963
  22. 22. Doublet B, Boyd D, Mulvey MR, Cloeckaert A. The Salmonella genomic island 1 is an integrative mobilizable element. Mol Microbiol. 2005;55: 1911–1924. pmid:15752209
  23. 23. Hall RM. Salmonella genomic islands and antibiotic resistance in Salmonella enterica. Future Microbiol. 2010;5: 1525–1538. pmid:21073312
  24. 24. Siebor E, de Curraize C, Amoureux L, Neuwirth C. Mobilization of the Salmonella genomic island SGI1 and the Proteus genomic island PGI1 by the A/C2 plasmid carrying blaTEM-24 harboured by various clinical species of Enterobacteriaceae. J Antimicrob Chemother. 2016;71: 2167–2170. pmid:27150396
  25. 25. Boyd DA, Shi X, Hu Q, Ng LK, Doublet B, Cloeckaert A, et al. Salmonella genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in Proteus mirabilis clinical and food isolates from China. Antimicrob Agents Chemother. 2008;52: 340–344. pmid:18025121
  26. 26. Boyd D, Cloeckaert A, Chaslus-Dancla E, Mulvey MR. Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother. 2002;46: 1714–1722. pmid:12019080
  27. 27. Levings RS, Lightfoot D, Partridge SR, Hall RM, Djordjevic SP. The genomic island SGI1, containing the multiple antibiotic resistance region of Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars. J Bacteriol. 2005;187: 4401–4409. pmid:15968049
  28. 28. Kiss J, Nagy B, Olasz F. Stability, entrapment and variant formation of Salmonella genomic island 1. PLoS One. 2012;7: e32497. pmid:22384263
  29. 29. Mulvey MR, Boyd DA, Olson AB, Doublet B, Cloeckaert A. The genetics of Salmonella genomic island 1. Microbes Infect Inst Pasteur. 2006;8: 1915–1922.
  30. 30. Klimke WA, Frost LS. Genetic analysis of the role of the transfer gene, traN, of the F and R100-1 plasmids in mating pair stabilization during conjugation. J Bacteriol. 1998;180: 4036–4043. pmid:9696748
  31. 31. Klimke WA, Rypien CD, Klinger B, Kennedy RA, Rodriguez-Maillard JM, Frost LS. The mating pair stabilization protein, TraN, of the F plasmid is an outer-membrane protein with two regions that are important for its function in conjugation. Microbiol Read Engl. 2005;151: 3527–3540.
  32. 32. Arutyunov D, Arenson B, Manchak J, Frost LS. F plasmid TraF and TraH are components of an outer membrane complex involved in conjugation. J Bacteriol. 2010;192: 1730–1734. pmid:20081027
  33. 33. Harris RL, Silverman PM. Tra proteins characteristic of F-like type IV secretion systems constitute an interaction group by yeast two-hybrid analysis. J Bacteriol. 2004;186: 5480–5485. pmid:15292150
  34. 34. Kiss J, Papp PP, Szabó M, Farkas T, Murányi G, Szakállas E, et al. The master regulator of IncA/C plasmids is recognized by the Salmonella genomic island SGI1 as a signal for excision and conjugal transfer. Nucleic Acids Res. 2015;43: 8735–8745. pmid:26209134
  35. 35. Murányi G, Szabó M, Olasz F, Kiss J. Determination and Analysis of the Putative AcaCD-Responsive Promoters of Salmonella Genomic Island 1. PLOS ONE. 2016;11: e0164561. pmid:27727307
  36. 36. Garcillán-Barcia MP, de la Cruz F. Why is entry exclusion an essential feature of conjugative plasmids? Plasmid. 2008;60: 1–18. pmid:18440635
  37. 37. Hedges RW. R factors from Providence. J Gen Microbiol. 1974;81: 171–181. pmid:4362618
  38. 38. Cabezón E, Sastre JI, de la Cruz F. Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation. Mol Gen Genet MGG. 1997;254: 400–406. pmid:9180693
  39. 39. Bellanger X, Payot S, Leblond-Bourget N, Guédon G. Conjugative and mobilizable genomic islands in bacteria: evolution and diversity. FEMS Microbiol Rev. 2014;38: 720–760. pmid:24372381
  40. 40. Harmer CJ, Hamidian M, Ambrose SJ, Hall RM. Destabilization of IncA and IncC plasmids by SGI1 and SGI2 type Salmonella genomic islands. Plasmid. 2016; 87–88:51–57.
  41. 41. Höppner C, Liu Z, Domke N, Binns AN, Baron C. VirB1 orthologs from Brucella suis and pKM101 complement defects of the lytic transglycosylase required for efficient type IV secretion from Agrobacterium tumefaciens. J Bacteriol. 2004;186: 1415–1422. pmid:14973016
  42. 42. Schmidt-Eisenlohr H, Domke N, Baron C. TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli. J Bacteriol. 1999;181: 5563–5571. pmid:10482495
  43. 43. de Paz HD, Sangari FJ, Bolland S, García-Lobo JM, Dehio C, de la Cruz F, et al. Functional interactions between type IV secretion systems involved in DNA transfer and virulence. Microbiol Read Engl. 2005;151: 3505–3516.
  44. 44. Gillespie JJ, Phan IQH, Scheib H, Subramanian S, Edwards TE, Lehman SS, et al. Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems. mBio. 2015;6: e01867–01815. pmid:26646013
  45. 45. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97: 6640–6645. pmid:10829079
  46. 46. Grenier F, Matteau D, Baby V, Rodrigue S. Complete Genome Sequence of Escherichia coli BW25113. Genome Announc. 2014;2. (5). pii: e01038–14.
  47. 47. Singer M, Baker TA, Schnitzler G, Deischel SM, Goel M, Dove W, et al. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989;53: 1–24. pmid:2540407
  48. 48. Miller VL, Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988;170: 2575–2583. pmid:2836362
  49. 49. Datta S, Costantino N, Court DL. A set of recombineering plasmids for Gram-negative bacteria. Gene. 2006;379: 109–115. pmid:16750601
  50. 50. Cherepanov PP, Wackernagel W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 1995;158: 9–14. pmid:7789817
  51. 51. Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995;177: 4121–4130. pmid:7608087
  52. 52. Haldimann A, Wanner BL. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol. 2001;183: 6384–6393. pmid:11591683
  53. 53. Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988;16: 6127–6145. pmid:3041370
  54. 54. Miller JH. A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. CSHL Press, Plainview NY. 1992;