Cis-Acting Relaxases Guarantee Independent Mobilization of MOBQ4 Plasmids

Plasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. In this work, we studied a group of plasmids from enterobacteria that encode phylogenetically related mobilization functions that populate the previously non-described MOBQ4 relaxase family. These plasmids encode two transfer genes: mobA coding for the MOBQ4 relaxase; and mobC, which is non-essential but enhances the plasmid mobilization frequency. The origin of transfer is located between these two divergently transcribed mob genes. We found that MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative dissemination among clinically relevant enterobacteria. While highly similar in their mobilization module, two sub-groups with unrelated replicons (Rep_3 and ColE2) can be distinguished in this plasmid family. These subgroups can stably coexist (are compatible) and transfer independently, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such a strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.


INTRODUCTION
Mobilizable plasmids are small genetic elements transmissible by conjugation with the assistance of a helper conjugative plasmid. They encode a relaxase, and usually a relaxase accessory protein (RAP), which are in charge of the conjugative DNA processing at a specific site of the origin of transfer (oriT) called nic. Mobilizable plasmids lack the transfer genes required for establishing a conjugative bridge (mating pair formation system, MPF) to the recipient cell, as well as the type IV coupling protein (T4CP) that puts in contact relaxosome and MPF and thus depend on conjugative plasmids to be transferred (Garcillán-Barcia and de la Cruz, 2013).
According to their relaxase, transmissible plasmids were phylogenetically classified into MOB families (Francia et al., 2004;Garcillán-Barcia et al., 2009). Currently, nine relaxase MOB classes are defined, and five of them (MOB P , MOB F , MOB Q , MOB H , and MOB C ) are prevalent in transmissible plasmids hosted in γ-Proteobacteria. Plasmids gathered in a relaxase MOB family share similar genomic traits. Relaxase MOB classification has thus shown to be a good predictor of the plasmid backbone (Garcillán-Barcia and de la Cruz, 2013;Fernandez-Lopez et al., 2017). Mobilizable plasmids resident in γ-Proteobacteria form phylogenetically related clusters mainly within two relaxase MOB classes: MOB P and MOB Q (Garcillán- . Relevant examples are ColE1-like plasmids, grouped in family MOB P5 ; IncQ1 plasmids, such as RSF1010/R1162, gathered in MOB Q11 ; and IncQ2 plasmids, such as pTC-F14, in family MOB P14 (Garcillán- Garcillán-Barcia and de la Cruz, 2013). An additional clade of small plasmids encoding MOB Q relaxases, previously classified as MOB Qu , and here redefined as MOB Q4 , was observed in a phylogenetic reconstruction of this relaxase family .
A pair of degenerate primers specific for MOB Q4 plasmids was implemented in the Degenerate PCR MOB Typing (DPMT) approach developed by Alvarado et al. (2012) to detect and classify transmissible plasmids. This method revealed the abundance of MOB Q4 plasmids in clinical isolates of enterobacteria (Alvarado et al., 2012;Garcillán-Barcia et al., 2015), previously unnoticed by other plasmid typing methods. Whole-genome sequencing of clinical E. coli isolates also uncovered the presence of this kind of plasmids (Brolund et al., 2013;de Toro et al., 2014;Lanza et al., 2014). Prototype plasmids pIGWZ12 and ColE9-J (ColE2-like) cluster within the MOB Q4 clade. They are stable, theta-replicating, high copynumber, narrow host-range plasmids, whose replication systems have been extensively studied (Yasueda et al., 1989Zaleski et al., 2006Zaleski et al., , 2015. Here, we uncovered the diversity of MOB Q4 plasmids, determined the helper conjugative plasmids responsible for their dissemination, and established their behavior in terms of stability and transfer.
Additional plasmids were constructed to delimit the oriT region. A schematic representation of the fragments included in each construction is depicted in Figure 1. Such fragments were individually assembled to coordinates 1-1030 and 1360-3001 of vector pSEVA631 (GenBank Acc. No. JX560348). Plasmids pRC5 and pRC6 contained a fragment including the mobC gene, the 178bp intergenic region between mobC and mobA and the first 400 nucleotides of the mobA gene from pRC1 and pRC2, respectively. Plasmids pRC7 and pRC8 included only the 178bp intergenic fragment (Supplementary Figure S1), located between genes mobA and mobC of pRC1 and pRC2, respectively. Plasmids pRC14 and pRC15 contain the oriT regions of pRC7 and pRC8 but cloned in the inverse orientation. Plasmids pRC11 and pRC9, respectively included portions 1-70 and 71-178 of the intergenic fragment between genes mobA and mobC of pRC1, while the same portions from pRC2 were included in pRC12 and pRC10, respectively. A pSEVA631 fragment containing coordinates 1-1030 and 1360-3001 was self-ligated, generating the non-mobilizable vector pRC13, which was used as a control in the mating experiments.

Stability Assays
Plasmids pRC1 and pRC2 were introduced in the recA + and recA − isogenic strains UB1636 (F − lys his trp rpsL) (Achtman et al., 1971) and UB1637 (F − lys his trp rpsL recA56) (de la Cruz and Grinsted, 1982), either independently to check for their stability or both together to check for their compatibility. Single colonies were inoculated in Lysogeny-Broth (LB) supplemented with kanamycin at 50 µg/ml (for pRC1-containing strains) or chloramphenicol at 25 µg/ml (for pRC2-containing strains) and grown to saturation at 37 • C with agitation (150 rpm). A volume of 9.7 µl was transferred from saturated cultures to 10 mL of fresh LB media without antibiotics and grown to saturation in the same conditions. Rounds of transfer and growth were repeated up to 80 generations. The proportion of plasmid-bearing cells in the population was monitored by replica-plating 100 colonies in LB-agar supplemented with the appropriate antibiotics every 10 generations. A larger number of cells was inspected by fluorescence microscopy and, in the case of pRC1-containing cells, also by flow cytometry. Live cells were visualized using a Leica AF6500 microscope at 63x magnification. CFP and mCherry signals were monitored using BP filters (Excitation 434/17 -Emission 479/40 for CFP, Excitation 562/40 -Emission 641/75 for mCherry). Images were obtained using an iXon885 EM CCD Camera (Andor) and up to 1000 cells were analyzed in each case. Fluorescence emission was measured by flow cytometry using a FACS Canto II flow FIGURE 1 | Schematic representation of the MOB Q4 DNA segments included in a series of recombinant plasmids. The mobilization region of MOB Q4 plasmids includes mobC and mobA genes, represented by large, horizontal gray arrows. The extent of the mobilization region included in each construction is represented by a gray bar. The plasmid names for the MOB Q41 -based constructions are listed in the left column, while those for MOB Q42 -based constructions are in the right column. Plasmids pRC1, pRC2, pRC3, and pRC4 also include the replication module of MOB Q41 or MOB Q42 plasmids.
cytometer (Becton Dickinson) equipped with a 488 nm solid state laser for excitation. The cyan fluorescence of 20,000 events was detected using a 525/20 filter.

Mating Assays
Conjugative plasmids used in this work are listed in Supplementary Table S1. They were tested as helpers of the MOB Q4 plasmids in surface mating experiments, following the procedure described by del Campo et al. (2012). E. coli strain DH5α (F − endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 ϕ80dlacZ M15 (lacZYA-argF)U169, hsdR17(rK − mK + (), λ − ) (Grant et al., 1990) containing different plasmid combinations was used as donor and BW25113 (lacI q rrnBT14 lacZWJ16 hsdR514 araBADAH33 rhaBADLD78), BW25993 (lacI q hsdR514 araBADAH33 rhaBADLD78) (Datsenko and Wanner, 2000) as recipient. Donor and recipient strains were mixed in a 1:1 ratio, deposited onto an LB-agar surface and incubated for 1 h at 37 • C (except when drR27 was used as a helper, in which case matings were carried out at 25 • C). Then, the mixture was resuspended in LB and plated in the presence of appropriate antibiotics. Conjugation frequencies were expressed as the number of transconjugants per donor cell.

Phylogenetic Analysis
The 300 N-terminal residues of the MobA relaxase of plasmid ColE9-J were used as a query in a BLASTP search (Altschul et al., 1997) (e-value: 1xE-3). The homologous sequences were aligned using MUSCLE (Edgar, 2004). TrimAl v1.4 was used to calculate the average identity between sequences in the alignment (Capella-Gutiérrez et al., 2009). ProtTest 3 was used to estimate the best model of protein evolution for our set (Guindon and Gascuel, 2003;Darriba et al., 2011). RAxML version 7.2.7 (Stamatakis, 2006) was used for phylogenetic reconstruction. Using the JTTGAMMA model 10 maximum likelihood (ML) searches trees were inferred and support values were assigned to each node of the best tree from 1000 bootstrap searches. Relaxase of the pXF5847 plasmid (GenBank Acc. no. YP_009076807.1) was used as outgroup.

Analysis of MOB Q4 Plasmids
MOB Q is a broad relaxase class that encompasses several families, each of which includes related plasmid backbones: MOB Q1 comprises relaxases of mobilizable broad host-range IncQ1-like plasmids; MOB Q2 , conjugative relaxases of pTi and many rhizobial plasmids; MOB Q3 , conjugative broad host-range plasmids resident in gram-positive, such as pIP501 (Garcillán- . In this previous study, many MOB Q plasmids were not ascribed to a specific subclassification due to either low resolution of the clades or lack of information on the plasmid members. Here, we focused on one of these poorly defined clades, now named MOB Q4 , prompted by the fact that these relaxases have been recurrently detected in enterobacterial clinical isolates (Alvarado et al., 2012;Brolund et al., 2013;de Toro et al., 2014;Lanza et al., 2014;Garcillán-Barcia et al., 2015).
The phylogenetic reconstruction, based on the first N-terminal 300 residues of MOB Q4 relaxases produced two clusters, MOB Q41 and MOB Q42 (Figure 2A and Supplementary Table S2). This relaxase domain contains the three relaxase motifs ( Figure 2B) and share 84% average amino acid identity (97 and 90% for individual MOB Q41 and MOB Q42 groups, respectively). The 3D structure prediction of the relaxase domain of MOB Q41 and MOB Q42 plasmids rendered MOB Q relaxases NES [plasmid pLW1043, PDB Acc. No. 4HT4 (Edwards et al., 2013)] and MobA [plasmid R1162/RSF1010, PDB Acc. No. 2NS6, (Monzingo et al., 2007)] as best hits (100% confidence). The superimposed structures pointed to MOB Q4 amino acids Y25 (motif I), E87 and E89 (motif II), and H125, H133 and H135 (motif III) as homologs of the MobA_R1162 catalytic residues Y25, E74 and E76, and H112, H120 and H122, respectively ( Figure 2C). Contrary to the high conservation of the N-terminal domain among members of both MOB Q4 subgroups, the amino acid identity of the C-terminal part of the MOB Q4 relaxases dropped to 35%. This C-terminal domain exhibited low homology to SogL primases of IncI1 plasmids.
Each MOB Q4 subclade groups highly related backbones (Figure 2A). MOB Q41 are cryptic, small-size plasmids (Supplementary Table S2). Their backbone contains only four genes encoding a replication initiation protein (Rep), a relaxase (MobA), a putative relaxase accessory protein (MobC) and a hypothetical protein. The genes for the last two are generally not annotated. Besides the above-mentioned replication and mobilization genes, MOB Q42 plasmids also contain a colicin operon, including colicin, immunity and lysis genes, following the synteny of Group A nuclease colicins (Cascales et al., 2007). Plasmids ColE9-J and pO111_4 contain a second, partial colicin operon.
The MOB Q4 subdivision in two relaxase groups matches with the presence of two different replicons (Supplementary Table S2) and this family thus encompasses at least two plasmid species as defined by Fernandez-Lopez et al. (2017). MOB Q41 plasmids encode a replication initiation protein that belongs to the Rep_3 superfamily [PF01051 in the Pfam classification (Finn et al., 2016)], with no defined group in the PlasmidFinder classification (Carattoli et al., 2014). MOB Q42 plasmids encode ColE2-like initiators (Pfam PF03090 + PF08708), classified as Col156 by PlasmidFinder. Plasmids pIGWZ12 and ColE9-J exemplify each cluster. They are stable, theta-replicating, high copy number plasmids (15 and 10 copies per chromosome molecule, respectively Zaleski et al., 2012). The origin of replication of plasmid pIGWZ12 was located upstream the rep gene. It contains iterons, an A+T rich region and four DnaA boxes (Zaleski et al., 2006(Zaleski et al., , 2015. The iterons were found to be the incompatibility determinants (Zaleski et al., 2015). ColE2like plasmids, such as ColE9-J, form a group of closely related elements that share an identical priming mechanism, mediated by the plasmid-encoded Rep protein (Horii and Itoh, 1988;Itoh and Horii, 1989;Yasueda et al., 1989;Hiraga et al., 1994). The origin of replication consists of 32 bp located downstream of the rep gene, containing two directly repeated sequences (Kido et al., 1991;Nomura et al., 1991;. In ColE2-like plasmids, the rep gene expression is post-transcriptionally controlled by a plasmid-encoded RNA (RNAI), which binds the untranslated 5 region of the rep mRNA, preventing its translation (Sugiyama and Itoh, 1993;Takechi et al., 1994;Yasueda et al., 1994). MOB Q42 plasmids contain a cer-like site (Hiraga et al., 1994), an indication that they use a host site-specific recombination system for resolving multimers to monomers as ColE1-like plasmids do (Summers andSherratt, 1984, 1988;Summers, 1998).

Stability and Co-residence of MOB Q4 Plasmids
To study the MOB Q4 plasmids, two derivatives were constructed, pRC1 and pRC2. They included the replication and mobilization modules of the MOB Q41 and MOB Q42 backbones, respectively. Antibiotic-resistance and fluorescent protein genes were also included as reporters. Plasmid stability and compatibility were assayed in recA + and recA − E. coli strains by propagating the plasmids either alone or in combination during 80 generations. Despite the cargoes loaded in plasmids pRC1 and pRC2, the percentage of plasmid retention in the bacterial population was 100%, suggesting that the MOB Q4 backbone confers a minimized fitness cost to its enterobacterial host (San Millan and MacLean, 2017). Besides stability in E. coli, both MOB Q4 plasmid species also exhibited full compatibility (100% retention of both after 100 generations), as could be expected due to their different replicons (Novick, 1987), and ruling out other plasmid-encoded traits out of the replication module that could interfere with the stable vertical inheritance of each other.
We looked for reports providing indirect evidence on MOB Q4 plasmid mobilization through conjugation. In a survey for the presence of transmissible plasmids in a multidrug E. coli collection, MOB Q4 transconjugants were obtained from seven out of the eight MOB Q4 containing clinical isolates (Garcillán-Barcia et al., 2015). In all cases, a MOB P12 -MPF I plasmid, presumptively the helper, was also present in both, donor and transconjugant cells. Similarly, the MOB Q41 plasmid pSD4.0 and the IncI1 plasmid pSD107 were found in E. coli transconjugants arisen from a mating with Salmonella enterica (Bleicher et al., 2013).
On the other hand, MPF F -type plasmids [e.g., IncF-MOB F12 (F) or IncHI1-MOB H11 (R27) plasmids], which show high prevalence in enterobacteria, were not appropriate for MOB Q4 mobilization. MPF T plasmids behaved unevenly as MOB Q4 mobilizers. IncP1-MOB P11 (RP4 and R751) and IncX2-MOB P3 (R6Kdrd1) plasmids rendered MOB Q4 transconjugants, while IncW-MOB F11 (R388), IncN-MOB F11 (pKM101) or IncX1-MOB P3 (pOLA52) did not. Contrary to IncP, IncW and IncN plasmids, most IncF, IncI1, IncH, and IncX plasmids are naturally repressed for conjugation. In this study, we used derepressed variants of IncF (pOX38 and R100-1), IncI1α (R64drd11), IncHI1 (drR27), and IncX2 (R6Kdrd1) plasmids, but not a derepressed IncX1. IncX1 and IncX2 plasmids are highly similar in their conjugation genes. Taking into account that the IncX2 derepressed plasmid R6Kdrd1 was not efficient at mobilizing MOB Q4 plasmids ( Figure 3A and Supplementary  Table S3), and that the IncX1 plasmid pOLA52 self-transfers at low frequency (around 10 −4 per donor) (Sørensen et al., 2003), the lack of mobilization of the MOB Q4 plasmids pRC1 and pRC2 by pOLA52 is not surprising. The widely different mobilization efficiencies displayed by the two IncP1-MOB P11 helpers used is more curious. RP4 and R751 are prototypes of the α and β divisions of the IncP1 backbones, respectively. Despite the high conservation of their transfer genes, the kanamycin-sensitive RP4 derivative, pRL443, was 100-1000 times more efficient than R751 as a MOB Q4 helper. Noticeable differences were also observed for these two conjugative plasmids at transferring IncQ2-MOB P14 mobilizable plasmids pTC-F14 and pTF-FC2 (van Zyl et al., 2003). The common characteristic of the MOB Q4 mobilizers was their belonging to the MOB P relaxase class. This could indicate that the MOB Q4 relaxosomes interact more efficiently with the T4 encoded by these MOB P plasmids.

Effect of Co-residence in the MOB Q4 Plasmid Mobilization
Bacterial co-infection with multiple plasmids is common in nature (San Millan et al., 2014). Co-residence of compatible plasmids may lead to intracellular interactions that negatively or positively affect plasmid transfer rates (Gama et al., 2017a,b,c;Getino et al., 2017). Among them, plasmid-encoded fertility inhibition systems that block transmission of unrelated plasmids from the same donor cell have been intensively studied (Maindola et al., 2014;Gama et al., 2018;Getino and de la Cruz, 2018). Besides, competition of two relaxosomes for the same T4CP-MPF can result in the preponderance of one them (Cascales et al., 2005), a fact relevant for any mobilizable plasmid. Cohabitation of two or more mobilizable plasmids that use the same mating apparatus could affect each other's transfer. To test whether the mobilization of the MOB Q41 plasmid was affected by coresidence with a MOB Q42 plasmid and vice versa, pRC1 and pRC2 were introduced conjointly with the helper plasmid (either pRL443 or R64drd11) in the same cell ( Figure 3B). Curiously, presence of pRC1 did not produce a significant variation in pRC2 transfer. In turn, pRC2 produced one-log decrease in pRC1 transfer by pRL443. However, this moderate negative effect was not exhibited when using R64drd11 as a helper: on the contrary, pRC2 presence resulted in one-log increase in pRC1 transfer. Testing different combinations of MOB Q41 , MOB Q42 and helpers would be necessary to deeper assess the impact of residing together in MOB Q4 horizontal propagation.

mobC Deletion Effect in the Mobilization Efficiency
Many conjugative and mobilizable plasmids encode RAPs that recognize and bind their cognate oriT sequence probably favoring a single-stranded state around the nic site . Deletion of RAP genes trwA of R388 (Moncalián et al., 1997), nikA of R64 (Furuya et al., 1991), mobB and mobC of plasmids pTC-F14 and pTF-FC2 (van Zyl et al., 2003), traJ and traK of RP4 (Guiney et al., 1989), mobC of R1162/RSF1010 (Brasch and Meyer, 1986), and mbeC of ColE1 (Varsaki et al., 2009) resulted in drastic decrease of plasmid transfer. All MOB Q4 plasmids encode a gene, called mobC, which is located adjacent to oriT and transcribed opposite to the mobA relaxase gene (Figure 1). Most of the mobC genes are not annotated, so we updated their annotation, as listed in Supplementary Table S2. The MobC proteins of MOB Q4 plasmids are small (less than 100 amino acids) and showed no homology to other RAPs (by using PSI-Blast). To check whether MobC plays a role in the MOB Q4 plasmid mobilization, mobC deletion mutants were constructed from pRC1 and pRC2, respectively producing pRC3 and pRC4 (Figure 1). A moderate decrease in mobilization was observed in the mobC − variants: 1.5-log reduction for pRC3 and 0.6-log for pRC4, when using R64drd11 as a helper ( Figure 3B). MobC is thus not absolutely essential for MOB Q4 plasmid mobilization. This is an interesting difference to other plasmid groups, which should be further investigated. It is conceivable that some MOB Q4 plasmids can be found, the mobilization of which is independent of RAPs.

In trans Mobilization of oriT_MOB Q4 -Containing Vectors
The 178 bp intergenic region comprised between the mobC and mobA genes of MOB Q4 plasmids was assembled with an oriT-lacking fragment of vector pSEVA631. The resulting constructions, pRC7 (for MOB Q41 ) and pRC8 (for MOB Q42 ) (Figure 1), were introduced in donor strains to check for their mobilization. The transfer proteins were supplied in trans: the corresponding mobilizable plasmid (pRC1 or pRC2) provided the relaxosomal proteins, while the conjugative plasmid (R64drd11) supplied the T4CP and MPF. Plasmids pRC7 and pRC8 were transferred to the recipient population, but 1000-fold less efficiently than their corresponding mobA + mobC + partners (pRC1 and pRC2) (Figure 4). This result was confirmed by using plasmids pRC14 and pRC15, instead of pRC7 and pRC8, in the mobilization experiments. Plasmids pRC14 and pRC15 contained the same oriT region present in pRC7 and pRC8, but cloned in the inverse orientation. Besides, to avoid losing any oriT-related function, larger segments including also the mobC gene and the first 431 bp of the mobA gene [pRC5 and pRC6 (Figure 1)], were analyzed. Here again relaxase, T4CP and MPF components were provided in trans. Plasmids pRC5 and pRC6 behave similarly to pRC7 and pRC8, and were mobilized at least 500-fold less than pRC1 and pRC2 (Figure 4).
MOB Q4 relaxases showed thus a cis-acting preference for their oriTs, performing at least 500-fold better on a cis than on a trans oriT substrate. The cis-acting preference is a characteristic exhibited by some DNA-binding proteins, such as the TnpA transposases of Tn10, Tn5 and Tn903 (Morisato et al., 1983;Derbyshire et al., 1990;DeLong and Syvanen, 1991). Relaxases generally lack a cis preference for their oriTs. There are only a few examples of relaxases that show preference for a cisencoded substrate. The MOB P relaxase of transposon Tn1549 was found to be cis-acting (Tsvetkova et al., 2010). Notably, all plasmid-encoded cis-acting relaxases have been reported in members of the MOB Q class: TraA of plasmid pRetCFN42d (MOB Q2 ) (Pérez-Mendoza et al., 2006) and TraA of plasmid pIP501 (MOB Q3 ) (Arends et al., 2012). Nevertheless, other MOB Q relaxases, such as Nes_pSK41 (Pollet et al., 2016), as well as MobA of plasmids R1162/RSF1010 and pSC101 (Brasch and Meyer, 1986;Derbyshire and Willetts, 1987;Meyer, 2000) worked efficiently in trans.
The MOB Q4 relaxases were also tested for their specificity to act on a non-cognate MOB Q4 oriT. The oriTs of MOB Q41 and MOB Q42 plasmids differ in 10 nucleotides along their 178bp sequence (Supplementary Figure S1). Mobilization frequencies of oriT_MOB Q42 plasmids pRC6 or pRC8 by the MOB Q41 plasmid pRC1 + R64drd11, as well as oriT_MOB Q41 plasmids pRC5 or pRC7 by the MOB Q42 plasmid pRC2 + R64drd11, were similar to that obtained for the cognate systems, varying no more than one log (Figure 4).
To further delimit the oriT of MOB Q4 plasmids, the 178bp oriT fragments cloned in pRC7 and pRC8 (see Supplementary Figure S1) were subdivided in two portions, one containing oriT nucleotides 1-70 (pRC11 and pRC12) and the other containing oriT nucleotides 71-178 (pRC9 and pRC10) (Figure 1 and Supplementary Figure S1). Disruption of the 178bp oriT region resulted in a drastic loss of conjugation efficiency of the oriT-containing plasmid (Figure 4), as previously reported for pIGWZ12 (Zaleski et al., 2015).
The cis-acting preference of the MOB Q4 relaxases shown here is an example of biological orthogonality (de Lorenzo, 2011), that is, a mechanism to avoid interference. It implies that when two MOB Q4 plasmids are present in the same cell, the contribution of oriT cross-recognition by the heterologous MOB Q4 relaxase to plasmid transfer is not substantial. This feature could be essential to guarantee their efficient transfer, given the fact that both types of MOB Q4 plasmids use the same repertoire of conjugative helpers and share the same hosts. CONCLUSION MOB Q41 and MOB Q42 plasmids are able to coexist and spread in the E. coli population without affecting each other largely. They disseminate through bacterial conjugation, aided specially by MPF I conjugative plasmids, but neither of the MOB Q4 plasmids dominates the horizontal transfer process. Co-residence of MOB Q41 and MOB Q42 plasmids in the same host neither hindered nor boosted considerably their respective mobilization frequencies. Since both plasmids (MOB Q41 and MOB Q42 ) have a narrow host-range (they circulate among enterobacteria), their coexistence in natural environments is likely. In such ecological setting, specific discrimination among their highly similar oriT sequences would be guaranteed by the preferential cis activity of the MOB Q4 relaxase. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

FUNDING
This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2017-86378-P, AEI/FEDER, UE, to FC) and Consejo Superior de Investigaciones Científicas (201820I143 to MG-B). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

ACKNOWLEDGMENTS
The authors want to thank María Aramburu and Raúl Fernández-López for their technical assistance with the flow cytometer and the fluorescence microscopy, respectively. This manuscript has been released as a Pre-Print at bioRxiv (Garcillán-Barcia et al., 2019).