Interplay between the Xer recombination system and the dissemination of antibioresistance in Acinetobacter baumannii

Antibiotic-resistant infections pose a pressing challenge in clinical settings. Plasmids are widely recognized for hastening the emergence of resistance by facilitating horizontal gene transfer of antibiotic resistance genes among bacteria. We explore this inquiry in Acinetobacter baumannii , a globally emerging nosocomial pathogen responsible for a wide array of infections with worrying accumulation of resistances, notably involving plasmids. In this specie, plasmids of the Rep_3 family harbor adaptive genes within variable regions edged by potential site-specific recombination sites recognized by the XerCD recombinase. We first show that the Xer system of Acinetobacter baumannii functions as described in Escherichia coli , resolving chromosome dimers at the dif site as well as recombining plasmid-borne sites. The multiple Xer recombination sites found in Rep_3 plasmids do not, however, allow excising plasmid fragments. They rather recombine to co-integrate plasmids, which may then further evolve to exchange genes. Co-integrates represent


Introduction
In bacteria, most replicons are circular and during their replication, an odd number of homologous recombination events leads to their dimerization.This impairs the even distribution of replicons between daughter cells (segregation) during cell division.To resolve this problem, prokaryotes have evolved replicon dimer resolution sites acted on by dedicated Xer recombinases (XerC and XerD in most cases) (1,2), that we call xrs (Xer Recombination sites).The importance of this function for faithful replicon segregation explains its high conservation and the Xer system is now considered as one of the most conserved structural features of circular chromosomes in Bacteria and Archaea (3,4).
The XerCD recombination process is particularly well described for the unique xrs located on bacterial chromosomes, known as the dif site (Figure 1A).The dif site is composed of two protein-binding arms separated by a central region.XerC and XerD respectively bind specifically to their binding arm (5) and two XerCD-dif complexes then interact to form the XerCD-dif synapse.Within this synapse, only one type of recombinase, either XerC or XerD, is expected to be active and each of the two units of that recombinase cuts the DNA strand at the dif site to which it is bound (5).This nucleophilic attack of DNA, mediated by a conserved tyrosine residue, forms a covalent link between the recombinase and the dif site.The second step of the reaction is a strand exchange between the two dif copies in the central region followed by ligation, which creates a Holliday junction.This intermediate isomerizes, thereby activates the second pair of recombinases, which cut and exchange the second pair of strands, finishing the recombination reaction (5).In this process, the two pairs of recombinases are sequentially activated to catalyze the exchange of the two DNA strands (6).Therefore, the selection of the first active pair of recombinases controls the reaction and it has been proposed that, within the XerCD-dif synapse, XerC is the one initially active whilst XerD is initially inactive.Consequently, the reaction is blocked at the Holliday Junction step and thus tends to be reversible, without recombination (7).To catalyze a complete recombination process, XerD must be activated by FtsK, a division septum-associated DNA translocase, which is essential for cell division (8)(9)(10).By recognizing KOPS sequences, which are oriented toward dif on each chromosome replichores, FtsK translocates toward dif (11)(12)(13).Upon reaching the dif site, FtsK activates XerCD-dif recombination through a specific contact with the carboxy-terminal part of XerD (9).This FtsK control of the XerCD-dif recombination permits a spatio-temporal control of chromosome dimer resolution: at midcell during septation.
Plasmids are also affected by this dimerization problem (14).Most of them use the Xer system to resolve their dimeric forms and some have evolved xrs to be independent of the bacterial cell cycle (e.g.: cer or psi are FtsK-independent xrs; for review (15)).
While it is expected that plasmid carry a single xrs site to resolve dimers (15), an increasing number of plasmids harboring multiple xrs has been described (called Re27, pdif or pXerC/D, (16)(17)(18)(19)(20)).The functional significance of xrs multiplicy remains unresolved.Most cases are found in Acinetobacter baumannii (Ab), a human pathogen involved in nosocomial diseases which are difficult to treat due their resistance to multiple antibiotics.For instance, the pABV01 plasmid contains two xrs flanking the bla oxa-24 gene that confers resistance to carbapenems (17).This plasmid is related to two others (p2ABAYE and pAB0057) that are very similar but for the region contained between the two xrs (Fig. 1B).From these observations, emerged the concept of xrs-cassettes (adaptative genes flanked by two xrs) suggesting the implication of Xer system in antibiotic resistant genes (ARG) dissemination (also called pdif-modules; (18,20,21).Recent work clearly suggests that xrs present on Ab plasmids are hot spot of recombination between plasmids (19,22,23) and that they may be processed by Xer recombinases (24).However, very little is known about the activity of these sequences and/or cassettes and whether they are bona fide xer recombination sequences.We present here a functional study of the Xer system of Ab on chromosomal (dif) and plasmidic xrs.This allowed us to test models of mobility of xrs-cassettes.Our results support a model of gene exchange between Ab plasmids via an original cointegration/resolution mechanism.

In vitro experiments
Xer proteins were purified as described previously (25,28).To do EMSA experiments, 28-bp 5′-end-labeled [CY3] DNA fragments carrying dif Ab or xrs sites were obtained by hybridization of complementary strands (TableSup1).EMSA reactions were performed as previously described (25,28,31) and analyzed with a typhoon TRIO GE.Cleavage assay were performed as previously described (32)(see Table Sup).DNA substrates used were produced by hybridization of different oligonucleotides (TableSup1) and results of the assay were analyzed on SDSPAGE (Mini-PROTEAN TGX gels -4-20%).
Serial dilution of the transformants were plate on LB agar medium complemented with 5µg/mL chloramphenicol, 40µg/mL XGal and 0.4% Arabinose or Glucose and grown overnight at 37°C as previously describe (31).
For plasmid-based deletion assay, CB50 was transformed with pROUT29, pROUT25, pROUT31 or pROUT32, together with a pLN1-based xer expression plasmid.Transformants were plated on LB agar complemented with 5µg/mL chloramphenicol and 25µg/mL ampicillin.After overnight culture in selective LB medium with 0.4% arabinose, plasmid content was extracted (QIAprep Spin Miniprep Kit) and analyzed on 1% agarose gel electrophoresis.

Characterization of Xer system of A. baumannii
We first investigate the functionality of A. baumannii's Xer system (Xer Ab ).To this end, full-length XerC and XerD proteins from strain AB5075 (subsequently referred to as XerC Ab and XerD Ab ) were purified in heterologous system.Binding of purified proteins to their expected substrate, dif Ab -containing DNA, was assessed using Electrophoretic Mobility Shift Assay (EMSA, Fig. 2).Increasing concentration of XerC Ab led to two shifted bands (C1 and C2).Based on previous observations in E.
coli Xer system (Blakely), we inferred that these bands correspond to XerC Ab bound to its binding site (C1) and to both XerC Ab and XerD Ab binding sites (C2).Equivalent results were obtained for XerD Ab .XerD Ab appeared to bind dif Ab with higher affinity than XerC Ab .The C1 complexes migrates differently depending on the protein used, suggesting different conformation of these complexes (e.g.DNA bending ( 25)).When both XerC Ab and XerD Ab were added, only a complex equivalent to C2 was observed.
We inferred that this complex corresponds to the binding of both XerC Ab and XerD Ab on their binding sites on dif Ab .Taken together, these results show that that Xer recombinases of Ab form a tripartite complex with dif Ab , as described for the E. coli system (5,28).
We then checked if XerC Ab and XerD Ab can catalyze the first step of DNA recombination on dif Ab , i.e., DNA cleavage by the catalytic tyrosine residue, leading to the formation of a covalent link between the cleaved DNA and the Xer protein.To do so, we used a "suicide assay" into which the covalent DNA-protein intermediate is trapped (32)(Materials & Methods; Figure 2B).We observed that XerC Ab cleaved dif Ab -containing DNA when in presence of XerD Ab (Fig. 2B; lanes 9, 10 & 11).This cleavage was not observed when a catalytic residue of XerC Ab was mutated (lane12).
To further characterize the Xer Ab system, we replaced the dif Ec site on the E. coli chromosome by a dif Ab -lacI-dif Ab cassette in a strain deleted for the xerC and lacI genes (26)(Material & Methods).The resulting strain made white colonies when plated on LB in presence of the beta-galactosidase substrate (Xgal), LacI effectively repressing the beta-galactosidase-encoding gene lacZ (Fig. 2C, empty vector).When this strain was transformed with a plasmid expressing both xerC Ab and xerD Ab genes, blue colonies appeared, indicating that recombination events occurred (Fig. 2C, Table Sup2).Recombination was 5 time more efficient when xerDγ Ab was substituted to xerD Ab (Fig. 2C, Table Sup2), further demonstrating that Xer Ab recombination is controlled by FtsK.
To bring evidence of Xer Ab functionality in A. baumannii replication, we deleted the xerC Ab gene in strain AB5075.The resulting strain formed filament (Fig. 1D), similarly to observations performed in E. coli strains inactivated for the resolution of chromosome dimers do (33).
Taken together, all these results demonstrate that the XerCD/dif system of A.
baumannii is canonical, behaving as site-specific recombination system involved in chromosome dimer resolution under the control of the division protein FtsK.

Plasmids of A. baumannii carry multiple Xer Recombination Sites
We next checked if the xrs found in multiple copies in Ab plasmids are active.To do so we studied a set of xrs from three related plasmids of the Rep_3 family reported in clinical strains: pABV01; p2ABAYE; and p2AB5075.Results are summarized in Fig. 3A (Fig. Sup1 for raw data).All sequences assayed, but xrs107 which is strongly degenerated, were cooperatively bound by XerC Ab and XerD Ab .XerC Ab alone bond poorly to these sequences whereas XerD Ab exhibited a better apparent affinity.This is consistent with the fact that the predicted xerC-binding sites of these xrs is more divergent from the dif consensus than their xerD-binding sites (21).Most xrs were cleaved by XerC Ab and XerDγ Ab but not by XerD Ab (Fig. 3, xrs6 is presented as a typical example).We conclude that most xrs found in Ab plasmid appear active for Xer recombination.Interestingly, xrs38, which is the only xrs to be present on the three plasmids presented on Fig. 1B, displays a different property.Indeed, we found that xrs38 is bound and cleaved by XerC Ab but also, and unexpectedly by XerD Ab (Fig. 3B).
Likewise, xrs66, closely related to xrs38 but present on the plasmid p1ABAYE (see below) also exhibit this property.This suggests that xrs38 and xrs66 can recombine independently of FtsK, hinting at a role in plasmid dimer resolution.

xrs-cassettes found on A. baumannii plasmids do not form excisable modules.
Because xrs are frequently found flanking adaptative genes and because related Ab plasmids exhibit variability for sequences flanked xrs, it was proposed that pairs of xrs form a new type of mobile element, or modules, that can be exchanged between plasmids (17,18,21).These modules could excise from a plasmid and integrate into another one using XerCD Ab recombination.To test this hypothesis, we assayed module excision in an E. coli strain deleted for xerC and xerD and expressing different version of the xerC and xerD genes from a plasmid (Fig. 4A).These strains were transformed with plasmids (S) in which the lacI gene is flanked by the assayed pair of xrs in direct orientation.A deletion product (P) was observed between repeated dif Ab sites in strains expressing xerCD genes, either E. coli or A. baumannii ones (Fig. 4B).This shows that dif Ab can be recombined by XerCD either from E. coli or from Ab and that E. coli FtsK can activate XerD Ab (lanes CD Ab and CD γAB ).
Recombination was not detected when the strain expresses a mutated (loss of catalytic activity) version of xerD Ab (Fig. 4B, lane CD* Ab ).
We then assayed pairs of xrs mimicking genetic organization found in the pABVO1, p2AB5075 and p2ABAYE plasmids (Fig. 4C).No deletion was detected using any combination of module and recombinases.This strongly suggest that the xrs found in Ab plasmid do not form excisable modules.
Interestingly this experiment also revealed that the expression of xerCDγ Ab in these strains give rise to the formation of multimeric form of the xrs-containing plasmids (Fig. 4C).This confirms that xrs are recognized end recombined in vivo by XerCDγ Ab , leading to recombination between plasmid molecules (intermolecular), rather than within the plasmids (intramolecular).

High recombination rate between xrs-carrying replicons in A. baumannii
Xer recombination usually allows excision of a DNA fragment flanked by pairs of xrs in direct orientation with identical central region (2,15).Noteworthy, in Ab plasmids most pairs of xrs flanking adaptative genes are frequently in opposite orientation and/or have diverging central regions (18,19,21).This latter observation rules out intramolecular Xer recombination of Ab plasmid carrying xrs.However, gene exchange may results from recombination between xrs carried by co-existing plasmids (20).Recombination between compatible xrs with closely related central regions would co-integrates plasmids, allowing the exchange of genes by resolution of the co-integrate at a second pair of compatible xrs.
To assay intermolecular recombination between different plasmids, we took advantage of the ABAYE strain containing two different Rep_3-family plasmids that both carry multiple xrs: p1ABAYE (p1) and p2ABAYE (p2) (Fig. 5).Central regions of Sequencing data demonstrated that recombination occurred within the xrs (Fig. Sup2) whereas recombination was abrogtated in a ΔxerC derivative (Fig. 5: lanes 7-12).Note that xrs107 was poorly recognized by XerCDAb (Fig. 2), which may explain the absence of recombination.Site specific recombination was not observed between xrs that does not share closely related central region (Fig. Sup2).Therefore, Ab plasmids form co-integrates structures by recombination between xrs provided closely related central regions.
We attempted to quantify the ratio of co-integrates using ddPCR (Fig. SUP3).
Co-integrate formed by xrs6 -xrs105 recombination represented 0.25% of the DNA matrix leading to the PCR observed from p2 (Fig. 5B lane 1).Considering that p2 appears ≈ 1,5 time more abundant than p1, we estimate a rate of co-integrate of 0.45% for p1.Consistent results were obtained for recombination between xrs38 and xrs66, confirming a steady-state rate of .1 to 1% of co-integrate between the two plasmids.

Discussion
When xrs-cassettes were first described in Ab plasmids (17), authors immediately proposed that the Xer system may be involved in the mobility of these genetic structures.However, given that xrs flancking each cassette have different central regions and that they are organized as inverted sequences, it was not clear how the Xer system may ensure the mobility of these xrs-cassettes.
We first characterized the Xer system of Ab to describe a highly canonical chromosome dimer resolution system (2).It catalyzes site specific recombination between xrs sharing the same central region, in a FtsK dependent manner.This is yet another example of the remarkable conservation and control of the Xer system among bacteria (31,34).Furthermore, while deletion is indeed observed between directly repeated dif Ab sequences, this phenomenon does not occur between xrs arranged as they are on Ab plasmids.This invalidates the hypothesis that the Xer system of Ab (i.e.: XerC Ab , XerD Ab and FtsK Ab ) would catalyze the mobility xrs-cassettes from one plasmid to another by a simple excision/insertion mechanism.
Our results rather suggest that xrs present on different Ab plasmids recombines between each other, leading to the formation of cointegrates.Such cointegrate of Rep_3 plasmids in Ab were described in previous work when plasmid encounter is triggered by their artificial transfer (19,22).In this work, we have used the ABAYE strain which contains two Rep_3 plasmids (p1ABAYE and p2ABAYE), each containing multiple xrs.All the tested xrs are active, except xrs107 (p1ABAYE).This xrs is however mutated in the highly conserved motif "ATAA-Central Region-TTAT", which seems to be essential for Xer binding and cutting activity (5).Two pairs of xrs appeared to be compatible for recombination: xrs6 (p1ABAYE) and xrs105 (p1ABAYE), which share de GGTGTA central region; xrs66 (p1ABAYE) and xrs38 (p1ABAYE), which share de T/CCGCCA central region.This suggests that xrs are compatible when their central region regions are identical or closely related, consistent with the known Xer recombination mechanism (2,35).This shall be taken into account for future xrs annotation on Ab plasmids in order to predict all the possible recombination events between Ab plasmids.
We measured less that 1% recombination rate between active and compatible xrs on p1-and p2ABAYE.This could be considered low compared to Xer activity in the context of plasmid dimer resolution system where 100% of dimers are resolved (15).However, if plasmid dimer resolution described previously depends on XerCD, it is not activated by FtsK.Plasmid dimer resolution is activated thanks to the binding of DNA proteins (i.e.: PepA, ArgR or ArcA) upstream of the xrs allowing it to be independent of the bacterial cell cycle (2,15).Here, we show that recombination between xrs found on Ab plasmids depends on XerCD Ab but also on FtsK Ab activation.As FtsK Ab is only present at the septum when cells divide, recombination between xrs would only occur in the vicinity of the septum of dividing cells, explaining why we only observe few cointegrates (36,37).
Taken together, our results show that co-integrates are dynamics entities occurring between co-residing plasmids in Ab, in a XerCD system-dependent manner.This strongly support a mechanistic model where Xer recombination between one pair of active and compatible xrs present on two different plasmids lead to cointegrate formation and that resolution of this cointegrate by recombination between another pair of active and compatible xrs lead cassette exchange between these plasmids (20).
Although we observed recombination between each pair of active and compatible xrs, we did not observe any xrs-cassette exchange between these plasmids.This suggest that cointegrates are more frequently formed and resolved by recombination between the same pair of xrs than between different pairs of xrs.The xrs-cassette exchange may thus be too rare to be detected in our experiments (30 generations per experiment), but sufficiently frequent to explain the variability observed in Ab plasmids in the different Ab strains, exposed to different selective pressure.Furthermore, p1-and p2ABAYE coexist in the same strain (ABAYE) and may have reached a stable state.It is not clear if a cassette swap between these plasmids has any advantage for the ABAYE cell.In the future, experiment will have to be designed to address this question.For instance, it could be interesting to mutate maintenance genes of p1-or p2ABAYE to destabilize them and be able "select" for an eventual xrs-cassette exchange.This work demonstrates that Ab Rep_3 plasmids harbor multiple active Xer recombination sites and raises questions about stability of these plasmids.Indeed, if intermolecular recombination between compatible xrs present on different plasmid molecules leads to cointegrate formation (this work, (19,22)), it will also lead to multimerization of these plasmids, which may be deleterious for their stability (14).This multimerization of Rep_3 plasmids have not been reported and we have not detected it in our studies.This suggests that even though compatible xrs present on different plasmids results in cointegrates, recombination between compatible xrs present on different copies of the same plasmid is somehow inhibited.It could be possible that Ab Rep_3 plasmids carry a dimer resolution system, like described for pSC101 or pColE1 in E. coli (15,38).In the plasmid investigated in this study, this later mechanism could potentially explain the activity of xrs38 and xrs66.Both of these xrs appear to be recognized and cleaved by both XerC Ab and XerD Ab , even in the absence of FtsK, aligning with one of the characteristics of plasmid dimer resolution sites (15).This hypothesis will have to be tested in the future.
Taken together, these results bring experimental evidence of gene exchange between Rep_3 Ab plasmids could occur via a unique cointegration-resolution mechanism.It would be interesting to know if this mechanism has only been evolved by Acinetobacter and what are the advantage of such a system, for the plasmids and/or for the host bacteria.

Figure 3 :
Figure 3: Activity of xrs sequences present on Ab plasmids.A) summary of EMSA and cleavage assays performed on different xrs.pABV01 contain xrs38, xrs43 and xrs10.P1ABAYE contains xrs06, xrs107 and xrs66.P2ABAYE contains xrs38, xrs105 and xrs49.P2AB5075 contains xrs38, xrs06 and xrs49.Experiment were performed like in figure 2. Raw data are presented in figures SUP1&2.B) SDS-PAGE analysis of cleavage assays (top diagram) on xrs6 and xrs38.DNA molecules (xrs Top or Bottom nicked) are 10mM whereas proteins are 2 mM.XerC Ab is indicated as

Figure 4 :Figure 5 :BFigure 1 :
Figure 4: xrs-cassette deletion.A) The XerCD recombinase, when produced, should delete the lacI gene from the substrate plasmid (S) to give a deleted plasmid (P) and a non-replicative circle containing lacI, lost during divisions.B) Gel electrophoresis of the plasmid extraction after overnight culture of different strains transformed with a substrate plasmid containing a dif Ab -lacI-dif Ab cassette: S, substrate plasmid used for cells transformation; V, plasmids extracted from a strain which does not express any xer genes; CD Ec , plasmids extracted from a strain expressing xerCD from E. coli ; CD Ab , plasmids extracted from a strain expressing xerCD from Ab; CDg Ab , plasmids extracted from a strain expressing xerC Ab and a fusion between xerD Ab and g domain of ftsK Ab .Substrate (S) and product (P) plasmids are indicated, as well as multimeric forms of substrate and product plasmids (M).C) Gel electrophoresis of the plasmid extraction after overnight culture of different strains transformed by substrate plasmids (S) containing different xrs-lacIxrs cassettes.The xrs43-38 cassette represents the cassette found in pABV01, the xrs06-38 cassette represents the cassette found in p2AB5075, and the xrs105-38 cassette represents the cassette found in p2ABAYE,

Figure 3 :Figure 4 :
Figure 3: Activity of xrs sequences present on Ab plasmids.A) summary of EMSA and cleavage assays performed on different xrs.pABV01 contain xrs38, xrs43 and xrs10.P1ABAYE contains xrs06, xrs107 and xrs66.P2ABAYE contains xrs38, xrs105 and xrs49.P2AB5075 contains xrs38, xrs06 and xrs49.Experiment were performed like in figure 2. Raw data are presented in figures SUP1&2.B) SDS-PAGE analysis of cleavage assays (top diagram) on xrs6 and xrs38.DNA molecules (xrs Top or Bottom nicked) are 10μM whereas proteins are 2 μM.XerC Ab is indicated as C, XerD Ab as D, XerDγ Ab as Dγ and covalent products made between DNA and proteins as cov.Note that the covalent product XerD Ab -xrs38 migrate like XerDγAb (Lane 3 bottom gel).

Figure 5 :
Figure 5: Xer-dependent plasmid recombination.A) simplified genetic map of p1ABAYE (p1) and p2ABAYE (p2).The xrs present on these plasmid are indicated and their sequences are aligned.Small black square on plasmid represent the Rep_3 replication origin of these plasmids B) Gel electrophoresis of the PCR reactions performed on plasmid DNA extracted from wt or ΔxerC ABAYE strains.Lanes correspond to the amplified xrs.For instance: lane xrs105 corresponds to the PCR product obtained when the two primers flank xrs105 (p2); lane xrs6/105 corresponds to the PCR product obtained when one primer flanks xrs6 (p1) whereas the other flanks xrs105 (p2).

Figure Sup2 :
Figure Sup2: Xer-dependent plasmid recombination implicating xrs38 (p2ABAYE).A) Simplified genetic map of p1ABAYE (p1) and p2ABAYE (p2).B) Gel electrophoresis analysis of the PCR reaction performed on plasmid DNA extracted from ABAYE strains.First primer hybridizes upstream of the xerC-arm of xrs38.The second primer hybridizes upstream of the xerD-arm of xrs105 or xrs6 or xrs38 or xrs66.C) Sequencing revealed that PCR products of lane 1 corresponds to a non recombined p2ABAYE and that PCR products of lane 2 corresponds to recombination between p1 and p2ABAYE within xrs38.PCR product obtained lane 3 and 4 does not correspond to recombination between xrs38 and one of the other tested xrs.

Table Sup2 :
Deletion rate of a dif Ab -lacI-dif Ab cassette inserted in the E. coli chromosome at dif (Deghorain et al. 2011).A) The strain is deleted for xerC and complemented with pLN1::xerCD Ab .B) The strain is deleted for xerC and complemented with pLN1::xerCDγ Ab