# Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

Narimane Dahmane, Virginie Libante, Florence Charron-Bourgoin, Eric Guédon, Gérard Guédon, Nathalie N. Leblond-Bourget, Sophie Payot

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## ► To cite this version:

Narimane Dahmane, Virginie Libante, Florence Charron-Bourgoin, Eric Guédon, Gérard Guédon, et al.. Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer. Applied and Environmental Microbiology, 2017, 83 (13), pp.e00337-17. 10.1128/AEM.00337-17. hal-01543490

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1

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3

4 Diversity of integrative and conjugative elements of *Streptococcus salivarius* and their intra- and interspecies transfer

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7

8 Running title: Diversity and functionality of ICEs in *S. salivarius*

9

10

11 Narimane Dahmane ¹, Virginie Libante ¹, Florence Charron-Bourgoin ¹, Eric Guédon ², Gérard

12 Guédon ¹, Nathalie Leblond-Bourget ¹ and Sophie Payot ¹#

13

14

15 ¹ DynAMic, Université de Lorraine, INRA, 54506, Vandœuvre-lès-Nancy, France;

16 ² STLO, Agrocampus Ouest, INRA, 35000, Rennes, France

17

18 # Corresponding author

19 Tel: +33 3 72 74 56 81

20 Fax: +33 3 72 74 53 56

21 E-mail address: sophie.payot-lacroix@inra.fr
22 **ABSTRACT:**

23 Integrative and Conjugative Elements (ICEs) are widespread chromosomal mobile genetic elements,
24 which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation
25 module closely related to that of ICESt3 of *Streptococcus thermophilus* were characterized in
26 *Streptococcus salivarius* by whole genome sequencing. Sequence comparison highlighted ICE
27 evolution by shuffling of 3 different integration/excision modules (for integration in the 3' end of *fda*,
28 *rpsI* or *rpmG* genes) with the conjugation module of the ICESt3 subfamily. Sequence analyses also
29 pointed out a recombination occurring at *oriT* (likely mediated by the relaxase) as a mechanism of
30 ICE evolution. Despite a similar organization in 2 operons including three conserved genes, the
31 regulation modules show a high diversity (about 50% of amino acid sequence divergence for the
32 encoded regulators and presence of unrelated additional genes) with a probable impact on the
33 regulation of ICE activity.

34 Concerning the accessory genes, ICEs of the ICESt3 subfamily appear particularly rich in restriction-
35 modification systems and orphan methyltransferase genes. Other cargo genes that could confer a
36 selective advantage to the cell hosting the ICE were identified, in particular genes for bacteriocin
37 synthesis and cadmium resistance.

38 The functionality of 2 ICEs of *S. salivarius* was investigated. Autonomous conjugative transfer to
39 other *S. salivarius* strains, to *S. thermophilus* and to *Enterococcus faecalis* was obtained. The
40 analysis of the ICE-*fda* border sequence in these transconjugants allowed the localization of the
41 DNA cutting site of the ICE integrase.

42

43 **IMPORTANCE**

44 The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse
45 chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective
46 advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part
47 on self-encoded restriction-modification systems.

48 Here intra and interspecies transfer were demonstrated for 2 ICEs of *S. salivarius*. Closely related
49 ICEs were also detected *in silico* in other *Streptococcus* species (*S. pneumoniae* and
50 *S. parasanguinis*) thus indicating that diffusion of ICESt3-related elements probably plays a
51 significant role in HGT occurring in the oral cavity but also in the digestive tract where *S. salivarius*
52 is present.

53

54
55 INTRODUCTION

Acquisition of genes by horizontal gene transfer (HGT) is a major driving force for evolution of
bacterial genomes (1, 2). The main mechanism is conjugation, a process that enables transfer of large
DNA fragments without requiring any similarity between sequences thus being naturally broad host
range (3). Besides conjugative transfer of extrachromosomal elements (plasmids), recent analyses
revealed that many integrated mobile elements, called Integrative and Conjugative Elements (ICEs),
also encode their own transfer by conjugation (4, 5). Like all other mobile genetic elements (MGEs)
(6), ICEs have a modular structure. Three modules, a recombination module, a conjugation module
and a regulation module, altogether control and ensure the excision and transfer of the element (7).
ICEs are able to excise from the bacterial chromosome generally by site-specific recombination, to
transfer using their own conjugative machinery and to integrate in the chromosome of a recipient cell
(4, 5). Moreover, various ICEs can promote the transfer of large fragments of the bacterial
chromosome by an Hfr-like mechanism (8). They can also mobilize non autonomous integrated
transferable elements such as (i) Integrative and Mobilizable Elements (IMEs), genetic elements
unrelated to ICEs that can excise/integrate from the chromosome but need to hijack the transfer
machinery of a conjugative element or (ii) Cis Mobilizable Elements (CIMEs), elements that derive
from ICEs and IMEs by deletion of the conjugation/mobilization and recombination modules but
retain recombination sites *att* (4). In this latter case, an ICE that integrates in a recombination site of
a CIME can mobilize the non autonomous element by mediating the excision of the whole composite
element (process of accretion-mobilization) (9, 10). Multiplication of bacterial genome sequencing
projects in the last few years provides a remarkable opportunity to explore the pool of bacterial
genetic mobile elements ("mobilome"). These *in silico* analyses revealed the high abundance of ICEs
in bacteria (11-13). In addition to the genes involved or controlling their mobility, ICEs also carry
78 cargo genes, which can provide new properties (virulence, antibiotic resistance for example) to the
79 recipient cell (7).

80 *Streptococcus salivarius* is a Firmicute that is a major constituent of the human oral cavity
81 microbiota (14) and is commonly detected in the human gastrointestinal tract of healthy individuals
82 (15, 16). Few strains have also been associated with opportunistic infections in particular in cases of
83 meningitis (17), endocarditis (18) and bacteremia in immunocompromised patients (19, 20).
84 Analyses of *S. salivarius* genomes pointed out the considerable variability of gene content and the
85 differences in adaptive traits (21). Evidence of widespread HGT was obtained in link with the
86 presence of diverse MGEs (21) and the competence for natural transformation of the species (22).

87 We recently screened a collection of 138 strains of S. salivarius for the presence of MGEs (23). This
88 led to the identification of 60 strains (belonging to 39 MLST groups) with a positive PCR signal for
89 the relaxase gene of ICESt3, an ICE previously characterized in the closely related species
90 *Streptococcus thermophilus* (24-26). This indicated the presence in these strains of putative ICEs
91 belonging to the ICESt3 subfamily. However, not all these strains showed a positive PCR signal for
92 the ICESt3 integrase gene that catalyzes the integration into the fda site. This suggested the presence
93 of other recombination modules associated with the ICESt3 conjugation module in S. salivarius
94 ICEs.

95 In this work, we selected 13 strains with different MLST patterns (including 6 strains with a negative
96 PCR signal for the ICESt3 integrase gene) and sequenced their genome in order to gain access to
97 their ICE sequences. ICEs with a conjugation module closely related to the one of ICESt3 were
98 searched in other NCBI available genomes of S. salivarius and in other genomes of Firmicutes. ICE
99 sequences were compared and the putative function encoded by their genes was analyzed in silico.
100 Two putative ICEs from S. salivarius were analyzed experimentally to test their excision and their
101 autonomous intraspecies and interspecies conjugative transfer.
102 MATERIAL AND METHODS

103 Bacterial strains, plasmids and culture conditions

104 The strains and plasmids used in the experimental part of this study are listed in Table 1.

105 Thirteen *S. salivarius* strains including 4 commensal (F1-4, F1-8, F4-2 and F6-1) and 9 clinical (B35, B57, L22, L50, L60, L64, N5, N20 and T93) were selected on the basis of a previous work (23).

107 These strains were chosen according to 3 criteria. Firstly, the selected strains likely harbor an ICE with a conjugation module related to ICESt3 (as suggested by a positive PCR signal for the relaxase and coupling protein genes of ICESt3). Secondly, to increase the diversity of ICEs included in the analysis, half of the strains were selected according to the absence of a PCR signal for the integrase gene of ICESt3 thus suggesting the presence of a different recombination module. Lastly, the selected strains are distributed all over the phylogenetic tree built from MLST data (23). They belong to different MLST groups and differ by at least 3 MLST alleles (out of 6 alleles analyzed) and can thus be considered as unrelated, except two of them (strains L22 and L64) which differ by only 1 allele (23). These 13 strains are available upon request.

116 Modified strains were named according to the modification. For example, *S. salivarius* F1-8 (pMG36e) corresponds to a derivative of *S. salivarius* F1-8 carrying the plasmid pMG36e. pMG36e is a small plasmid (3611 pb) that carries only genes required for its replication and an erythromycin resistance gene. It is a non conjugative plasmid derived from pWV01 that is a broad spectrum plasmid replicating in *E. coli* and in Firmicutes (27) and has been successfully used for labeling recipient strains in conjugation experiments with *S. thermophilus* (24). *S. salivarius*, *E. faecalis* and *S. thermophilus* were grown in M17 broth supplemented with 0.5% lactose (LM17) at 37°C (*S. salivarius*, *E. faecalis*) or 42°C (*S. thermophilus*) without shaking. Selective mitis salivarius agar (MSA; Difco) containing a 1% (wt/vol) tellurite solution, brain heart infusion (BHI; Difco) and
reconstituted skim milk broths were also used for mating assays. Solid cultures were made in oxygen-free environment induced by GasPak utilization (BioMérieux).

When required, cultures were supplemented with the following antibiotics: chloramphenicol (5 µg ml⁻¹ for *S. thermophilus* or 8 µg ml⁻¹ for *S. salivarius* and *E. faecalis*); erythromycin (5 µg ml⁻¹ for *S. thermophilus* or 10 µg ml⁻¹ for *S. salivarius* and *E. faecalis*).

## DNA sequencing and sequence analysis

The 13 selected *S. salivarius* strains were subjected to whole genome sequencing. Sequencing was made using Illumina HiSeq2000 sequencer by Beckman Coulter genomics (2×100 pb after paired-end library construction, expected at least 60 × coverage). Observed sequencing depth was higher than 200 × for all the 13 genomes. *De novo* assembly was performed using CLC Genomics Workbench (CLC Bio) using default parameters. Scaffold of the genomes was built by using the Genome Finishing module of CLC Genomics Workbench with *S. salivarius* JIM8777 genome as reference. Some assembly gaps were filled by doing PCR and sequencing. This enabled to obtain genomes fragmented in less than 75 contigs (less than 30 contigs for two thirds of the genomes; median size of the contigs higher than 40 kb for half of them). Contigs were first annotated using the RAST annotation server (http://rast.nmpdr.org/) (28, 29). Then, contig(s) containing genes closely related to conjugation genes of ICESt3 were identified by BlastN analysis (using *orfO-orfA* genes as query by megaBlast analysis with default parameters and filter disabled) and annotation was completed manually.

In NCBI sequence databases, ICEs closely related to ICESt3 were searched by using Microbial nucleotide blast (using *orfO-orfA* genes as query by megaBlast analysis with default parameters and filter disabled) on complete (n=816, last accessed 2016/07/29) and draft (n=9573, last accessed 2016/07/29) genomes of Firmicutes. Hits with more than 90% of identity with the whole query sequence were further analyzed.
149 Pairwise comparisons of elements were performed with Artemis Comparison Tool provided by the
150 Sanger Centre using comparison files generated by Double Act (available at: http://www.hpa-
151 bioinfotools.org.uk/pise/double_act.html) (30). Manual editing of comparison figures was performed
152 using Inkscape.

153 Nucleotide sequence accession numbers

154 The sequences of ICEs have been deposited in the EMBL Nucleotide Sequence Database under
155 accession numbers LT622825-LT622837.

156 Phylogenetic tree construction

157 Proteins of twenty-seven ICEs were included in the analysis. These ICEs correspond to (i) the 19
158 ICEs of the ICESt3 subfamily with closely related conjugation modules and (ii) 8 additional ones
159 previously reported to belong to the same ICE subfamily but showing more distantly related
160 conjugation modules (11, 26). The sequences of signature proteins were aligned using Clustal omega
161 with default parameters (31). The trees were built with MEGA (32) using (i) maximum likelihood
162 (ML) based on JTT (Jones-Taylor-Thornton) model including amino acid empirical frequencies
163 (partial deletion of gaps and missing data at 80% cutoff, Gamma distribution in 5 categories,
164 allowance for invariant sites), and (ii) BioNJ methods with the Poisson model (33). The branch
165 support of the groupings was estimated using bootstrap (100 replicates).

166 ICE tagging

167 ICE_SsaF1-4_fda and ICE_SsaF4-2_fda were tagged by a chloramphenicol resistance cassette
168 originating from pSET5s plasmid. The resistance cassette was inserted in an intergenic region
169 located between convergent coding sequences in the adaptive module to avoid impacting ICE
170 functionality. Two DNA fragments of about 1,000 bp corresponding to the upstream and the
171 downstream regions of the integration locus were amplified by PCR using specific primers that
172 present an extended sequence matching with the 5' and the 3'ends of the chloramphenicol resistance
cassette. A second PCR amplification was carried out using these two PCR fragments and the resistance gene as a template to synthesize a fragment carrying the antibiotic resistance cassette flanked by the upstream and downstream chromosomal regions of the gene. Natural competence of S. salivarius cells was induced by addition of the synthetic peptide (H2N - LPYFTGCL - COOH) (22) and the overlap PCR product was then added for transformation. The cross-over events, upstream and downstream from the tagged region, were positively selected by the newly acquired antibiotic resistance of the transformed clones. The integrity of the regions flanking the antibiotic cassette was confirmed by PCR.

PCRs were done using the Phusion high-fidelity DNA polymerase (Thermo Scientific). PCRs were performed with 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate (dNTP), 0.5 µM of each primer (for primer sequences, see Table 2) and 0.02 U µl⁻¹ of Phusion DNA polymerase in appropriate buffer per 50 µl reaction volume. Cycling conditions for the overlap PCR were: 3 min at 98°C, 30 s at annealing temperature (with 1°C of incrementation at each cycle), 30 s/kb at 72°C, followed by 30 additional cycles with an annealing temperature of 55°C and a final extension of 10 min at 72°C.

## Excision tests with or without mitomycin C (MMC)

PCR amplifications of *attB*, *attI*, *attR* and *attL* fragments were carried out in a 25 µl volume containing 1 µl of overnight culture, 200 µM of each dNTP (Thermo Scientific, France), 0.5 µM of each primer (for primer sequences, see Table 2), and 0.025 U µl⁻¹ of DreamTaq DNA polymerase in an appropriate buffer (Thermo Scientific, France). PCR amplifications were performed using the following cycling parameters: 10 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 50°C, and 1 min/kb at 72°C, with a final 5-min extension at 72°C. Amplified products were analyzed by electrophoresis on a 1.5% agarose gel.
To test the impact of mitomycin C on the ICE excision, PCR amplifications of the attachment sites were done after a 2.5 h-treatment of the cells with MMC. To select the MMC concentration that gives the highest induction of excision, a range of MMC concentrations (0.025 to 0.4 mg ml⁻¹) was first tested using 1 ng of genomic DNA as template. Semi-quantitative PCR (performed with 30 cycles) were then done at the selected concentration of MMC (0.05 mg ml⁻¹) using different genomic DNA quantities (0.1 µg to 1 pg). Amplifications of the *fda* gene were done in parallel as controls.

## Mating experiments

Donor and recipient strains were grown overnight with an appropriate antibiotic. 15 ml of broth medium were inoculated with 150 µl of donor or recipient stationary phase cultures. Cultures were grown until mid-exponential phase (optical density at 600 nm of 0.4), then were mixed and centrifuged for 15 min in a prewarmed centrifuge at 4,500 × g to pellet cells. The pellet was resuspended in 1 ml of LM17 broth and 150 µl were spread on 0.45 µm pore-size cellulose nitrate filters (Sartorius stedim biotech) deposited on LM17 soft agar (0.8 %) plates. Plates were then incubated at 37°C (for *S. salivarius/S. salivarius* and *S. salivarius/E. faecalis* mating pairs) or at 39°C (for *S. salivarius/S. thermophilus* mating pairs). After an overnight incubation, the filters were removed from the agar plates and placed into 10 ml of LM17 liquid media. Bacteria were recovered by vortexing for 30 s. The suspension was then directly spread on agar plates supplemented with the appropriate antibiotics or concentrated 10 times by centrifugation at 4,500 × g for 15 min to enable counting the CFUs of the donor, the recipient, and the transconjugants cells after a 24h-incubation. Transconjugants clones obtained after *S. salivarius* intraspecies mating were typed by DNA sequencing of PCR products corresponding to the *ddlA* gene (for primer sequences, see Table 2).

Treatment of donor cells with mitomycin C (MMC) was done as follows: cells carrying ICE_SsaF1-4_fda were grown in LM17 liquid medium at 37°C to an optical density at 600 nm of 0.4. The culture was then diluted 10-fold in 15 ml of prewarmed LM17 containing MMC at the
concentration that showed the maximum ICE excision level (0.05 mg ml⁻¹). A 10-fold dilution without MMC was used as a control. After 1 hour of culture, the cells were harvested by centrifugation in a prewarmed centrifuge and washed once with 15 ml of prewarmed LM17. The donor cells treated with MMC were mixed with recipient cells grown at optical density at 600 nm of 0.4 and centrifuged for 15 min in a prewarmed centrifuge at 4,500 × g to pellet cells. The pellet was resuspended in 1 ml of LM17 broth, and 150 µl were spread on nitrocellulose filter on LM17 soft agar plates before incubation for 4h or overnight at appropriate temperatures. The filters were then treated as previously described.

Mating frequencies were calculated by dividing the number of transconjugants by the number of donor cells, except in the case of donor cells treated with MMC, where mating frequencies were calculated relative to recipients. At least three independent biological replicates were done.

## Plasmid curing

Transconjugants carrying plasmid pMG36e were cultured overnight without erythromycin, and were then spread on LM17 plates at different dilutions. One hundred isolated clones were then streaked on LM17 plates with or without erythromycin. Erythromycin sensitive clones were then confirmed by PCR for the absence of plasmid (see Table 2 for primer sequences).

## Random Amplification of Polymorphic DNA (RAPD)

RAPD (34) was carried out with DreamTaq enzyme (for primer sequences see Table 2). Cycling conditions were: 40 cycles consisting of 94°C for 1 min, 31°C for 1 min, and 72°C for 2 min; the final extension was continued for 7 min at 72°C. 1 µl of liquid culture was used as DNA template. The PCR products were separated by electrophoresis on a 1.5% agarose gel.

241
242 **RESULTS AND DISCUSSION**

243

244 **Diversity of Integrative and Conjugative Elements of the ICESt3 subfamily identified in**
245 **S. salivarius and in other streptococci**

246 **Conjugation modules**

247 To explore the diversity of the ICEs of the ICESt3 subfamily found in S. salivarius, 13 strains (F1-4, F1-8, F4-2, F6-1, B35, B57, L22, L50, L60, L64, N5, N20 and T93) with different MLST patterns were selected from a previous work (23) (Table 1). These strains likely encode an ICE of the ICESt3 subfamily as suggested by a positive PCR signal for the ICESt3 relaxase gene (23). Their genome was sequenced and assembled in order to gain access to their ICE sequences. Except for 2 ICEs (ICE_SsaF6-1_rpsI and ICE_SsaB57_fda) split into 2 contigs, the sequences of the ICEs appear on a single contig (Fig. 1). As shown in Fig. 1, ICEs found in these 13 S. salivarius strains all display a conjugation module that is closely related to that of ICESt3 from S. thermophilus (>90% of nucleic sequence identity). ICEs with a closely related full conjugation module were also detected in 2 genomes of S. salivarius available in NCBI (those of strains JF and 1270) (Fig. 1). The element of S. salivarius NCTC 8618 (GenBank accession number: CP009913.1) also has a closely related full conjugation module but is not shown in the figure since it harbors a truncated integrase gene, and is therefore defective. Elements with a closely related conjugation module (partial in most of the genomes due to gaps in the assembly) were also detected in the genomes of strains GED7778A (GenBank accession number LRQS00000000), 140 (GenBank accession number: JVSQ01000000), 20-02 S1 (GenBank accession number: LXMB00000000), 20-12 S2 (GenBank accession number: LXMC00000000) and UC3162 (GenBank accession number: JYOY01000000). These elements are not shown in Fig. 1 because their sequence is incomplete. ICEs with a closely related conjugation module (92-94% of nucleic sequence identity with that of ICESt3) were also identified in the genome
of *Streptococcus pneumoniae* 2842STDY5753514 (mitis group) and *Streptococcus parasanguinis* DD19 (sanguinis group) (Fig. 1). Phylogenetic analysis of the relaxase OrfJ (Fig. 2) and the coupling protein OrfK (Fig. 3) of these ICEs indicates that they group with OrfJ and OrfK from *S. salivarius* ICEs. Similar results were obtained for the other proteins of the conjugation module (data not shown). Taken as a whole, these phylogenetic analyses and sequence comparisons clearly showed that transfers of ICEs closely related to ICESt3 have occurred between distinct species from different streptococcal groups.

The comparison of the clustering groups obtained for the relaxase (OrfJ) and the coupling protein (OrfK) indicated that OrfJ of ICE_Sma33MO_fda is closely related to the relaxases of ICESt3 and *S. salivarius* ICEs whereas OrfK is clearly different from the coupling proteins of these ICEs, but is closer to that of ICE_SparauNCFD2020_rpsI (see phylogenetic trees in Fig. 2 and 3). Nucleic sequence alignments indicated that the left extremity of the conjugation module (including *orfN* to *orfK* genes) of ICE_Sma33MO_fda is closely related to the corresponding sequence of ICE_SparauNCFD2020_rpsI (86% of nucleic sequence identity compared to 64% of nucleic sequence identity with the corresponding genes of ICESt3) whereas the right extremity of the conjugation module of ICE_Sma33MO_fda (corresponding to *orfJ* to *orfA* genes) is closely related to those of ICESt3 (96% of nucleic sequence identity compared to 64% of nucleic sequence identity with the corresponding genes of ICE_SparauNCFD2020_rpsI) and *S. salivarius* ICEs. The putative *nic* site of the transfer origin of ICEs of the ICESt3 subfamily, predicted by comparison with the *oriT* characterized for ICEBs1 of *Bacillus subtilis* (35), is located between *orfK* and *orfJ* genes (supplementary Fig. S1). Sequence alignment of this region indicated that the drop in nucleic sequence identity between ICE_Sma33MO_fda and ICE_SparauNCFD2020_rpsI occurs at the *nic* site (supplementary Fig. S1). This suggests that a recombination event occurred at *oriT* in ICE_Sma33MO_fda. This *oriT* recombination was likely mediated by the relaxase of the ICE that is
able to recognize and nick this sequence. Such oriT site-specific recombination has already been demonstrated for several canonical relaxases of plasmids (belonging to MobF, MobP, MobC and MobM families) (4, 36, 37) and has been suggested to occur in ICEs of the SXT/R391 family and for ICEclc (both encoding canonical relaxases of the MobH family) (38, 39). This mechanism that enables to create hybrid ICEs could thus also be mediated by relaxases of the non-canonical MobT family, which are related to rolling circle replication initiators and are found in ICEs of the Tn916/ICESt3/ICEBs1 family, a family of ICEs widespread in streptococci (11) and other Firmicutes (5).

298

299 *Integration modules*

300 ICESt3 conjugation modules found in S. *salivarius* ICEs are associated with 3 different recombination modules (see supplemental Figure S2) enabling integration of the corresponding ICEs in the 3' end of three different genes: *fda* (encoding the fructose-1-6-diphosphate aldolase, as for ICESt3), *rpsI* (encoding the S9 ribosomal protein) and *rpmG* (encoding the L33 ribosomal protein).
301
302
303
304 This confirms our previous results showing a positive PCR signal for ICESt3 relaxase gene but not for ICESt3 integrase gene for 22 strains (23). Closely related integrases were found in ICEs of S. *salivarius* strains that are distant in the phylogenetic tree built on the basis of MLST data (for example in strains F1-4, L22 and N5 or in strains B35/L50) (23) but also in ICEs found in other species (for example in S. *pneumoniae* 2842STDY5753514 or in S. *parasanguinis* DD19 and S. *macedonicus* 33MO) (supplemental Figure S2). This is in agreement with the previously reported exchanges of recombination modules in ICESt3 subfamily (11, 26).
305
306
307
308
309
310

311

312 *Regulation modules*
Comparison of the regulation modules of all ICEs of the ICESt3 subfamily showed their high diversity (Fig. 1). The only common characteristic is the presence, in most of the ICEs, of homologs of *arp1*, *orfQ* and *arp2* genes. This strongly suggests that these genes participate to the same regulation cascade and likely interact/interfere with each other. Exceptions are ICE_SsaL60_rpsI devoid of *arp1* gene, ICE_SsaB35_rpsI lacking the *orfQ* gene and ICE_SpaDD19_fda carrying truncated *arp1* and *orfQ* genes. ICE_SsaN20_rpsI also displays a peculiar regulation organization, with full copies of *arp1* and *orfQ* genes separated by truncated copies of *orfQ* and *arp1* genes. This is due to an insertion of genes (including one encoding a transposase) in this region of the ICE (Fig. 1). The *arp1*, *orfQ* and *arp2* genes encode respectively a cI-related repressor, a ImmA-related putative protease (40) and a putative transcriptional regulator (41) that are related to those of ICESt3 from *S. thermophilus* (26, 42). Arp1 and Arp2 homologs all share an N-terminal helix-turn helix PF01381 domain that is found in several regulatory proteins including Cro and cI regulators of the λ phage. Arp1 proteins also display a C-terminal PF00717 peptidase S24-like domain involved in the autocleavage of the cI repressor induced by DNA damage. OrfQ homologs are characterized by a COG2856 Zn-dependent peptidase ImmA domain, also found in the ImmA protease of ICEBs1 of *B. subtilis* that cleaves the ImmR repressor in response to DNA damage (40). A high diversity is observed in the nucleotide (Fig. 1) and amino acid primary sequences of the encoded regulators. Up to 45%, 58% and 53% of divergence was observed between the sequences of Arp1, OrfQ and Arp2 proteins encoded by ICEs of *S. salivarius*. If considering only the ICEs that harbor the *arp1*, *orfQ* and *arp2* genes, the sequence divergence goes from 5 to 37% for Arp1, 7 to 58% for OrfQ and 7 to 37% for Arp2.

In support of a modular evolution of ICEs that was already noticeable from the exchanges in the recombination modules mentioned previously, some ICEs harbor regulators which are distantly related to those found in the other ICEs of *S. salivarius* despite having a closely related conjugation
module (deduced from the phylogenetic trees of relaxases and coupling proteins). This is the case of
ICE_SsaF4-2_fda that has a conjugation module closely related to the one of ICESt3 (94% of nucleic
sequence identity over the whole conjugation module) but encode more distant regulators (with 63,
46 and 73% of amino acid identity with those encoded by ICESt3 for Arp1, OrfQ and Arp2
respectively).

### *Accretions*

Accretion of ICEs with other genetic elements was observed: ICEs of strains F1-8 and L60 are in
accretion with a putative IME, whereas those of strains F6-1 and L50 are adjacent to 1 or 2 CIMEs
(data not shown).

Evidences of accretion or recombination events were also found in the sequence of several ICEs of
*S. salivarius*. A truncated supplementary copy of the *orfA* gene was found between *orfA* and the
excisionase genes in *S. salivarius* L22 and B57 ICEs and in the closely related ICE found in
*S. parasanguinis* DD19. The presence of supplementary truncated *arp1* and *orfQ* genes and of a
transposase gene was also detected in the regulation module of *S. salivarius* N20 ICE. These
structures could result from (i) the integration of an ICE within an *att* site flanking a resident element
(accretion) or (ii) by recombination between ICEs, followed by internal deletion(s) in both cases.
These mechanisms contribute to the plasticity and evolution of ICEs and potentiate gene transfer
mediated by these MGE.

### Cargo genes found on ICEs of the ICESt3 subfamily in *S. salivarius* and other streptococci
A diversity of cargo genes was found in the left part of the ICEs of the ICESt3 subfamily explaining the variation of their size ranges: from 25.8 kb for the smallest one (ICE_SsaL64_fda) to 37.3 kb for the largest one (ICE_SsaN20_rpsI) (Fig. 1). The 2 closely related strains included in the analysis (L22 and L64) harbor ICEs with unrelated cargo genes (Fig. 1). In addition, ICEs found in unrelated strains of S. salivarius (for example in strains JF and L22 or F4-2 and F1-8) or in different species (in S. salivarius T93 and S. parasanguinis DD19) harbor the same cargo genes (Fig. 1). This distribution is due to the horizontal transfer of these genes (exchange of module between ICEs or transfer of the whole ICE).

Two thirds (13/19 if including ICESt3 (43)) of the ICEs analyzed in this work carry a restriction-modification (RM) system (n=6) or an orphan methyltransferase (n=7) (Fig. 1). This is in accordance with a previous study indicating that the abundance of RM systems correlates with the presence of MGEs in small genomes (44). RM systems were first described as bacterial innate immune systems allowing protection against foreign unmethylated DNA (45). Unmethylated incoming DNA will be degraded by restriction enzymes produced by the cell while the genome of the host remains protected due to methylation by the cognate methyltransferase. RM systems carried by ICEs of S. salivarius could protect their host from invasion by other genetic elements such as phages, thus playing a role in "cellular defense" as described for other bacteria (46-48). RM systems also turn out to be selfish mobile elements themselves (49). RM systems that carry the restriction enzyme activity and modification enzyme activity on separate proteins (as observed for the ICEs of the ICESt3 subfamily) can have an impact on the maintenance of the MGE carrying them (mechanism of genetic addiction). The MGE encodes the poison endonuclease activity and its "antidote", the methyltransferase (45). Post-segregational killing would occur if the whole RM system is lost. Dilution of the modification enzyme by cell division will lead to the exposure of unmethylated recognition sites on newly replicated chromosomes that will be targeted by the restriction enzyme.
Only a few remaining molecules of restriction enzymes are sufficient to kill the cell. Concerning orphan methyltransferases (encoded by 7 of the ICEs of the ICESt3 subfamily analyzed in this work), if these enzymes target the same DNA sequence as a resident RM system, they could protect the host from post-segregational killing thus participating in the displacement of the resident MGE (50).

Two ICEs found in *S. salivarius* L60 and N20 carry a cadmium resistance cluster (*cadD/cadX*) (Fig. 1). Cadmium is a widespread heavy-metal air pollutant which is commonly released into the environment from industrial processes (in particular glass manufacturing) and urban activities, as well as from the widespread application of fertilizers, manures and sewage sludge (51). The *cadD/cadX* genes also appear in a CIME in accretion with the ICE from strains F6-1 and L50 (data not shown). In these latter ones, full recombination sites are still present and thus the CIME and the ICE are considered separately. In these strains, the whole CIME-ICE composite element could excise and transfer by a process of accretion-mobilization already demonstrated with other ICEs of this family (9, 10). Sequence comparison revealed 98% of nucleic identity over 2683 bp between the CIME located upstream of the ICE in strain L50 and the region that includes *cadD/cadX* genes in ICE_SsaL60_rpsI. This suggests that in strain L60 the cadmium resistance cluster was acquired by the ICE after deletion of the recombination site delimiting the ICE and the adjacent CIME. This mechanism contributes to the plasticity and evolution of ICEs as previously suggested (4).

ICE_SsaF6-1_rpsI carries a cluster of genes (10.8 kb) that could be involved in membrane lipid synthesis. It includes in particular a *fabF*-like gene encoding a beta-ketoacyl-[acyl-carrier-protein] synthase II (KASI/IIcd00834 domain with E=1.4e-144) and a KBL_like gene encoding a serine palmitoyltransferase involved in sphingolipid synthesis (cd06454 domain with E=3.7e-151).

Two ICEs (found in the unrelated strains *S. salivarius* JF and L22) harbor a closely related cluster of genes (*slv* cluster, 15.7 to 15.9 kb) involved in the biosynthesis of a bacteriocin (salivaricin D). These clusters differ from the one previously described in the commensal strain 5M6c of *S. salivarius*
409 isolated from a healthy infant (52) by the number of copies of the *slvD* structural gene (2 and 3
410 identical copies respectively compared to 1 in strain 5M6c). Salivaricin D is a nisin-like lantibiotic
411 with a broad-spectrum that includes a large array of Gram positive bacteria in particular the
412 important pathogens *S. pneumoniae* and *S. pyogenes* (52). Thus, it could be used as a potential
413 weapon, for commensal species of the oral cavity such as *S. salivarius*, to compete with
414 oronasopharynx-colonizing streptococci including pathogens (52-54). Bacteriocins could also play a
415 role of addiction system by killing neighboring cells that do not encode resistance to the bacteriocin
416 and thus contribute to the maintenance of the MGE in the population (55).

417 The ICE found in *S. macedonicus* 33MO carries genes that encode cell-envelope proteins: one with a
418 PF03780 domain with cell envelope-related function (E=7.93e-23), and 3 membrane proteins. Lastly,
419 ICEs of *S. salivarius* T93 and *S. parasanguinis* DD19 harbor the same cargo genes, in particular (i) a
420 two-component system with a signal transduction histidine kinase (nitrate/nitrite-specific COG3850
421 NarQ domain, E=5.3e-13) and a NarL DNA-binding response regulator (COG2197 domain, E=6.1e-
422 48) and (ii) a cluster of genes encoding a putative LolCDE complex which catalyzes the release of
423 lipoproteins from the cytoplasmic membrane (LolC PF13521 AAA_28 domain, E=1.2e-09; LolD
424 cd03255 ABC_MJ0796_LolCDE_FtsE domain, E=1.2e-108 and LolE COG4591 domain, E=4.4e-
425 14) (Fig. 1).

426
427 Excision tests of 2 putative ICEs: ICE_SsaF1-4_fda and ICE_SsaF4-2_fda

428 Experiments were carried out to test the functionality of 2 putative ICEs of *S. salivarius*. The first
429 one, ICE_SsaF1-4_fda, has a conjugation module showing the highest percentage of identity with
430 that of ICESt3 of *S. thermophilus*, an ICE whose transfer was previously demonstrated (24) (Fig. 1).
431 Arp1, OrfQ and Arp2 regulators encoded by this ICE are also closely related to those of ICESt3 (85,
432 71 and 84% of amino acid identity respectively). As mentioned before, the second one, ICE_SsaF4-
433

2_fda, has a conjugation module closely related to ICE_SsaF1-4_fda and ICESt3 but harbors a
distantly related regulation module.

434

435

Excision of these 2 ICEs was tested by PCR. Amplifications were obtained for *attI* and *attB*
corresponding to the circular excised form and the empty chromosomal site respectively, indicating
the functionality of their recombination module. Recombination sites (*attL* and *attR*) flanking the
ICEs were also amplified (data are shown only for ICE_SsaF1-4_fda, Fig. 4).

436

437

438

439

440

Test of intraspecies conjugative transfer of 2 putative ICEs of S. salivarius

441

Both ICE_SsaF1-4_fda and ICE_SsaF4-2_fda were first tagged by a chloramphenicol resistance
cassette (Table 1). Donor strains were then used in filter mating experiments with two S. salivarius
recipient strains: JIM8777 and F1-8, displaying different genotypes (Table 1 and 3). Both strains
carry an empty fda integration site and strain F1-8 carries an ICE belonging to the ICESt3 subfamily
(Fig. 1) in accretion with an IME integrated in the 3' end of rpmG. Some putative transconjugants
were recovered when JIM8777 or F1-8 S. salivarius strains were used as recipients (Table 3). After
sub-culturing, transconjugants were confirmed by detection of the integrase gene (data are shown
only for ICE_SsaF1-4_fda, Fig. 5a, 5b). For JIM8777 derivative clones, this screen was combined
with detection of the pMG36e plasmid and RAPD (Fig. 5a). F1-8 transconjugants were confirmed by
sequencing of an MLST locus (ddlA gene). The site specific insertion in the fda gene and the
excision of the newly acquired ICEs were also confirmed by the PCR detection of attL, attR, attI and
attB sites for both F1-8 (data are shown only for ICE_SsaF1-4_fda, Fig. 5c), and JIM8777 (data not
shown). This suggests that the elements are still active in these transconjugants (at least for excision).
Mating experiments were repeated with at least 3 different cultures of donor and recipient cells
(biological repetitions) but very few transconjugants (2 clones for the whole experiment) were
obtained thus preventing any calculation of transfer frequency. Attempts to increase ICE_SsaF1-

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456
457 4_fda transfer frequency were made: (i) test of different media (milk, BHI, mitis, M17 broth with 1%
458 final glucose instead of 0.5% final lactose), (ii) test of different donor/recipient ratio ranging from
459 1:1, 2:1, 10:1, 50:100 to 100:1, (iii) mix of donor and recipient at the end of exponential growth
460 phase or stationary phase instead of mid-exponential phase. None of the tested conditions enabled to
461 increase transfer frequency which remained inferior to 10⁻⁸ transconjugants per donor cells.
462

462

463 Test of interspecies conjugative transfer of ICEs of S. salivarius

464 To evaluate ICE interspecies transfer, mating assays were carried out with two other *Firmicutes* as
465 recipients: the closely related *S. thermophilus* and *Enterococcus faecalis*. Recipient strains were
466 *S. thermophilus* LMG18311 and *E. faecalis* JH2-2 both carrying pMG36e and described as recipients
467 strains of ICESt3 from *S. thermophilus* (transfer frequency of 3.4 × 10⁻⁶ ± 0.5 × 10⁻⁶ and 3.9 × 10⁻⁷ ±
468 0.9 × 10⁻⁷ respectively (24)). Some putative transconjugants (2 clones for the whole experiment)
469 were recovered when using strains F1-4 or F4-2 as donors in mating experiments (Table 3) and
470 confirmed by PCR detection of the ICE integrase gene (data are shown only for ICE_SsaF1-4_fda,
471 Fig. 6). LMG18311 genetic background was confirmed by PCR amplification of the internal
472 transcribed spacer (ITS) followed by *Hae*III DNA digestion, which allows discriminating
473 *S. salivarius* strains and *S. thermophilus* LMG18311 (data are shown only for ICE_SsaF1-4_fda, Fig.
474 6a). JH2-2 genetic background was confirmed by the PCR amplification of the ITS and by the
475 amplification of *fda* gene with primers specific to *E. faecalis* (data are shown only for
476 ICE_SsaF1-4_fda, Fig. 6b).
477

478 ICE_SsaF1-4_fda carries 27 bp showing identity with the 3' end region of the fda gene of
479 *S. salivarius* (Fig. 7). This sequence (present in *attI*) enables specific recombination with the
480 chromosomal integration site of the recipient cell (*attB* site) and generates a 27 bp-imperfect direct
481 repeat (DR) in intraspecies transconjugants (Fig. 7). Sequencing of the *attR* site obtained after
482
481 integration of ICE_SsaF1-4_fda in E. faecalis JH2-2 recipient cells provided relevant information on
482 the DNA localization of one cutting site of fda integrase. This *attR* site is a hybrid between the
483 sequence found in the *attI* of ICE_SsaF1-4_fda and the *attB* of E. faecalis JH2-2 recipient cells.
484 Since the *attB* sequence in the recipient strains is different from the *attI* of ICE_SsaF1-4_fda (Fig. 7),
485 the presence of a T nucleotide at position 20 of the DR in the *attR* of the transconjugants, as in
486 ICE_SsaF1-4_fda, indicates that integrase cuts downstream of this nucleotide to allow strand
487 exchange during recombination. A previous work aiming at studying CIME-ICE accretion also
488 identified the same cutting position as well as the position of a second staggered cutting site located
489 6 bp downstream (Fig. 7) (56).

490 Intra- and interspecies conjugative transfer of two different ICEs found in S. salivarius was obtained
491 in laboratory conditions. These ICEs could thus play a significant role in HGT occurring both in the
492 oral cavity (as exemplified by the presence of a closely related ICE in S. parasanguinis) and in the
493 digestive tract.

494

495 **Impact of mitomycin C on excision and transfer of ICE_SsaF1-4_fda**

496 A previous work demonstrated that excision and transfer of ICESt3 of S. thermophilus can be
497 increased by treating donor cells with mitomycin C (42). Hence we examined the impact of such
498 treatment on excision and transfer of an ICE from S. salivarius (ICE_SsaF1-4_fda). Induction of ICE
499 excision by mitomycin C was tested by PCR amplification of *attI* and *attB* fragments using different
500 amounts of genomic DNA (0.1 µg to 1 pg) after MMC treatment or not of the cells (Fig. 8). The
501 minimal quantity of DNA producing a positive result for *attI* and *attB* was 0.1 ng for non-treated
502 cells whereas 10 pg of DNA were sufficient to detect a signal after MMC treatment of the cells (Fig.
503 8). Furthermore, when cells were treated with MMC, a higher intensity of the PCR signal was
504 observed for *attI* and *attB* using 0.1, 1 and 10 ng of DNA compared to cells without MMC treatment
505 (Fig. 8). Such difference was not observed for the *fda* control (Fig. 8). Thus, as for ICESt3 (42),
506 MMC treatment leads to an increase of ICE_SsaF1-4_fda excision.

507 To evaluate the effect of MMC on ICE transfer, strain F1-4 was treated with MMC (at the
508 concentration inducing the maximum level of ICE excision) and used in filter mating experiments
509 with *S. salivarius* strain F1-8 as recipient. No difference of ICE transfer was detectable between cells
510 treated or not with MMC. This is in contrast to what was observed for ICESt3 (42). Thus, it appears
511 that for ICE_SsaF1-4_fda excision is not a limiting step for transfer.

512

513 Retransfer of ICE_SsaF1-4_fda from transconjugants

514 To test whether ICE_SsaF1-4_fda transfers autonomously, retransfer was tested by using
515 transconjugants (obtained as described above) as donor cells. Plasmid pMG36e was removed by
516 curing transconjugants prior to the retransfer assays. F1-8 (ICE_SsaF1-4_fda) was used as donor
517 cells in filter mating experiments with either F1-8 (pMG36e) or LMG18311 (pMG36e) as recipient
518 cells. LMG18311 (ICE_SsaF1-4_fda) and JH2-2 (ICE_SsaF1-4_fda) were used as donor cells with
519 LMG18311 (pMG36e) and JH2-2 (pMG36e) respectively (Table 3). For each mating assay,
520 transconjugants were recovered and confirmed by PCR detection of both the ICE integrase gene and
521 the plasmid pMG36e present in the recipients (data not shown). A small number of transconjugant
522 colonies (as for intraspecies transfer of ICE_SsaF1-4_fda i.e. 2 clones for the whole experiment) was
523 observed when using *S. salivarius* as donor, and strains with the same genetic background as
524 recipients (F1-8/F1-8 mating pairs) or *S. thermophilus* LMG18311 as recipient cells. By contrast,
525 more transconjugant colonies (at least 20 clones for the whole experiment) were obtained using
526 *S. thermophilus* LMG18311 (ICE_SsaF1-4_fda) and *E. faecalis* JH2-2 (ICE_SsaF1-4_fda) as donors
527 and LMG18311 (pMG36e) and JH2-2 (pMG36e) as recipient cells. These results confirm that
528 ICE_SsaF1-4_fda is able to transfer autonomously but also suggest that (i) host factors could impact
529 ICE replication or/and assembly of the conjugation machinery in *S. salivarius* as already suggested
530 for some strains of *S. thermophilus* (26), (ii) molecules displayed at the cell surface of *S. salivarius*
531 could impact the donor/recipient cell contacts and thus the assembly of the conjugative machinery.
532 These hypotheses are consistent with the fact that no transconjugant was recovered when
533 *S. thermophilus* CNRZ368 donor cells carrying ICESt3 were mated with *S. salivarius* JIM8777 or
534 F1-8 recipient cells (data not shown in this study) whereas transconjugants were obtained using
535 *S. thermophilus* LMG18311 as recipient cells as already reported (24).

536 It should be kept in mind that *S. salivarius* encounters different physiological conditions in the oral
537 cavity and along the digestive tract. The impact on ICE regulation of these complex interactions
538 between bacteria and of changing environment in these ecological niches should be explored in close
539 future.

540

541 ACKNOWLEDGMENTS

542 We thank Stéphane Bertin and Emilie Robert for their technical help.

543

544 FUNDING INFORMATION

545 N.D. is recipient of a scholarship funded by INRA and Région Grand Est (formerly Région
546 Lorraine).

547 This work received financial support from the Région Lorraine and Université de Lorraine (2011-
548 2013) and ANR (MATICE project <ANR-15-CE21-0007>).

549
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710 55. **Rankin DJ, Rocha EP, Brown SP.** 2011. What traits are carried on mobile genetic elements, and why? *Heredity* **106**:1-10.

712 56. **Bellanger X.** 2009. Transfert, accrétion et mobilisation des éléments intégratifs conjugatifs et des îlots génomiques apparentés de *Streptococcus thermophilus*: Un mécanisme clef de l'évolution bactérienne ? *Ph. D. thesis*. Nancy-Université, Nancy.

715 57. **Guédon E, Delorme C, Pons N, Cruaud C, Loux V, Couloux A, Gautier C, Sanchez N, Layec S, Galleron N, Almeida M, van de Guchte M, Kennedy SP, Ehrlich SD, Gibrat JF, Wincker P, Renault P.** 2011. Complete genome sequence of the commensal *Streptococcus salivarius* strain JIM8777. *J Bacteriol* **193**:5024-5.

719 58. **Jacob AE, Hobbs SJ.** 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in *Streptococcus faecalis* var. zymogenes. *J Bacteriol* **117**:360-72.

721 59. **Takamatsu D, Osaki M, Sekizaki T.** 2001. Thermosensitive suicide vectors for gene replacement in *Streptococcus suis*. *Plasmid* **46**:140-8.

723 60. **Delorme C, Poyart C, Ehrlich SD, Renault P.** 2007. Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group. *J Bacteriol* **189**:1330-41.

725

726
727 LEGENDS OF FIGURES

728

729 FIG 1 Comparison of the Integrative and Conjugative Elements (ICEs) found in *S. salivarius* and in
730 other streptococci.

731 ICEs are named according to their host strain and integration site. ICEs of *S. salivarius* are indicated
732 in bold. For more clarity, elements in accretion with ICEs are not shown. Nucleic sequence identity
733 higher than 80% between sequences is indicated in light grey and higher than 90% in dark grey.

734 Direct repeats (DR) delimiting ICEs are shown as a triangle, circle or square depending on their
735 sequences. ORFs appear as arrows (truncated genes are indicated by Δ). Modules of recombination
736 (integrase *int* and excisionase *xis* genes), conjugation (*orfA-orfO* genes) and regulation (including
737 *arp1*, *arp2* and *orfQ* genes) appear in red, blue and green respectively. The three different integration
738 genes (*fda*, *rpsI* or *rpmG*) targeted by the integrase are indicated by distinct symbols in the integrase
739 gene and is stated in the ICE name. Genes from the adaptation module encoding proteins with
740 putative function inferred from *in silico* analysis are indicated in color: in pink for RM systems and
741 orphan methyltransferase genes, in dark grey for cadmium resistance genes (*cadD/X*), in yellow for
742 the membrane lipid synthesis cluster, in orange for the bacteriocin synthesis cluster, in light green for
743 genes encoding cell envelop proteins, in light pink for the cluster of genes for putative lipoprotein
744 transport, in brown for the two component system (TCS), in black for the *tnp* transposase gene(s).

745 Sequences used for this analysis are: ICESt3 of *S. thermophilus* [AJ586568] and sequenced genomes
746 of *S. pneumoniae* 2842STDY5753514 [FDNK01000013], *S. macedonicus* 33MO [JNCV01000015]
747 and *S. parasanguinis* DD19 [LQNY01000339-LQNY01000340]. Gaps in the assembly found in
748 ICE_SsaF6-1_rpsI, ICE_SsaB57_fda and ICE_SpaDD19_fda are indicated by a double slash.

749
FIG 2 Phylogenetic BioNJ tree obtained for relaxases (OrfJ) of ICEs belonging to the ICESt3 subfamily. The relaxase protein sequence of twenty-seven ICEs (the 19 ICEs of the ICESt3 subfamily with closely related conjugation modules and 8 additional ones previously reported to belong to the same ICE subfamily but showing more distantly related conjugation modules) were included in the analysis. ICEs of *S. salivarius* are indicated in bold and *ICE_Sma33MO_fda* is indicated by a star. Bootstrap values supporting main branches are given for BioNJ/ML respectively.

FIG 3 Phylogenetic BioNJ tree obtained for coupling proteins (OrfK) of ICEs of the ICESt3 subfamily. The sequence of the coupling protein of twenty-seven ICEs (the 19 ICEs of the ICESt3 subfamily with closely related conjugation modules and 8 additional ones previously reported to belong to the same ICE subfamily but showing more distantly related conjugation modules) were included in the analysis. ICEs of *S. salivarius* are indicated in bold and *ICE_Sma33MO_fda* is indicated by a star. Bootstrap values supporting main branches are given for BioNJ/ML respectively.

FIG 4 PCR detection of integrated and excised forms of ICE_SsaF1-4_fda.

The sizes of the PCR fragments obtained for the amplification of the *attB*, *attI*, *attR* and *attL* sites (586 bp, 887 bp, 900 and 523 bp respectively) were confirmed by parallel migration of a DNA ladder. The primer pairs used for these amplifications are indicated in Table 2.

FIG 5 Characterization of transconjugants carrying ICE_SsaF1-4_fda after intraspecies transfer.

F1-4 (*ICE_SsaF1-4_fda*) was used as donor in mating experiments with (a) JIM8777 (pMG36e) and (b, c) F1-8 (pMG36e) as recipients. Tc. indicates transconjugants; integrase, amplification of the integrase gene of *ICE_SsaF1-4_fda*; pMG36e, amplification of an internal fragment of the pMG36e plasmid; RAPD, random amplification of polymorphic DNA; *attL*, *attR*, *attI* and *attB*, amplification
774 of fragments carrying these attachments sites in F1-8 transconjugants (c). The primer pairs used for
775 these amplifications are indicated in Table 2.

776

777 FIG 6 Characterization of transconjugants carrying ICE_SsaF1-4_fda after interspecies transfer.

778 F1-4 (ICE_SsaF1-4_fda) was used as donor in mating experiments with (a) LMG18311 (pMG36e)
779 and (b) JH2-2 (pMG36e) as recipients. Tc. indicates transconjugants; integrase, amplification of the
780 integrase gene of ICE_SsaF1-4_fda; ITS16S/23S, amplification of the internal transcribed spacer;
781 ITS16S/23S+HaeIII digestion, amplification of the internal transcribed spacer followed by HaeIII
782 digestion of the obtained fragment; EF fda, amplification of fda fragment using E. faecalis specific
783 primers.

784

785 FIG 7 Localization of the DNA cutting site of integrase by sequencing of *attR* site in *E. faecalis* JH2-2 transconjugants.

787 ICE_SsaF1-4_fda is shown in green in its integrated form in donor S. salivarius strain F1-4 (upper
788 part of the figure), its excised form (middle part of the figure) and its integrated form in E. faecalis
789 JH2-2 recipient strain after transfer and integration (lower part of the figure). Nucleotide differences
790 in *att* sequences are indicated in green for ICE_SsaF1-4_fda and in red for E. faecalis JH2-2. The
791 position of the DNA cutting site of integrase deduced from this analysis is indicated by a black
792 arrow. A previous work aiming at studying CIME-ICE accretion also identified the same cutting
793 position as well as the position of a second staggered cutting site located 6 bp from the first one
794 (indicated in grey in the figure) (56).

795

796 FIG 8 Impact of mitomycin C on ICE_SsaF1-4_fda excision.
797 Fragments corresponding to the recombination sites *attB* and *attI* of non-treated (MMC-) or treated

798 (MMC+) cells were amplified by PCR using template DNA quantities ranging from 0.1 µg to 1 pg.

799 The amplification of the *fda* gene was used as a control.

800
<table><caption>TABLE 1 Bacterial strains and plasmids used in this study (to be continued)</caption><thead><tr><th>Strains and plasmids</th><th>Relevant phenotype or genotype</th><th>Source or reference</th></tr></thead><tbody><tr><td><strong>Strains</strong></td><td></td><td></td></tr><tr><td><em>S. salivarius</em></td><td></td><td></td></tr><tr><td>B35</td><td>Wild-type (WT) strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>B57</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>F1-4</td><td>Wild-type (WT) strain carrying a putative ICE of the ICESt3 subfamily (ICE_SsaF1-4_fda)</td><td>(23)</td></tr><tr><td>F1-4(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>F1-4 carrying ICE_SsaF1-4_fda tagged with a Cm<sup>r</sup> cassette</td><td>This work</td></tr><tr><td>F1-8</td><td>WT strain carrying a putative ICE in accretion with an IME integrated in the 3' end of rpmG, no element in fda</td><td>(23)</td></tr><tr><td>F1-8(pMG36e)</td><td>F1-8 carrying pMG36e, a plasmid conferring erythromycin resistance</td><td>This work</td></tr><tr><td>F4-2</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>F6-1</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>JIM8777</td><td>WT strain carrying a putative IME in the 3' end of rpmG and a putative CIME in the 3' end of rpsI, no element integrated in the fda gene</td><td>(57)</td></tr><tr><td>JIM8777(pMG36e)</td><td>JIM8777 carrying pMG36e, a plasmid conferring erythromycin resistance</td><td>This work</td></tr><tr><td>L22</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>L50</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>L60</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>L64</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>N5</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>N20</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td>T93</td><td>WT strain carrying a putative ICE of the ICESt3 subfamily</td><td>(23)</td></tr><tr><td><strong>S. thermophilus</strong></td><td></td><td></td></tr><tr><td>CNRZ385 ICESt3cat</td><td>CNRZ385 carrying ICESt3 with cat gene inserted in orf385J pseudogene, Cm<sup>r</sup></td><td>(24)</td></tr><tr><td>LMG18311</td><td>WT strain with no element in fda</td><td>BCCM/LMG</td></tr><tr><td>LMG18311(pMG36e)</td><td>LMG18311 carrying pMG36e, a plasmid conferring erythromycin resistance</td><td>(24)</td></tr></tbody></table>
<table><caption>TABLE 1 Bacterial strains and plasmids used in this study</caption><thead><tr><th>Strains and plasmids</th><th>Relevant phenotype or genotype</th><th>Source or reference</th></tr></thead><tbody><tr><td><em>E. faecalis</em></td><td></td><td></td></tr><tr><td>JH2-2</td><td>WT strain with no element in <em>fda</em></td><td>(58)</td></tr><tr><td>JH2-2(pMG36e)</td><td>JH2-2 carrying the plasmid pMG36e, conferring erythromycin resistance</td><td>(24)</td></tr><tr><td>Plasmids</td><td></td><td></td></tr><tr><td>pSET5s</td><td>pWV01-type thermosensitive replication origin from pVE6002, LacZ, Cm<sup>r</sup></td><td>(59)</td></tr><tr><td>pMG36e</td><td>3.6 kb, replication origin from pWV01, Ery<sup>r</sup></td><td>(49)</td></tr></tbody></table>
<table><caption>TABLE 2. Primers used in this work (to be continued).</caption><thead><tr><th>Primer use</th><th>Primer name</th><th>Sequence (5'-3')</th><th>Reference</th></tr></thead><tbody><tr><td>ICE labeling</td><td>F1-4 CDS7 F<br>F1-4 CDS8 R_Bis<br>F1-4 CDS9 F_Bis<br>F1-4 CDS9 R<br>ICE F4-2 fragment I_F<br>ICE F4-2 fragtI_R-catbis<br>ICE F4-2 fragtII_F-catbis<br>ICE F4-2 fragment II_R<br>Cat_F<br>Cat_R</td><td>GAGATTGAGCATATCCTTCC<br>GGTGACTAGTTATCTACACGCGAGATTCGTGGACTAACTT<br>CCATATCCTTCTTTTTCTGCTCACTATCTTGTTCGTTTTGT<br>GGAGAGTTTAGCTGGGAGG<br>GGAAATATCCTGTTGTCATC<br>GGTGACTAGTTATCTACACGCCTATAAAGTTGTTAAGTTCACT<br>CCATATCCTTCTTTTTCTGGCGTGTAATTGAAGAGTGA<br>GTCTAAACTGAGCCAAGAAG<br>GCCTCCTAAATTCACTTTAG<br>GTAAAAAGTACAGTCGGCAT</td><td>This study</td></tr><tr><td>Detection of ICE integrated and excised forms</td><td></td><td></td><td></td></tr><tr><td>attB amplification</td><td>attBfdaSsal<br>attB ST3 Rev</td><td>GCCCAACCAAATAACACTAAA<br>CTCTTCGACCCACGTAAATTC</td><td>(23)</td></tr><tr><td>attI amplification</td><td>intST3 For<br>attI ST3 Rev</td><td>AGGGCTTTCTGACGAATTAG<br>CGGTGTAATGGGAAGTATGG</td><td>(23)</td></tr><tr><td>attL amplification</td><td>attL Rv CDS gwlG<br>attBfdaSsal</td><td>CGGTGTAATGGGAAGTATGG<br>GCCCAACCAAATAACACTAAA</td><td>This study<br>(23)</td></tr><tr><td>attR amplification</td><td>intST3 for<br>intICESt3-fdaRev</td><td>AGGGCTTTCTGACGAATTAG<br>ACCAGGTTTCGATGCTATTACAG</td><td>(23)<br>(43)</td></tr><tr><td>Integrase gene</td><td>intST3 for<br>intST3 Rev</td><td>AGGGCTTTCTGACGAATTAG<br>GAGTTCTAATAACTGAGGCTA</td><td>(23)</td></tr></tbody></table>
<table><caption>TABLE 2. Primers used in this work</caption><thead><tr><th>Primer use</th><th>Primer name</th><th>Sequence (5'-3')</th><th>Reference</th></tr></thead><tbody><tr><td>Semi-quantitative PCR<br>attI amplification</td><td>intICEF1-4 For<br>attIICEF1-4 Rev</td><td>AGGTCTTTCTGACGAATTAG<br>CGGCGTAATGGGAAGTATGG</td><td>This study</td></tr><tr><td>attB amplification</td><td>attBfdaSsal<br>attB ST3 Rev</td><td>GCCCAACCAAATAACACTAAA<br>CTCTTCGACCCACGTAAATTC</td><td>(23)</td></tr><tr><td>fda amplification</td><td>Fda1<br>Fda2</td><td>TTCAAGAATTTACGTGGG<br>AGATGCTAAAGCTATGGTTG</td><td>This study<br>(34)</td></tr><tr><td>Donor / Recipient discrimination<br>ITS16S/23S</td><td>16SITS<br>23SITS</td><td>TTGTACACACCGCCCGTCA<br>GGTACCTTAGATGTTTCAGTTC</td><td>(24)</td></tr><tr><td>ddlA gene</td><td>ddlA up<br>ddlA dn</td><td>TCAAGTGTGGCTATGGA<br>GTAGATGGCTCCATCCTC</td><td>(60)</td></tr><tr><td>E. faecalis fda<br>specific amplification</td><td>Efa fba1b<br>Efa fba2</td><td>ATGTGTTCTTCTGCATCTTT<br>CCCATTGATTACGATTTTT</td><td>(24)</td></tr><tr><td>RAPD</td><td>XD9</td><td>GAAGTCGTCC</td><td>(34)</td></tr><tr><td>pMG36e</td><td>pMG36e Fwd<br>pMG36e_R</td><td>GCCTCCTCATCCTCTTCAT<br>ACAGAACCGTTTCTACTCAATGAAC</td><td>(24)<br>This study</td></tr></tbody></table>
<table><caption>TABLE 3 Mating pairs tested in filter experiments</caption><thead><tr><th></th><th>Donor cells with AB<sup>r a</sup></th><th>Recipient cells with AB<sup>r a</sup></th></tr></thead><tbody><tr><td rowspan="2">Intraspecies transfer</td><td>F1-4(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>JIM8777 (pMG36e) Ery<sup>r</sup><br>F1-8 (pMG36e) Ery<sup>r</sup></td></tr><tr><td>F4-2(ICE_SsaF4-2_fda) Cm<sup>r</sup></td><td>JIM8777 (pMG36e) Ery<sup>r</sup><br>F1-8 (pMG36e) Ery<sup>r</sup></td></tr><tr><td rowspan="2">Interspecies transfer</td><td>F1-4(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>LMG18311 (pMG36e) Ery<sup>r</sup><br>JH2-2 (pMG36e) Ery<sup>r</sup></td></tr><tr><td>F4-2(ICE_SsaF4-2_fda) Cm<sup>r</sup></td><td>LMG18311 (pMG36e) Ery<sup>r</sup><br>JH2-2 (pMG36e) Ery<sup>r</sup></td></tr><tr><td rowspan="3">ICE retransfer from<br>transconjugants</td><td>F1-8(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>F1-8 (pMG36e) Ery<sup>r</sup><br>LMG18311 (pMG36e) Ery<sup>r</sup></td></tr><tr><td>LMG18311(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>LMG18311 (pMG36e) Ery<sup>r</sup></td></tr><tr><td>JH2-2(ICE_SsaF1-4_fda) Cm<sup>r</sup></td><td>JH2-2 (pMG36e) Ery<sup>r</sup></td></tr></tbody></table>

<footnote>a AB<sup>r</sup> antimicrobial resistance of the strain</footnote>
<figure><img src="image_3.png" /><figcaption>FIG 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIG 2</figcaption></figure>
FIG3

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>FIG 4</figcaption></figure>
a

<figure><img src="image_7.png" /></figure>

b

<figure><img src="image_8.png" /></figure>

c
a

<figure><img src="image_9.png" /></figure>

b

<figure><img src="image_10.png" /></figure>
FIG7

<figure><img src="image_11.png" /></figure>

Excision

<figure><img src="image_12.png" /></figure>

Transfer

<figure><img src="image_13.png" /></figure>

fda

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

attI

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

attB

<figure><img src="image_18.png" /></figure>

E. faecalis JH2-2

<figure><img src="image_19.png" /></figure>

fda

<figure><img src="image_20.png" /></figure>

Integration

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

attR

<figure><img src="image_23.png" /></figure>

TC
JH2-2

<figure><img src="image_24.png" /></figure>

fda

<figure><img src="image_25.png" /></figure>
FIG8

<figure><img src="image_26.png" /></figure>
<figure><img src="image_27.png" /><figcaption>**Fig. S1:** sequence comparison of *orfK*, intergenic sequence *orfK-orfJ* and beginning of the *orfJ* gene in ICEs found in *S. parasanguinis* NCFD2020, *S. macedonicus* 33MO, *S. salivarius* F1-4 compared to ICESt3 of *S. thermophilus*. Start and stop codons are indicated as blue rectangles and the *nic* site of *oriT* is indicated as a green arrow.</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>**Fig. S2.** Phylogenetic BioNJ tree obtained for integrases of ICEs belonging to the ICESt3 subfamily. The integrase protein sequence of twenty-seven ICEs (the 19 ICEs of the ICESt3 subfamily with closely related conjugation modules and 8 additional ones previously reported to belong to the same ICE subfamily but showing more distantly related conjugation modules) were included in the analysis. ICEs of *S. salivarius* are indicated in bold. Bootstrap values supporting main branches are given at nodes.</figcaption></figure>