Narimane Dahmane

Virginie Libante

Florence Charron-Bourgoin

Eric Guédon

Gérard Guédon

Nathalie N Leblond-Bourget

Sophie Payot
email: sophie.payot-lacroix@inra.fr

S Salivarius

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

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT:

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

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

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

IMPORTANCE

The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective

INTRODUCTION

Acquisition of genes by horizontal gene transfer (HGT) is a major driving force for evolution of bacterial genomes [START_REF] Abby | Lateral gene transfer as a support for the tree of life[END_REF][START_REF] Ochman | Lateral gene transfer and the nature of bacterial innovation[END_REF]. 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 [START_REF] Llosa | Bacterial conjugation: a twostep mechanism for DNA transport[END_REF]. 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 [START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF][START_REF] Roberts | Bacterial Integrative Mobile Genetic Elements[END_REF]. Like all other mobile genetic elements (MGEs) [START_REF] Toussaint | Mobile elements as a combination of functional modules[END_REF], 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 [START_REF] Burrus | Conjugative transposons: the tip of the iceberg[END_REF].

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 [START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF][START_REF] Roberts | Bacterial Integrative Mobile Genetic Elements[END_REF]. Moreover, various ICEs can promote the transfer of large fragments of the bacterial chromosome by an Hfr-like mechanism [START_REF] Brochet | Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae[END_REF]. 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 [START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF]. 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) [START_REF] Bellanger | Site-specific accretion of an Integrative Conjugative Element and a related genomic island leads to cismobilization and gene capture[END_REF][START_REF] Puymège | Conjugative transfer and cis-mobilization of a genomic island by an Integrative and Conjugative Element of Streptococcus agalactiae[END_REF]. 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 [START_REF] Ambroset | New insights into the classification and integration specificity of Streptococcus Integrative Conjugative Elements through extensive genome exploration[END_REF][START_REF] Guglielmini | The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation[END_REF][START_REF] Puymège | Analysis of Streptococcus agalactiae pangenome for prevalence, diversity and functionality of integrative and conjugative or mobilizable elements integrated in the tRNA(Lys CTT) gene[END_REF]. In addition to the genes involved or controlling their mobility, ICEs also carry cargo genes, which can provide new properties (virulence, antibiotic resistance for example) to the recipient cell [START_REF] Burrus | Conjugative transposons: the tip of the iceberg[END_REF].

Streptococcus salivarius is a Firmicute that is a major constituent of the human oral cavity microbiota [START_REF] Aas | Defining the normal bacterial flora of the oral cavity[END_REF] and is commonly detected in the human gastrointestinal tract of healthy individuals [START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF][START_REF] Van Den Bogert | Comparative genomics analysis of Streptococcus isolates from the human small intestine reveals their adaptation to a highly dynamic ecosystem[END_REF]. Few strains have also been associated with opportunistic infections in particular in cases of meningitis [START_REF] Wilson | Clinical and laboratory features of Streptococcus salivarius meningitis: a case report and literature review[END_REF], endocarditis [START_REF] Kitten | Oral streptococcal bacteremia in hospitalized patients: taxonomic identification and clinical characterization[END_REF] and bacteremia in immunocompromised patients [START_REF] Corredoira | Clinical characteristics and significance of Streptococcus salivarius bacteremia and Streptococcus bovis bacteremia: a prospective 16year study[END_REF][START_REF] Han | Viridans streptococci isolated by culture from blood of cancer patients: clinical and microbiologic analysis of 50 cases[END_REF].

Analyses of S. salivarius genomes pointed out the considerable variability of gene content and the differences in adaptive traits [START_REF] Delorme | Genomics of Streptococcus salivarius, a major human commensal[END_REF]. Evidence of widespread HGT was obtained in link with the presence of diverse MGEs [START_REF] Delorme | Genomics of Streptococcus salivarius, a major human commensal[END_REF] and the competence for natural transformation of the species [START_REF] Fontaine | A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius[END_REF].

We recently screened a collection of 138 strains of S. salivarius for the presence of MGEs [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF]. This led to the identification of 60 strains (belonging to 39 MLST groups) with a positive PCR signal for the relaxase gene of ICESt3, an ICE previously characterized in the closely related species Streptococcus thermophilus [START_REF] Bellanger | Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus[END_REF][START_REF] Burrus | The ICESt1 element of Streptococcus thermophilus belongs to a large family of integrative and conjugative elements that exchange modules and change their specificity of integration[END_REF][START_REF] Carraro | Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus[END_REF]. This indicated the presence in these strains of putative ICEs belonging to the ICESt3 subfamily. However, not all these strains showed a positive PCR signal for the ICESt3 integrase gene that catalyzes the integration into the fda site. This suggested the presence of other recombination modules associated with the ICESt3 conjugation module in S. salivarius

ICEs.

In this work, we selected 13 strains with different MLST patterns (including 6 strains with a negative PCR signal for the ICESt3 integrase gene) and sequenced their genome in order to gain access to their ICE sequences. ICEs with a conjugation module closely related to the one of ICESt3 were searched in other NCBI available genomes of S. salivarius and in other genomes of Firmicutes. ICE sequences were compared and the putative function encoded by their genes was analyzed in silico.

Two putative ICEs from S. salivarius were analyzed experimentally to test their excision and their autonomous intraspecies and interspecies conjugative transfer.

MATERIAL AND METHODS

Bacterial strains, plasmids and culture conditions

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

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 [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF].

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 [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF]. 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 [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF]. These 13 strains are available upon request.

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 [START_REF] Van De Guchte | Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis[END_REF] and has been successfully used for labeling recipient strains in conjugation experiments with S. thermophilus [START_REF] Bellanger | Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus[END_REF]. 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 pairedend 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/) [START_REF] Aziz | The RAST Server: rapid annotations using subsystems technology[END_REF][START_REF] Overbeek | The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST)[END_REF]. 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 databanks, 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.

Pairwise comparisons of elements were performed with Artemis Comparison Tool provided by the Sanger Centre using comparison files generated by Double Act (available at: http://www.hpabioinfotools.org.uk/pise/double_act.html) [START_REF] Carver | Artemis and ACT: viewing, annotating and comparing sequences stored in a relational database[END_REF]. Manual editing of comparison figures was performed using Inkscape.

Nucleotide sequence accession numbers

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

Phylogenetic tree construction

Proteins of twenty-seven ICEs were included in the analysis. These ICEs correspond to (i) the 19 ICEs of the ICESt3 subfamily with closely related conjugation modules and (ii) 8 additional ones previously reported to belong to the same ICE subfamily but showing more distantly related conjugation modules [START_REF] Ambroset | New insights into the classification and integration specificity of Streptococcus Integrative Conjugative Elements through extensive genome exploration[END_REF][START_REF] Carraro | Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus[END_REF]. The sequences of signature proteins were aligned using Clustal omega with default parameters [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF]. The trees were built with MEGA (32) using (i) maximum likelihood (ML) based on JTT (Jones-Taylor-Thornton) model including amino acid empirical frequencies (partial deletion of gaps and missing data at 80% cutoff, Gamma distribution in 5 categories, allowance for invariant sites), and (ii) BioNJ methods with the Poisson model [START_REF] Gouy | SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. The branch support of the groupings was estimated using bootstrap (100 replicates).

ICE tagging

ICE_SsaF1-4_fda and ICE_SsaF4-2_fda were tagged by a chloramphenicol resistance cassette originating from pSET5s plasmid. The resistance cassette was inserted in an intergenic region located between convergent coding sequences in the adaptive module to avoid impacting ICE functionality. Two DNA fragments of about 1,000 bp corresponding to the upstream and the downstream regions of the integration locus were amplified by PCR using specific primers that 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) [START_REF] Fontaine | A novel pheromone quorum-sensing system controls the development of natural competence in Streptococcus thermophilus and Streptococcus salivarius[END_REF] 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.

RESULTS AND DISCUSSION

Diversity of Integrative and Conjugative Elements of the ICESt3 subfamily identified in

S. salivarius and in other streptococci

Conjugation modules

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 [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF]. 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 and3). 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 [START_REF] Lee | Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis[END_REF], 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) [START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF][START_REF] Kishida | Host range of the conjugative transfer system of IncP-9 naphthalene-catabolic plasmid NAH7 and characterization of its oriT region and relaxase[END_REF][START_REF] Wang | The resolution and regeneration of a cointegrate plasmid reveals a model for plasmid evolution mediated by conjugation and oriT site-specific recombination[END_REF] and has been suggested to occur in ICEs of the SXT/R391 family and for ICEclc (both encoding canonical relaxases of the MobH family) [START_REF] Ceccarelli | Identification of the origin of transfer (oriT) and a new gene required for mobilization of the SXT/R391 family of integrating conjugative elements[END_REF][START_REF] Miyazaki | A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13[END_REF]. 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 [START_REF] Ambroset | New insights into the classification and integration specificity of Streptococcus Integrative Conjugative Elements through extensive genome exploration[END_REF] and other Firmicutes [START_REF] Roberts | Bacterial Integrative Mobile Genetic Elements[END_REF].

Integration modules

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). This confirms our previous results showing a positive PCR signal for ICESt3 relaxase gene but not for ICESt3 integrase gene for 22 strains [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF]. 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) [START_REF] Chaffanel | Resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of Streptococcus salivarius[END_REF] 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 [START_REF] Ambroset | New insights into the classification and integration specificity of Streptococcus Integrative Conjugative Elements through extensive genome exploration[END_REF][START_REF] Carraro | Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus[END_REF].

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 [START_REF] Bose | A conserved anti-repressor controls horizontal gene transfer by proteolysis[END_REF] and a putative transcriptional regulator [START_REF] Auchtung | Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis[END_REF] that are related to those of ICESt3 from S. thermophilus [START_REF] Carraro | Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus[END_REF][START_REF] Bellanger | Derepression of excision of integrative and potentially conjugative elements from Streptococcus thermophilus by DNA damage response: implication of a cI-related repressor[END_REF]. 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 [START_REF] Bose | A conserved anti-repressor controls horizontal gene transfer by proteolysis[END_REF]. 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).

Furthermore, all the regulation modules include additional genes (1 to 3 depending on the ICE) between the arp2 and orfQ genes. These genes that encode proteins of unknown function contribute to the diversity of the regulation modules.

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 restrictionmodification (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 [START_REF] Oliveira | The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts[END_REF]. 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 [START_REF] Balado | Integrating conjugative elements of the SXT/R391 family from fish-isolated Vibrios encode restriction-modification systems that confer resistance to bacteriophages[END_REF][START_REF] Price | CRISPR-Cas and Restriction-Modification act additively against conjugative antibiotic resistance plasmid transfer in Enterococcus faecalis[END_REF][START_REF] Vasu | Diverse functions of restriction-modification systems in addition to cellular defense[END_REF]. RM systems also turn out to be selfish mobile elements themselves [START_REF] Rocha | Evolutionary role of restriction/modification systems as revealed by comparative genome analysis[END_REF]. 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 [START_REF] Mruk | To be or not to be: regulation of restriction-modification systems and other toxin-antitoxin systems[END_REF]. 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 [START_REF] Takahashi | A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex[END_REF].

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 [START_REF] Jarup | Current status of cadmium as an environmental health problem[END_REF]. 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 [START_REF] Bellanger | Site-specific accretion of an Integrative Conjugative Element and a related genomic island leads to cismobilization and gene capture[END_REF][START_REF] Puymège | Conjugative transfer and cis-mobilization of a genomic island by an Integrative and Conjugative Element of Streptococcus agalactiae[END_REF]. 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 isolated from a healthy infant ( 52) by the number of copies of the slvD structural gene (2 and 3 identical copies respectively compared to 1 in strain 5M6c). Salivaricin D is a nisin-like lantibiotic with a broad-spectrum that includes a large array of Gram positive bacteria in particular the important pathogens S. pneumoniae and S. pyogenes [START_REF] Birri | Salivaricin D, a novel intrinsically trypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a healthy infant[END_REF]. Thus, it could be used as a potential weapon, for commensal species of the oral cavity such as S. salivarius, to compete with oronasopharynx-colonizing streptococci including pathogens [START_REF] Birri | Salivaricin D, a novel intrinsically trypsin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a healthy infant[END_REF][START_REF] Dobson | Bacteriocin production: a probiotic trait?[END_REF][START_REF] Wescombe | Something old and something new: an update on the amazing repertoire of bacteriocins produced by Streptococcus salivarius[END_REF]. Bacteriocins could also play a role of addiction system by killing neighboring cells that do not encode resistance to the bacteriocin and thus contribute to the maintenance of the MGE in the population [START_REF] Rankin | What traits are carried on mobile genetic elements, and why?[END_REF].

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

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

Experiments were carried out to test the functionality of 2 putative ICEs of S. salivarius. The first one, ICE_SsaF1-4_fda, has a conjugation module showing the highest percentage of identity with that of ICESt3 of S. thermophilus, an ICE whose transfer was previously demonstrated (24) (Fig. 1).

Arp1, OrfQ and Arp2 regulators encoded by this ICE are also closely related to those of ICESt3 (85, 71 and 84% of amino acid identity respectively). As mentioned before, the second one, ICE_SsaF4-20 2_fda, has a conjugation module closely related to ICE_SsaF1-4_fda and ICESt3 but harbors a distantly related regulation module.

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).

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

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 and3). 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-integration of ICE_SsaF1-4_fda in E. faecalis JH2-2 recipient cells provided relevant information on the DNA localization of one cutting site of fda integrase. This attR site is a hybrid between the sequence found in the attI of ICE_SsaF1-4_fda and the attB of E. faecalis JH2-2 recipient cells.

Since the attB sequence in the recipient strains is different from the attI of ICE_SsaF1-4_fda (Fig. 7), the presence of a T nucleotide at position 20 of the DR in the attR of the transconjugants, as in ICE_SsaF1-4_fda, indicates that integrase cuts downstream of this nucleotide to allow strand exchange during recombination. A previous work aiming at studying CIME-ICE accretion also identified the same cutting position as well as the position of a second staggered cutting site located 6 bp downstream (Fig. 7) [START_REF] Bellanger | Transfert, accrétion et mobilisation des éléments intégratifs conjugatifs et des îlots génomiques apparentés de Streptococcus thermophilus : Un mécanisme clef de l'évolution bactérienne ?[END_REF].

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

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

A previous work demonstrated that excision and transfer of ICESt3 of S. thermophilus can be increased by treating donor cells with mitomycin C (42). Hence we examined the impact of such treatment on excision and transfer of an ICE from S. salivarius (ICE_SsaF1-4_fda). Induction of ICE excision by mitomycin C was tested by PCR amplification of attI and attB fragments using different amounts of genomic DNA (0.1 µg to 1 pg) after MMC treatment or not of the cells (Fig. 8). The minimal quantity of DNA producing a positive result for attI and attB was 0.1 ng for non-treated cells whereas 10 pg of DNA were sufficient to detect a signal after MMC treatment of the cells (Fig. 8). Furthermore, when cells were treated with MMC, a higher intensity of the PCR signal was observed for attI and attB using 0.1, 1 and 10 ng of DNA compared to cells without MMC treatment (Fig. 8). Such difference was not observed for the fda control (Fig. 8). Thus, as for ICESt3 [START_REF] Bellanger | Derepression of excision of integrative and potentially conjugative elements from Streptococcus thermophilus by DNA damage response: implication of a cI-related repressor[END_REF], MMC treatment leads to an increase of ICE_SsaF1-4_fda excision.

To evaluate the effect of MMC on ICE transfer, strain F1-4 was treated with MMC (at the concentration inducing the maximum level of ICE excision) and used in filter mating experiments with S. salivarius strain F1-8 as recipient. No difference of ICE transfer was detectable between cells treated or not with MMC. This is in contrast to what was observed for ICESt3 [START_REF] Bellanger | Derepression of excision of integrative and potentially conjugative elements from Streptococcus thermophilus by DNA damage response: implication of a cI-related repressor[END_REF]. Thus, it appears that for ICE_SsaF1-4_fda excision is not a limiting step for transfer.

Retransfer of ICE_SsaF1-4_fda from transconjugants

To test whether ICE_SsaF1-4_fda transfers autonomously, retransfer was tested by using transconjugants (obtained as described above) as donor cells. Plasmid pMG36e was removed by curing transconjugants prior to the retransfer assays. F1-8 (ICE_SsaF1-4_fda) was used as donor cells in filter mating experiments with either F1-8 (pMG36e) or LMG18311 (pMG36e) as recipient cells. LMG18311 (ICE_SsaF1-4_fda) and JH2-2 (ICE_SsaF1-4_fda) were used as donor cells with LMG18311 (pMG36e) and JH2-2 (pMG36e) respectively (Table 3). For each mating assay, transconjugants were recovered and confirmed by PCR detection of both the ICE integrase gene and the plasmid pMG36e present in the recipients (data not shown). A small number of transconjugant colonies (as for intraspecies transfer of ICE_SsaF1-4_fda i.e. 2 clones for the whole experiment) was observed when using S. salivarius as donor, and strains with the same genetic background as recipients (F1-8/F1-8 mating pairs) or S. thermophilus LMG18311 as recipient cells. By contrast, more transconjugant colonies (at least 20 clones for the whole experiment) were obtained using S. thermophilus LMG18311 (ICE_SsaF1-4_fda) and E. faecalis JH2-2 (ICE_SsaF1-4_fda) as donors and LMG18311 (pMG36e) and JH2-2 (pMG36e) as recipient cells. These results confirm that ICE_SsaF1-4_fda is able to transfer autonomously but also suggest that (i) host factors could impact ICE replication or/and assembly of the conjugation machinery in S. salivarius as already suggested for some strains of S. thermophilus [START_REF] Carraro | Differential regulation of two closely related integrative and conjugative elements from Streptococcus thermophilus[END_REF], (ii) molecules displayed at the cell surface of S. salivarius could impact the donor/recipient cell contacts and thus the assembly of the conjugative machinery.

These hypotheses are consistent with the fact that no transconjugant was recovered when S. thermophilus CNRZ368 donor cells carrying ICESt3 were mated with S. salivarius JIM8777 or F1-8 recipient cells (data not shown in this study) whereas transconjugants were obtained using S. thermophilus LMG18311 as recipient cells as already reported [START_REF] Bellanger | Conjugative transfer of the integrative conjugative elements ICESt1 and ICESt3 from Streptococcus thermophilus[END_REF].

It should be kept in mind that S. salivarius encounters different physiological conditions in the oral cavity and along the digestive tract. The impact on ICE regulation of these complex interactions between bacteria and of changing environment in these ecological niches should be explored in close future. 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. Fragments corresponding to the recombination sites attB and attI of non-treated (MMC-) or treated (MMC+) cells were amplified by PCR using template DNA quantities ranging from 0.1 µg to 1 pg.

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

FIG 2 FIG 3

 23 FIG 2 Phylogenetic BioNJ tree obtained for relaxases (OrfJ) of ICEs belonging to the ICESt3

FIG 5 FIG 6 FIG 7 FIG 8

 5678 FIG 5 Characterization of transconjugants carrying ICE_SsaF1-4_fda after intraspecies transfer.

Fig. S1 : 47 Fig. S2 .

 S147S2 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.

TABLE 1

 1 Bacterial strains and plasmids used in this study (to be continued)

	Strains and plasmids	Relevant phenotype or genotype

TABLE 2 .

 2 Primers used in this work (to be continued).

	Primer use	Primer name	Sequence (5'-3')

TABLE 2 .

 2 Primers used in this work

	Primer use	Primer name	Sequence (5'-3')

ACKNOWLEDGMENTS

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

FUNDING INFORMATION

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

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

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

Test of interspecies conjugative transfer of ICEs of S. salivarius

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

ICE_SsaF1-4_fda carries 27 bp showing identity with the 3' end region of the fda gene of S. salivarius (Fig. 7). This sequence (present in attI) enables specific recombination with the chromosomal integration site of the recipient cell (attB site) and generates a 27 bp-imperfect direct repeat (DR) in intraspecies transconjugants (Fig. 7). Sequencing of the attR site obtained after ICEs are named according to their host strain and integration site. ICEs of S. salivarius are indicated in bold. For more clarity, elements in accretion with ICEs are not shown. Nucleic sequence identity higher than 80% between sequences is indicated in light grey and higher than 90% in dark grey.

LEGENDS OF FIGURES

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


Guillaume Bouchez

Tushar Malica

Delphine Wolfersberger

Marc Sciamanna

Optimized properties of chaos from a laser diode

We perform an experimental parametric study of the chaos generated by a laser diode subjected to phase-conjugate feedback. In addition to the typical figure of merit , i.e. chaos bandwidth, the corresponding spectral flatness and permutation entropy at delay is analysed. Our experimental observations reveal that the chaos can be generated with a bandwidth of ≈29 GHz, a spectral flatness up to 0.75 and a permutation entropy at delay up to 0.99. These optimized performances are maintained over a large range of parameters and have not been achieved in the conventional optical feedback configuration. Interestingly, reducing the pump current reduces the chaos bandwidth while keeping the spectral flatness and the permutation entropy at delay same as observed for increased pump current. Our experimental findings are consistent with the presented numerical simulations produced using the Lang-Kobayashi model.

I. INTRODUCTION

Keeping a laser diode stable is usually considered as a priority. However, chaotic laser diodes can be employed in various fields, such as, encrypted communications, random number generation, and remote sensing [1][2][3]. The aforementioned stability of the semiconductor lasers has been perturbed to introduce chaos in the system through various popular techniques such as optical or optoelectronic feedback, optical injection, and current modulation [4]. One particular approach is the phaseconjugate feedback (PCF) in which the light reflected into the laser is the phase-conjugate of the laser output. This configuration has shown to exhibit a variety of rich dynamics in addition to chaos, in particular, restabilization under strong feedback strength [5][6][7] and external cavity modes (ECMs). The latter is characterised by the self-pulsating dynamics regularly oscillating close to a multiple of the frequency of the external cavity [7,8]. Previously, we have also experimentally demonstrated that the PCF-generated chaos significantly enhances the chaos bandwidth when compared to the case of conventional optical feedback (COF) [9] and can exhibit highly spatiotemporally-complex outputs [10].

To quantify the chaos in the acquired time series, one widely used indicator is the chaos bandwidth. It is defined as the span of frequencies than contains 80% of the laser output power spectrum total energy [11]. Broadband chaos improves the performances of the chaotic random number generation [12] and chaos cryptography [13]. In COF, the intrinsic limit of the chaos bandwidth is given by the relaxation oscillations (RO) frequency. The chaos bandwidth can be further enhanced by dualfeedback [14] successive injections into three different laser diodes [12], mutual coupling [15] or self-interferences in a fibered Mach-Zehnder interferometer [16]. In Ref. [15], the chaos bandwidth was shown to reach up to 38.6 GHz. * guillaume.bouchez@centralesupelec.fr Further analysis of the chaotic complexity can be performed using the spectral flatness, which quantifies the distribution of the frequencies [17]. Last, the permutation entropy quantifies the entropy and measures the predictability at operating condition under consideration [18]. Previous studies on the PCF systems were limited to the evolution of chaos bandwidth [9,19] or the permutation entropy [10] versus the feedback strength.

In this article, we extend prior analysis with an extensive parametric study of the chaos properties, i.e. the chaos bandwidth, the spectral flatness and the permutation entropy, as a function of the feedback strength, of the time-delay in the external cavity, and the laser pumping current. As a result, we experimentally and numerically demonstrate that the PCF approach is a robust method to generate a comprehensive chaotic system output which is broadband, flat and spatio-temporally complex. We show that the higher feedback strength improves the bandwidth of the chaos. Increasing the pump current increases the chaos bandwidth while the spectral flatness and the permutation entropy remain consistently high, independent of the other parametric changes. However, the time delay has negligible impact on the PCF-induced chaos. We, therefore, demonstrate that by tuning the experimental parameters the user can obtain highly complex chaos and simultaneously adjust its chaos bandwidth.

We observe an optimal bandwidth of 28.7 GHz, an entropy at delay up to 0.99 and a flatness up to 0.75, and these high values are maintained over a large range of parameters. Our experiment shows, best to our knowledge, the best chaos performance in the simplest configuration of a laser diode with a single optical feedback, i.e., without any additional coupling or injection.

II. EXPERIMENTAL FINDINGS

A. Principle of the experiment

Our experimental setup is presented in Fig. 1 and is similar to the one in Refs. [7,10,19]. We use a commercial Fabry-Pérot laser diode emitting at 852 nm, model JDS-Uniphase SDL-5420. Its threshold current is 13.9 mA and we operate it between 30 and 80 mA. At 80 mA, the laser output power is 54.4 mW. 20% of the laser output is sent to a high bandwidth photodiode, through an optical fiber. A Faraday isolator prevents parasitic reflections from light coupling into the fiber. The laser output light is then focused by a lens into a rhodium-doped barium titanate (BaT iO 3 ) crystal of dimensions 5 mm × 5 mm × 5 mm that works as a phaseconjugate mirror. The phase-conjugate feedback follows the inverse path of the laser output beam and enters back into the laser internal cavity. 20% of this feedback light is sent to a power meter to know the feedback strength. It is defined as the ratio of the free running laser output to the power of the light sent back into the laser facet. We set the distance between the mirror and the laser at two different values, at 53.2 and 14.7 cm. These values correspond to the round-trip delays of 3.55 ns and 0.98 ns and external-cavity frequencies of 282 MHz and 1.02 GHz respectively. These values were verified by an observation of the autocorrelation function of the time series. Due to the chosen experimental configuration, the delay length is always inferior to the period of the relaxation oscillations frequency implying our study lies in the so-called long external cavities [20].

The laser dynamics is analyzed by a high bandwidth photodiode (Newport 1474-A, AC coupled, 3dBbandwidth of 38 GHz) connected to an oscilloscope (Teledyne LeCroy 10-Zi-A) with a bandwidth of 36 GHz and a sampling rate of 80 GSample/s. The output of the power meter is also connected to the oscilloscope. We record 1-µ s long time series.

The phase-conjugate mirror principle is shown on the right of Fig. 1. The beam enters the crystal and part of it is deviated by beam fanning, which is a photorefractive effect taking place inside the crystal. The deviated light is reflected by the edges of the crystal. Thus, the light deviated at point 1 arrives at point 2. Similarly, the light deviated at point 2 arrives at point 1. At point 1, four wave-mixing occurs between the non-deviated beam, the beam deviated at point 2, and the beam deviating at point 1. The fourth beam is the counter-propagative phase-conjugate of the input beam. Similar wave-mixing occurs at point 2. This configuration is named "cat" mirror [21].

B. Various chaotic dynamics generated by the system

Figure 2 illustrates several dynamics of a PCF system. Figures 2 (a)-(e) show a 120 ns subset of the acquired time-series measured on 120 ns (on a total length of 1 µs). A zoom on a time-slot of 10 ns is also displayed in Figs. 2 (f)-(i).

Figures 2 (a) and (f) show the case of low feedback strength. Irregular pulsations are found with intensity peaks generally separated by a time-span of about 160 ps to 200 ps (see for instance the oscillations between 42 ns and 43 ns). However, faster dynamics can also be observed on the same time traces. They are particularly visible between 43 and 44 ns. Such dynamics match earlier observations of chaos from undamped relaxation oscillations [6,19,22]. Indeed, in case of an increased feedback strength, the system undergoes a first bifurcation into leading to a limit cycle at the relaxation oscillation frequency [5,23].

Figures 2 (b) and (g) are for higher feedback strengths. One can notice the time traces displays both "slow" (≈200 ps) and faster variations, hence the chaos encompasses a wider range of time scales. As the feedback is further increased in Figs. 2 (c) and (h), the laser is at the edge of a dynamical change and follows an intermittent destabilized limit cycle, mixing chaos and regular oscillations. Between 18 ns and 41 ns, the output power is close to periodicity, with a period of approximately 150 ps (6.8 GHz). While at longer times the signal is highly chaotic. The system goes back and forth between chaos and quasi-periodicity and an analysis of the full time series confirms it. Figure 2 (h) is centered on the bifurcation and shows that the quasi-periodic signal is interrupted for a few picoseconds every nanosecond. This duration of 1 ns is a signature of the delay time. The system, therefore, enters a dynamical regime where both relaxation oscillations and external cavity time-scales interplay.

The time series in Fig. 2 (d) shows an external cavity mode (ECM), i.e. a limit cycle solution that is signature of the PCF [7,8]. The output power of the laser oscillates at a period equal to 111 ps (9.02 GHz), which is about the ninth harmonic of the frequency of the external cavity (9.18 GHz). By comparison, the period of the ROs at that current is 208 ps (4.8 GHz). Such high-frequency pulsed solution is an example of the previously introduced ECMs, which are peculiar solutions of the PCF system.

Figures 2 (e) and (j) show oscillations at various timescales. These oscillations are often observed to be faster than in Fig. 2 (b). However, this time series displays a strong signature of the time delay. Short pulses are indeed sometimes generated at a repetition rate equal to 2.55 ns (the delay used for this specific measurement), for instance between 84 ns and 102 ns. A very limited number of time series shows such chaotic signatures and all of them simultaneously have broadband chaos, as confirmed in the following text.

C. Quantifying chaos properties

The large diversity of the chaos reported in Fig. 2 calls for a more quantitative analysis of the chaos properties. Therefore, several indicators have been introduced: the chaos bandwidth, the spectral flatness and the permutation entropy.

The chaos bandwidth is defined as the span of frequencies than contain 80% of the total energy of the RF spectrum [11]. With this definition, a broadband chaos will have strong preference to higher frequencies.

To verify whether the frequencies are broadly distributed, the spectral flatness SF is defined as the ratio of the arithmetic mean by the geometric mean of the 80% highest energy part of the spectrum [17]:

SF = N n=1 P (f n ) 1/N N n=1 P (fn) N (1)
where P (f n ) is the n-th discrete component of the power spectral density at the frequency f n and where f N is the frequency of the chaos bandwidth ( N n=1 P (f n ) contains 80% of the total spectrum energy). A spectral flatness of 1 is associated with a flat spectrum, for example a theoretical white noise, while the spectral flatness tends to 0 for spiky signals [24]. Another definition, which only considers the span in dB between the maximum and the minimum has been introduced in other works [12]. But such definition does not satisfy the definition in signal processing and does not apply to the time series analysis. The upper limit of the spectral flatness from the noise generated solely by the oscilloscope fluctuates between 0.83 and 0.84, and, therefore, the maximum value we can measure. Indeed, in practice, the power spectral density of a white noise is distributed around a constant value with a non-zero standard deviation. Therefore, the maximum spectral flatness is strictly inferior to 1.

Last, we introduce the permutation entropy (PE) as a precise indicator of the complexity of a time trace [25,26]. We quantify complexity by measuring the recurrence and the probability distribution of the carefully chosen much smaller subsets of the time-series referred to as ordinal patterns (∆). These ordinal patterns are essentially similar-patterned in relative amplitudes between adjacent data points grouped together. The algorithm, as explained in [25,26] and previously used in our work presented in [10], is calculated for two given user-defined parameters: the PE-delay (θ) and the ordinal pattern length (D). Mathematically, the normalized PE (ρ θ ) for a given probability distribution 'p' associated with 'i' integral number of ordinal patterns and timescale 'θ':

ρ θ = -1 ln(D!) D! i=1 p(∆ i )ln[p(∆ i )] (2)
A maximum of D! number of ordinal patterns may be constructed given the total length of the time-series is much larger than the ordinal pattern length, i.e. N >> D. The result will be a value of 0 ≤ ρ τ ≤ 1 with zero signifying complete predictability while one indicates complete stochasticity.

D. Analysis of PCF chaos

Figure 3 shows the power spectra of the time series we studied in Fig. 2. The drop-off of the spectra at a frequency of 38 GHz is due to the maximum bandwidth of the oscilloscope. Chaotic spectra from lasers with feedback display resonances at multiples of the frequency associated with the external cavity, 1.02 GHz for Figs. 3 (a)-(d) and 292 MHz for Fig. 3 (e). These resonances will impact on the complexity of the chaos. But the spectral flatness will indicate the strength of these resonances. We use the method of Ref. [19] to measure the chaos bandwidth. We removed the part of the spectrum whose components have a power spectral density less than 3-dB superior to the noise levels. The blue part (respectively red) of the spectra contain 80% (respectively 20%) of the remaining energy.

The ECM observed in Fig. 3 (d) oscillates at a frequency of 9.02 GHz. This frequency equals 8.84 times the frequency of the external cavity (1.02 GHz), which is about the ninth harmonics of the frequency of the external cavity. This observation agrees with earlier theoretical predictions that tell us that the frequency of the ECMs is close but not always equal to the harmonics of the external cavity frequency, and even more, varies with the feedback strength [27,28].

The chaos in Fig. 3 (a) is obtained at low feedback strength and its frequency spectrum is similar to the chaos obtained with COF [15]. Most of the energy is centered around the RO frequency (4.8 GHz) and the chaos bandwidth is 13.6 GHz. This is comparable to what one FIG. 3. Spectra of the laser output powers, corresponding to the time traces of Fig. 2, at several values of feedback strength, delay and pump current. (a) to (d), are obtained under a pumping current of 80 mA and a delay length of 0.98 ns, while (e) was measured with a pump of 50 mA and a delay length of 2.55 ns. Feedback strength of (e) is 5.6%. The blue part of spectrum contains 80% of the total energy and the chaos bandwidth is indicated in white. The magenta region is the low energy part of the spectrum and is therefore not taken in account in the calculations of chaos bandwidth.

would achieve if the PCF was replaced by a COF. However, the spectral flatness, 0.74, shows that the energy is well distributed in this limited range of frequencies. The chaos of Fig. 3 (b) shows higher frequencies. The chaos bandwidth rises to 26.1 GHz and the spectral flatness equals 0.71. It should be noted that these values are significantly high when considering the high bandwidth, since high bandwidths are generally associated with limited spectral flatness in older works [17]. The spectrum shown in Fig. 3 (c) is remarkable because it features clear peaks at 6.80 GHz and 7.86 GHz alongside standard chaotic spectrum. Its spectral flatness equals 0.71.

The chaos bandwidth of Fig. 3 (e) equals to 28.7 GHz and is even higher than in Fig. 3 (b), and equals 80% of the bandwidths of the oscilloscope (36 GHz), hence is the maximum value we can measure. The associated spectral flatness remains high, 0.72. The spectrum also exhibits a bump in the frequencies between 25 GHz and 30 GHz and that bump explains the exceptional value of chaos bandwidth we report. A similar bump was observed in some spectra of Ref. [10], where we reported values of chaos bandwidth up to 30 GHz.

Figure 4 shows the permutation entropies associated with the time traces of Fig. 3 (a)-(c). Values are calculated with a pattern length of D = 7, 80000-points long time series and the PE delay (θ) varies from 1 to 190. The permutation entropy of the ECM of Fig. 3 (d), at a feedback strength of 5.2%, is very low and oscillates around 0.6 when varying the PE delay. For the remaining operating conditions, signatures are observed when PE delay θ equals a submultiple of the external cavity delay: θ = τ /4, θ = τ /3, θ = τ /2, θ = τ and θ =2τ . The time series obtained at a feedback strength of 4.0% (in blue), has a lower chaos bandwidth and stronger temporal signatures than the time series obtained at a feedback strength of 1.6% (in red), while the PE remains high. However, the time series at a feedback strength of 4.4% (in green) presents a lower permutation than the other cases. The mixed ECM-chaos dynamics, therefore, reduces the complexity. In the following analysis of the permutation entropy, we will only consider the PE for θ close to the external cavity delay τ . Since for that θ, the PE shows the strongest signature of the external cavity delay, PE at θ ≈ τ will be referred simply as PE(τ ) here onwards.

E. Influence of the feedback strength and of the delay length

We show in Fig. 5 (a) the experimental trend of the chaos bandwidth versus the feedback strength. Furthermore, Figs. 5 (b)-(c) show the evolution of the spectral flatness and of the PE(τ ) with the feedback strength and for both the external cavity delays. The pump current is fixed at 80 mA. The natural slow variations of the reflectivity of the mirror due to nonlinear optics taking place inside the crystal allow us to address various values of feedback strength [7,19]. The range of the feedback strength variations can be tuned with the geometry of the crystal [29] and we have observed that in our experiment. The chaos properties are shown for the feedback strength values being separated by 0.15%. Each point is accompanied with a bar indicating the standard de-viation when computing the property for the different operating conditions. The PE(τ ) being longer to calculate, we selected a smaller number of time series and calculated their PE(τ ). The multi-stability between the different dynamics is indicated by error bars as it shows the variations in the chaos properties in the PCF system [23,30]. However, the standard deviation of the chaos bandwidth on each range of feedback is rather limited. The variations for the spectral flatness are stronger, especially for the 3.55 ns delay at low feedback strength. However, the overall trend indicates that the variations in the spectral flatness decreases as the feedback strength increases.

Both, the chaos bandwidth and the spectral flatness increases with the feedback strength until the feedback strength equals 3%. Following that, for values of feedback strength higher than 3%, the spectral flatness decreases with the feedback strength while the chaos bandwidth saturates at high values, up to 26 GHz. PE(τ ) rises with the feedback strength and reaches a plateau at a feedback strength of 1.5%, then decreases slightly. The maximum value of PE(τ ) is 0.994. These drops in the PE(τ ) and in the spectral flatness are related to the onset of the self-pulsing ECMs starting when the feedback strength is greater than 2.5%.

F. Influence of the pump current

Figure 6 shows the evolution of chaos bandwidth, spectral flatness, and the PE(τ ) when varying the pump current from 40 mA to 80 mA. We trace in Fig. 6 (a) the averaged value of the chaos bandwidth for all the time series whose feedback strengths are in the following ranges: 0.45 to 0.55% (blue diamonds), 0.95 to 1.05% (magenta diamonds), 1.45 to 1.55% (black diamonds), 2.45 to 2.55% (green diamonds) and 3 to 3.5% (red diamonds), versus the pump current. Figure 6 (b) similarly displays the averaged value of the spectral flatness on several intervals of feedback strengths. Finally, we give in Fig. 6 (c), the evolution of the PE(τ ) versus the pump current, for feedback strength in the ranges 0.45% to 0.55% and 2.45% to 2.55%. The delay length is here fixed at 52.2 cm (round-trip time of 3.55 ns).

We observe that the chaos bandwidth generally increases with the pump, with some exceptions, such as a remarkable value of 22 GHz of average chaos bandwidth measured at a pump of 40 mA and a feedback strength ≈ 2.5%. The unusual variations of the chaos bandwidth versus the pump current for a feedback strength of 2.5% can be explained by the apparition or not of the mixed chaos and ECMs dynamics described in section II B which impacts the chaos properties.

However, the evolution of the spectral flatness is more complex. For a pump current of 40 mA, the spectral flatness decreases with an increasing feedback strength. The high spectral flatness for a feedback strength of 0.45% at 40 mA is probably due to the smaller variations of the laser output power, since the pump current is closer to the threshold. In this case the measurement of the laser output power is close to the electronic noise of the oscilloscope.

The PE(τ ) always remains high for all pump currents. Slightly higher PE(τ ) values are observed for smaller feedback strengths and do not decrease when varying the pump current. The main finding here is that the complexity will not significantly change if the chaos bandwidth increases due to an increase in the pump current, suggesting that the chaos generated by PCF chaos is both complex and broadband. In COF, the chaos bandwidth is lower than the PCF chaos bandwidth for most of low values of feedback [9,17]. As the feedback increases, both simulations [17] and experiments [31], observe a decrease of the PE(τ ).

III. NUMERICAL ANALYSIS A. Numerical model

To simulate laser dynamics with phase-conjugate feedback, we use a Lang-Kobayashi model, modified for phase-conjugate feedback [32]:

Ė(t) = (1 + iα)E(t)N (t) + γF (t) (3)
T Ṅ (t) = P -N (t) -(1 + 2N (t))|E(t)| 2 (4) τ R Ḟ (t) = E * (t -τ ) -F (t) (5)
E(t) is the complex normalized electric field of the laser, F (t) is the complex normalized feedback field and N (t) is the real normalized carrier density. t is the time, τ is the round-trip time in the external cavity, T is the electron lifetime, all times being expressed as multiples of the photon lifetime τ p . γ is the dimensionless feedback strength, α is the linewidth enhancement factor and P is a pump parameter. There is no phase-shift in the PCF equations because the phase-shift of the forward propagation is canceled by the phase-conjugate backward propagation [33].

Phase-conjugate mirrors do not generate instantaneously the phase-conjugated beam. To account this in simulations, the finite depth penetration time, τ R , has been added [32,34]. It filters the phase-conjugate feedback field and is directly proportional to the interaction length in the mirror. We investigated the influence of τ R in Ref. [19] and demonstrated that increasing τ R forces the chaos bandwidth to saturate. We now extend this analysis to additional parameters. In the experiment that we presented in section II A, we are using a 5 mm × 5 mm × 5 mm cube and the associated finite depth penetration τ R is 50 [32]. We set T=1200 and τ p =1.4 ps, as in Ref. [32]. To match our experimental observations of the relaxation oscillations frequencies, we choose:

P = 0.2 I -I th I th (6)
I being the pump current and I th the threshold current (13.9 mA here). τ R is set to 50 [7]. We choose α = 3 as typical for a laser diode. We simulate 20000-points long time series, with an integration step equal to the photon lifetime τ p . We simulate the laser system for increasing values of feedback strength, each simulation being initialized with the result of the previous simulation. To avoid the system dwelling on seldom stable dynamics, we add a gaussian noise with a standard deviation of 10 -12 to the intensity of the output field. In the following, we focus on the properties of the chaotic outputs. We first compare our experimental results of Fig. 5 with Fig. 7 where the simulated chaos bandwidth (a), spectral flatness (b) and PE(τ ) (c) are displayed versus the feedback strength γ. To compare with the dynamical regimes found experimentally in Fig. 5, we note that the appearance of the ECMs is at γ ≈ 0.06 in simulations and at ≈ 2.5% in experiment. The restabilization to the steady state numerically occurs at γ ≈ 0.1 and experimentally at a feedback strength of approximately 5%.

Simulations predict a qualitatively similar evolution of the chaos bandwidth regardless the delay, with some values of feedback creating small variations in the bandwidth, especially for γ > 0.06. The evolution described in Ref. [19] is also confirmed by the argument that the chaos bandwidth of a filtered PCF system increases and then saturates when approaching the restabilization of the laser (here γ = 0.085). PE(τ ) and spectral flatness are found to be rather insensitive to the changes of the time delays, as consistent with our experimental observations. Furthermore, the PE(τ ) and flatness increase with γ and reach high values. They decrease to much smaller values for large γ compared to our observations in experiment. From γ = 0.04 onwards, both PE(τ ) and spectral flatness fall into low values. Similar to the experiment, this drop can be related to the onset of ECMs with a stronger drop and the spectral flatness of less than 0.01 with the PE(τ ) of 0.2. We explain this discrepancy by the mixed ECM+chaos dynamics found experimentally in that range of γ and not observed in simulations. The origin of such dynamics remains unclear and may relate to the multistability of ECMs driven by noise [23,30]. Intermittent switching between dynamical solutions like observed experimentally help distributing the energy among different frequencies and maintaining a large number of different patterns. Hence this helps maintaining a good flatness and PE(τ ). Simulations in that range of γ show instead fully chaotic states whose spectrum are centered around the frequency of the ECMs, reducing therefore flatness and PE(τ ).

C. Simulated influence of the pump

We show in Fig. 8 the simulated evolution of the chaos bandwidth versus the current I for the following values of feedback strength: γ = 0.1, γ = 0.06, γ = 0.04, γ = 0.06, γ = 0.08. To capture the main features, we averaged chaos bandwidth and spectral flatness from ten simulations with random initialization. Similarly, PE is calculated for two different simulations. The observations from the experiment of Fig. 6 are confirmed by the simulation. Thus, the chaos bandwidth increases with the pump current. In particular, the increase in the chaos bandwidth with the pump current is stronger when γ is higher. This was also observed in Fig. 6 (i.e., in experiment) for feedback strength between 3% and 3.5%. The spectral flatness is independent from the change of pump current. Similarly for lower feedback strengths, increasing the pump current even slightly, decreases the spectral flatness. This confirms our experimental findings. On the other hand, the pump current has little influence on the PE(τ ) at low values of γ. However, if γ ≥ 0.06, an increase of the current strongly enhances the PE(τ ). The value of γ associated with the onset of ECMs decreases with the pump current [28]. Hence the drop of spectral flatness and PE(τ ) described in section III B will occur at a lower value of γ as the pump current decreases. This explains why the PE(τ ) and the spectral flatness strongly increase with the pump current for γ > 0.06.

IV. CONCLUSIONS

Using an experiment with a self-pumped photorefractive phase-conjugate mirror and an accurate filtered model of PCF, we have been able to observe the influence of the external cavity length and of the pump current on the bandwidth and complexity of a laser with PCF. Large chaos bandwidth, spectral flatness and PE(τ ) are found experimentally, independently of the delay. On the other hand, the chaos bandwidth can be enhanced by an increase of the pump current while PE(τ ) and spectral flatness remain at high values. Our extensive parametric study of the chaos from PCF therefore show how PCF can be used to optimize the chaos properties of a laser system. Most of the experimental observations are well supported by numerical simulations.

There have been only few such in-depth analysis of chaos properties in a laser with feedback. The achieved performances are well beyond those found for a laser with COF, hence confirming the superior performance of chaos generated by PCF. It is also worth noting that the bandwidth recorded here reaches about 3-4 times the relaxation oscillation frequency. Furthermore, for the majority of the parameter range, the values reach the limit of what can be captured by our state-of-the-art measurement setup. The chaos flatness measured is also close to nature of white noise but is obtained through a deterministic chaos mechanism. Additional improvement of bandwidth is expected with an additional optical injection, i.e., a well-known technique used for COF and therefore, an interesting path to consider next for PCF.

FIG. 1 .

 1 FIG. 1. Setup of the experiment.

FIG. 2 .

 2 FIG. 2. Time traces of the laser output power at several values of feedback strength, delay and pump current. (a) to (d), are obtained under a pumping current of 80 mA and a delay length of 0.98 ns, while (e) was measured with a pump of 50 mA and a delay length of 2.55 ns. (f)-(j) are zooms on the region marked with a red square on (a)-(e). Feedback strength of (e) is 5.6%. The arbitrary units of (a)-(d)/(f)-(i) are similar but a different scale is used on (e) and (j).

2 FIG. 4 .

 24 FIG.4. Evolution of the permutation entropy as a function of PE delay for the three time traces obtained at feedback strengths of 1.6% (green dashed line), 4.0% (red dotted line) and 4.4% (green dashed line). The multiples or sub-multiples of the external cavity delay are indicated with arrows. PE delay is expressed in multiples of the sample time (12.5 ps) and τ is here the external cavity round-trip time.

Feedback 5 TFIG. 5 .

 55 FIG. 5. Evolution of (a) the average chaos bandwidth, (b) the average spectral flatness and (c) the PE(τ ) versus the feedback strength. The pump current is set at 80 mA. Experiments have been performed with two values of delay: 3.55 ns (blue triangles) and 0.98 ns (yellow circles).

FIG. 6 .

 6 FIG.6. Measured evolution of (a) the chaos bandwidth, (b) the spectral flatness and the (c) the PE(τ ) versus the pump current, for feedback strength in 5 various intervals, [0.45, 0.55%] (blue diamonds), [0.95%, 1.05%] (magenta diamonds), [1.45%, 1.55%] (black diamonds), [2.45%, 2.55%] (green diamonds) and [3%, 3.5%] (red diamonds). The delay is equal to 3.55 ns.

BFIG. 7 .

 7 FIG. 7. Theoretical evolution of (a) the chaos bandwidth, (b) the spectral flatness and (c) the PE(τ ) versus the feedback strength. The pump current I =80 mA. Simulations have been performed with two values of delay: 3.55 ns (yellow circles) and 0.98 ns (blue inverted triangles).

1 FIG. 8 .

 18 FIG. 8. Simulated evolution of (a) the chaos bandwidth, (b) the spectral flatness and (c) the PE(τ ), versus the pump current (I), for five values of feedback strength (γ). The delay is equal to 3.55 ns.

ACKNOWLEDGMENTS

Chaire photonique is funded by: Ministère de lEnseignement supérieur, de la Recherche et de lInnovation, Région Grand-Est, Département Moselle, European Regional Development Fund (ERDF), Metz Métropole, Airbus GDI Simulation; CentraleSupélec; Fondation Cen-traleSupélec


). Second, despite the title, paleobiology is actually not the main focus of the book; it could have been more accurately titled 'The Biology, Ecology, Evolution of Radiolarians Living and Fossil'. The only inaccuracy of such a title is that some might have expected the book to include Acantharians and Phaeodarians. Acantharia are not treated and Phaeodarians, alas, are no longer considered to belong within the Radiolaria. However, the polycystines alone are a large enough subject to easily fill this substantial volume.

 [START_REF] Haeckel | Kunstformen der Natur[END_REF][START_REF] Haeckel | Kunstformen der Natur[END_REF]). Haeckel's many illustrations of radiolarians were a major component of Kunstformen dur Natur (10 of the 100 plates), a book that introduced the general public to the beauty of protists and had a major impact on the Art Nouveau movement [START_REF] Dolan | Ernst Haeckel's Radiolarians and Medusa: The influence of his visits to Villefranche on his science and his art[END_REF]. Paleobiology of the Polycystine Radiolaria is aimed, in principle, at "advanced undergraduates to doctoral students and a broad range of professionals in biological and earth sciences". Such a wide audience requires an engaging text combined with presenting a fair amount of detail and the authors have done this quite well. The book is composed of 9 chapters. It begins with a chapter devoted to the history of radiolarian studies. It takes us on a tour from the days of Ehrenberg to the present with marvelous illustrations along the way. The second chapter, "Biology", introduces us to the physical world of planktonic protists and then covers basic biology, cell structure and skeleton formation and anatomy of radiolarians. The third chapter "Ecology" reviews our fairly considerable knowledge of the distribution of radiolarians relative to biogeographic zones, oceanographic features, depth, etc. The fourth chapter is titled "Genetics". One could say it is not really about radiolarian genetics about which we actually know little (we do not even know if sexual reproduction occurs in all groups). The chapter is a valuable review of genetic relationships among taxa based on recent work on the molecular phylogeny of radiolarians, including studies up to 2019. The fifth chapter is an odd beast as it tackles both taxonomy and the fossil record of radiolarians. First, we are introduced to general principles of taxonomy, then the many singular peculiarities and challenges of radiolarian taxonomy are presented, and finally we are given summaries of the characteristics of the amazingly diverse groups of polycystines.

The second part of Chapter 5, on the fossil record of radiolarians, begins the 'Paleo' part of the book. We are given a brief summary of the very long fossil record of the radiolaria. The following chapter on 'Preservation and Methods' tells us about how radiolarians are fossilized and the specialized methods used to study micro--fossils. Chapter 7 introduces us to the field of Paleoceanography and its reliance in part on radiolarian data. We learn, for example, that the ratio of Spumellaria to Nassularia in the sediment has been used as an indicator of the depth of the water column in the past as Nassularians are more common than Spumellarians in deep water sediments. The Paleoceanography chapter also provides prerequisite knowledge needed to understand chapter 8, 'Radiolarian Biostratigraphy'. Of all the chapters, the biostratigraphy is the one that puts some demands on the reader (at least this reader) as it not difficult to get lost in the long geological record of radiolarians. The final chapter is "Evolution" and it tackles radiolarian evolution and evolution in general, beginning with presentations of mechanisms of evolutionary changes and ending with discussion of radiolarian evolution throughout geological epochs.

The only previous comprehensive treatment of radiolarians is O.R. Anderson's 1983 book 'Radiolaria'. It remains a valuable standard reference but it is nearly 40 years old and a considerable amount of work has been done in the intervening years since its appearance. There are fairly recent reviews of radiolaria, for example by [START_REF] Suzuki | Biology and ecology of Radiolaria[END_REF] and [START_REF] Boltovskoy | Radiolaria and Phaeodaria[END_REF], and they are also quite valuable but they are not comprehensive treatments.

Bottom line? Paleobiology of the Polycystine Radiolaria is well worth the purchase price and should be in the personal library of all protistologists working on marine forms. It is worth noting that the book is very well illustrated and overall, well produced. There is central section of color versions of some of the key illustrations. As a final note, the authors are to be commended for going to the trouble of finding and dealing with a professional publisher, not having succumbed to the temptation of using a print on demand outfit to produce their book. John R. Dolan Station Zoologique Laboratoire d'Océanographie de Villefranche--sur--Mer Sorbonne Université/CNRS 06230 Villefranche--sur--Mer France LITERATURE CITED Adl, S.M., Bass, D., Lane, C.E., Lukeš, J., Schoch, C.L., Smirnov, A., Agatha, S., Berney, C., Brown, M.W., Burki, F., Cárdenas, P., Čepička, I., Chistyakova, L., del Campo, J., Dunthorn, M., Edvardsen, B., Eglit, Y., Guillou, L., Hampl, V., Heiss, A.A., Hoppenrath, M., James, T.Y., Karnkowska, A., Karpov, S., Kim, E., Kolisko, M., Kudryavtsev, A., Lahr, D.J., Lara, E., Le Gall, L., Lynn, D.H., Mann, D.G., Massana, R., Mitchell, E.A., Morrow, C., Park, J.S., Pawlowski, J.W., Powell, M.J., Richter, D.J., Rueckert, S., Shadwick, L., Shimano, S., Spiegel, F.W., Torruella, G., Youssef, N., Zlatogursky, V. and Zhang, Q. 2019

Figure 1 .

 1 Figure 1. Plates 11 and 71 showing spumullarian (left) and nassularian (right) radiolarians from Haeckel's Kunstformen dur Natur (Haeckel 1899--1904). Haeckel's many illustrations of radiolarians were a major component of Kunstformen dur Natur (10 of the 100 plates), a book that introduced the general public to the beauty of protists and had a major impact on the Art Nouveau movement (Dolan 2019).

  . Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes. J. Eukaryot. Microbiol., 66: 4--119. https://doi.org/10.1111/jeu.12691 Anderson, O.R. 1983. Radiolaria. Springer--Verlag, New York