Alicia Nevers
email: alicia.nevers@inrae.fr

Markus Kranzler
email: markus.kranzler@vetmeduni.ac.at

Stéphane Perchat
email: stephane.perchat@inrae.fr

Michel Gohar
email: michel.gohar@inrae.fr

Alexei Sorokin
email: alexei.sorokine@inrae.fr

Didier Lereclus
email: didier.lereclus@inrae.fr

Monika Ehling-Schulz
email: monika.ehling-schulz@vetmeduni.ac.at

Vincent Sanchis-Borja
email: vincent.sanchis-borja@inrae.fr

Plasmid -chromosome interplay in natural and non-natural hosts: global transcription study of three Bacillus cereus group strains carrying pCER270 plasmid

Keywords: Bacillus cereus group, pCER270 megaplasmid, RNA-seq, transcriptional regulation, plasmidchromosome interaction

The Bacillus cereus group comprises genetically related Gram-positive spore-forming bacteria that colonize a wide range of ecological niches and hosts. Despite their high degree of genome conservation, extrachromosomal genetic material diverges between these species. The discriminating properties of the B. cereus group strains are mainly due to plasmid-borne toxins, reflecting the importance of horizontal gene transfers in bacterial evolution and species definition. To investigate how a newly acquired megaplasmid can impact the transcriptome of its host, we transferred the pCER270 from the emetic B. cereus strains to phylogenetically distant B. cereus group strains. RNA-sequencing experiments allowed us to determine the transcriptional influence of the plasmid on host gene expression and the impact of the host genomic background on the pCER270 gene expression. Our results show a transcriptional cross-regulation between the megaplasmid and the host genome. pCER270 impacted carbohydrate metabolism and sporulation genes expression, with a higher effect in the natural host of the plasmid, suggesting a role of the plasmid in the adaptation of the carrying strain to its environment. In addition, the host genomes also modulated the expression of pCER270 genes. Altogether, these results provide an example of the involvement of megaplasmids in the emergence of new pathogenic strains.

Introduction

Plasmids are found throughout the bacterial kingdom and undergo a highly dynamic evolution. Plasmid maintenance requires favorable encoded-traits that can compensate for the associated fitness cost. On the other hand, their ability to be horizontally transferred and to establish themselves in other hosts determines their spread [START_REF] San Millan | Fitness Costs of Plasmids: a Limit to Plasmid Transmission[END_REF].

The Bacillus cereus group is composed of Gram positive, sporulating bacteria that includes several closely related species, such as Bacillus anthracis, Bacillus cereus sensu stricto (ss), Bacillus cytotoxicus, Bacillus thuringiensis, and Bacillus weihenstephanensis [START_REF] Ehling-Schulz | The Bacillus cereus Group: Bacillus Species with Pathogenic Potential[END_REF]. With the exception of B. cytotoxicus, all defined species within this group exhibit a high conservation of the core genome. However, these bacteria display a wide range of ecological lifestyles [START_REF] Guinebretière | Ecological diversification in the Bacillus cereus Group[END_REF][START_REF] Patiño-Navarrete | Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria[END_REF]. B.

thuringiensis is an insect pathogen broadly used as biopesticide. B. weihenstephanensis is a psychrotolerant species that can grow at temperatures ranging from 4°C to 37°C, whereas human pathogens include B. cereus ss, B. anthracis and B. cytotoxicus strains (4).

Megaplasmids, defined as plasmids larger than 100 kb [START_REF] Hall | What makes a megaplasmid?[END_REF], have substantially contributed to the diversity of the B. cereus group ecological niches. Plasmid-encoded genes, particularly toxin-coding genes, are frequently used to distinguish species within the B. cereus sensu lato (sl) group, as they often play a role in pathogenicity. B. thuringiensis, for example, harbors plasmids carrying the insecticidal cry genes [START_REF] Schnepf | Bacillus thuringiensis and Its Pesticidal Crystal Proteins[END_REF][START_REF] Josém | A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis[END_REF]. Cry toxins do form parasporal crystals which dissolve and take a major part in the infectious process, once ingested by their target insects. B. anthracis is a highly pathogenic agent for mammals, including humans. This bacterium causes anthrax disease due to the production of several toxins and a capsule, which are all encoded on the pXO1 and pXO2 plasmids [START_REF] Kolstø | What Sets Bacillus anthracis Apart from Other Bacillus Species?[END_REF]. B. cereus ss is the causative agent of foodborne diseases, characterized by diarrheic or emetic syndromes [START_REF] Rouzeau-Szynalski | Why be serious about emetic Bacillus cereus: Cereulide production and industrial challenges[END_REF]. While the causes of diarrheal syndromes are only partially understood, those responsible for emetic syndrome are better characterized [START_REF] Ehling-Schulz | The Bacillus cereus Group: Bacillus Species with Pathogenic Potential[END_REF]. Emetic strains of B. cereus harbor pCER270, a 270 kb megaplasmid, J o u r n a l P r e -p r o o f sharing sequence similarities with B. anthracis pXO1, particularly at the region of the replication origin [START_REF] Rasko | Complete Sequence Analysis of Novel Plasmids from Emetic and Periodontal Bacillus cereus Isolates Reveals a Common Evolutionary History among the B. cereus -Group Plasmids, Including Bacillus anthracis pXO1[END_REF]. pCER270, like pXO1, is a non-conjugative plasmid, whose transfer requires the assistance of a self-transmissible plasmid [START_REF] Makart | Horizontal transfer of chromosomal markers mediated by the large conjugative plasmid pXO16 from Bacillus thuringiensis serovar israelensis[END_REF][START_REF] Green | Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12[END_REF]. Unique regions in pCER270 include the cereulide synthetase gene cluster (ces), encoding an enzymatic complex required for the non-ribosomal synthesis of the emetic peptide toxin cereulide [START_REF] Ehling-Schulz | Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1[END_REF][START_REF] Ehling-Schulz | Identification and Partial Characterization of the Nonribosomal Peptide Synthetase Gene Responsible for Cereulide Production in Emetic Bacillus cereus[END_REF]. Transcription of cesPTABCD from one promoter generates a 23-kb polycistronic transcript, while cesH transcription initiates from its own promoter [START_REF] Dommel | Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods[END_REF]. So far, ces expression has been assessed in LB medium, and cereulide production has been monitored in LB and MOB medium (sporulation medium) [START_REF] Dommel | Transcriptional kinetic analyses of cereulide synthetase genes with respect to growth, sporulation and emetic toxin production in Bacillus cereus[END_REF][START_REF] Lücking | Ces locus embedded proteins control the non-ribosomal synthesis of the cereulide toxin in emetic Bacillus cereus on multiple levels[END_REF]. The ces locus is expressed during late exponential phase, allowing for cereulide accumulation which peaks during stationary phase. The timing of ces expression in response to nutrients abundance has been shown to be controlled by CodY global transcriptional regulator [START_REF] Frenzel | CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits: CodY modulates virulence of Bacillus cereus[END_REF]. AbrB, a transition-state regulator involved in repression of stationary-phase genes during exponential phase, has also been involved in the negative control of ces locus expression [START_REF] Lücking | Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR[END_REF]. More recently, a study showed that a pCER270-encoded transcriptional regulator similar to PagR in B. anthracis, repressed ces expression [START_REF] Kalbhenn | Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus[END_REF].

Cereulide is resistant to proteolysis, high temperatures and acidic pH, and therefore it accumulates in cooked food and causes vomiting when ingested [START_REF] Rouzeau-Szynalski | Why be serious about emetic Bacillus cereus: Cereulide production and industrial challenges[END_REF]. Many studies noticed that the low prevalence of emetic B. cereus in the environment is largely increased in food samples, questioning the role of pCER270 in this prevalence [START_REF] Ehling-Schulz | Food-bacteria interplay: pathometabolism of emetic Bacillus cereus[END_REF][START_REF] Hoton | Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains[END_REF]. Interestingly, some B. weihenstephanensis strains have been found as being able to produce cereulide. These psychrotolerant emetic strains harbor their ces genes either encoded on large plasmids or on the chromosome, most likely as a result of an horizontal gene transfer (HGT) event [START_REF] Hoton | Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains[END_REF][START_REF] Mei | The genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon Tnces in emetic Bacillus weihenstephanensis[END_REF][START_REF] Guérin | Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures[END_REF].

The importance of megaplasmids in defining B. cereus sl species raises questions about the emergence of new pathogens, especially when HGT and plasmid sharing are frequent in the environment. Indeed, previous studies on B. thuringiensis showed that conjugation can occur J o u r n a l P r e -p r o o f in soil with frequencies that are not neglectable, and that plasmid transfer is common in dead insect larvae [START_REF] Vilas-Bôas | Bacillus thuringiensis conjugation under environmental conditions[END_REF][START_REF] Jarrett | Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis[END_REF]. Furthermore, conjugative plasmid transfer appears to be more efficient in food matrices, especially milk, than in the widely used LB medium (Lysogeny Broth) [START_REF] Van Der Auwera | Plasmid exchanges among members of the Bacillus cereus group in foodstuffs[END_REF].

In this context, it seems particularly relevant to study the consequences of megaplasmids transfer within new hosts. Indeed, the initial establishment of a plasmid in its new genomic environment is the first step that is required for its maintenance and co-evolution with the core genome. Here, we focused on the transcriptional regulations associated with the transfer of for high efficiency conjugation with pXO16 containing donors. These streptomycin resistant strains were used as the reference "pCER270-'' plasmidless strains for the transcriptomic studies, and as recipient strains for conjugation. A spectinomycin resistance gene was inserted into pCER270 using CRISPR methodology based on the vector pJOE9282 [START_REF] Altenbuchner | Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System[END_REF] with modifications (unpublished), thus obtaining pCER270-spec. This non-conjugative plasmid was transferred into recipient strains, by a pXO16-assisted mobilization (unpublished). The details of conjugation experiments will be described elsewhere. The pCER270+ transconjugants were selected as resistant to spectinomycin (300 µg/ml), sensitive to tetracycline (pXO16 marker)

and further checked by PCR to ensure the presence of an essential part of pCER270, intrinsic host plasmids and the loss of pXO16.

For the three strains tested, the pCER270-and pCER270+ strains were grown in parallel in either LB medium, or HCT medium, a sporulation medium [START_REF] Lecadet | Generalized Transduction in Bacillus thuringiensis var. berliner 1715 using Bacteriophage CP-54Ber[END_REF]. The onset of the stationary phase is determined as the end of the exponential growth and corresponds to an inflection of the growth curve. For RNA preparation, LB cultures were collected two hours (early stationary phase) after the beginning of stationary phase, whereas in HCT medium the cultures were collected four hours after the onset of the stationary phase. At this bacterial growth phase, direct-targets of Spo0A, such as spoIIE, are induced [START_REF] Cardoso | Rap-Phr Systems from Plasmids pAW63 and pHT8-1 Act Together To Regulate Sporulation in the Bacillus thuringiensis Serovar kurstaki HD73 Strain[END_REF]. This time point corresponds to the onset of the sporulation, when the asymmetric septum is not yet formed. Three biological J o u r n a l P r e -p r o o f replicates of each strain were processed for RNA-seq experiments and the following statistical analysis.

RNA extraction

RNA extraction was performed using Trizol and 1-bromo-3-chloropropane as previously described [START_REF] Gélis-Jeanvoine | Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis[END_REF]. Total RNAs were treated with DNAse using the Invitrogen™ Kit TURBO DNA-free (AM1907). RNA quality was checked on agarose gel and on Bioanalyzer.

Ribosomal RNAs were depleted from total RNA samples using the RiboZero kit (Illumina)

according to the manufacturer's instructions.

Libraries preparation and sequencing

RNA-seq libraries were generated using the NextSeq 500/550 High Output Kit v2 (75 cycles). A single read -75 sequencing run was performed on a NextSeq instrument at the I2BC High-throughput sequencing facility (CNRS, Gif-sur-Yvette).

Data processing

Reads were demultiplexed using bcl2fastq2-2.18.12 and adapters trimmed using Cutadapt 3.2. The sequencing data were then uploaded to the Galaxy web platform, and data analysis was performed on the usegalaxy.org public server [START_REF] Afgan | The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update[END_REF]. The reads were aligned to each of three species genomes concatenated with the pCER270 sequence (accession number CP001179) using Bowtie2. MulticovBed was used to generate the read counts. Genomic sequences with accession numbers CP001177 (chromosome) and CP001178-CP001181

(plasmids) were used for B.ce F4810-72. CP004069 (chromosome) and CP004069-CP004076

(plasmids), excluding CP004072 (pAW63, not found in this strain) for B.th HD73. For B.we KBAB6, an in-house unpublished genome was used. The B.we KBAB6 wild type strain were deposited at NCBI as biosample SAMN14639501. Under the same biosample, there will soon be the assembly and annotation versions of this genome. Processed data were analyzed using R and Deseq2 differential analysis of total RNA-sequencing using SARTools R Pipeline [START_REF] Varet | SARTools: A DESeq2-and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-Seq Data[END_REF].

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Significantly differentially expressed genes are characterized by a p-value that is lower than 0.05 and a log2(fold change) higher than 0.7 or lower than -0.7 for up-regulated and downregulated genes, respectively. Integrative Genomic Viewer (IGV) software allowed to visualize sequencing reads distribution from Wig files, generated with Galaxy BAM-to-Wiggle tool and normalized with bigwigCompare tool.

Raw files and processed data are available at NCBI Gene Expression Omnibus

Database, with the accession number GSE220688.

Functional groups identification in the different hosts of pCER270

In order to define functional groups in the different strains SyntTax (https://archaea.i2bc.paris-saclay.fr/synttax/) was used to define orthologs in B.ce F4810-72, B.th HD73 and the published B.we KBAB4, highly similar to B.we KBAB6 [START_REF] Oberto | SyntTax: a web server linking synteny to prokaryotic taxonomy[END_REF].

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Results

B.ce F4810-72 is more sensitive to condition-specific gene regulations induced by the pCER270

To better understand how the pCER270 megaplasmid can establish itself in a new host and to estimate the consequences of its transfer on the transcriptional landscape of the recipient strain, we analyzed the transcription of B.th HD73 and B.we KBAB6 strains, in comparison to the natural pCER270-harboring strain (B.ce F4810-72). The spectinomycin-tagged pCER270 plasmid was transferred to its recipient strains via a pXO16-assisted conjugation. For reference, transfer of the plasmid was performed in the B.ce F4810-72 emetic strain previously cured of its pCER270. Recipient strains, referred to as "pCER270-" and validated transconjugants referred to as "pCER270+" strains, were grown in parallel in LB and HCT media. RNA-seq samples were collected two hours and four hours after the onset of stationary phase, in LB and HCT medium respectively. Indeed, since a number of adaptative processes (production of virulence factors, biofilm formation, sporulation) occurs at this time, the transition phase between the exponential phase and the stationary phase was chosen for this transcriptional study. ces expression, for example, starts at the end of exponential phase [START_REF] Dommel | Transcriptional kinetic analyses of cereulide synthetase genes with respect to growth, sporulation and emetic toxin production in Bacillus cereus[END_REF].

Deseq2 RNA-sequencing analysis enabled the identification of differentially expressed genes in pCER270+ versus pCER270-strains. Volcanoplots were generated for the different hosts of the megaplasmid as well as for the two conditions tested (Fig. 1A). These graphical representations of transcriptomic data demonstrate that pCER270 genes (in red) are expressed and have a similar profile across all genetic backgrounds. Moreover, Volcanoplots shapes suggest that the presence of the megaplasmid has a much greater impact on host gene expression when the strains were grown in sporulation (HCT) medium than in standard (LB) medium.

Indeed, in LB medium, only a few host genes are located above the statistical strength and fold change thresholds (dotted gray lines), while the number of differentially expressed genes in the All these findings suggest that the host transcriptome is less impacted by the pCER270 presence during LB-growth compared to HCT-growth. Furthermore, the B.ce F4810-72, the natural host of pCER270, showed a greatly increased number of pCER270-impacted genes compared with its two artificial hosts. B.th HD73 genome transcription seems to be less sensitive to pCER270 transfer.

pCER270 increases expression of TCA cycle genes in HCT medium but only in the B.ce F4810-72 strain

The tricarboxylic acid cycle (TCA cycle) is a metabolic pathway which plays a crucial role in bacterial energetic metabolism. From the 13 genes described for this metabolic pathway in B. subtilis (http://subtiwiki.uni-goettingen.de ( 35)), we have defined the sets of TCA cycle genes in each of the different strains. TCA cycle genes are up-regulated in the B.ce F4810-72 strain harboring pCER270. The boxplots for this functional group of genes show an overall increase of their expression in the natural host strain of the plasmid (B.ce F4810-72 pCER270+ strain), grown in the HCT medium (Fig. 2A). Indeed, the Deseq2 analysis tool identified 12 genes out of 13 of this functional group that were significantly up-regulated (Fig. 2B). However, pCER270 had no effect on the expression of these genes in B.th HD73 and B.we KBAB6 strains, suggesting they are less sensitive to pCER270-induced regulation than the B.ce F4810-J o u r n a l P r e -p r o o f 72 strain. In LB medium, pCER270 did not impact TCA cycle genes expression, even in its B.ce F4810-72 natural host (Fig. S1). In summary, pCER270 specifically increases TCA cycle genes expression in the HCT sporulation-inducing medium and in the emetic context, as shown by IGV profiles corresponding to some representative genes within this functional group (Fig. 2C).

pCER270 up-regulates the acetoin catabolism genes in B.ce F4810 and B.th HD73 strains

growing in HCT medium, but down-regulates them in B.we KBAB6.

Acetoin is an extracellular metabolite that can be metabolized as a carbon source in the absence of glucose [START_REF] Peng | Transcription in the acetoin catabolic pathway is regulated by AcoR and CcpA in Bacillus thuringiensis[END_REF]. The expression of acetoin breakdown genes acoL, acoA, acoB and acoC was increased in the pCER270+ B.ce F4810-72 natural host strain. In HCT conditions, all these genes were up-regulated compared to the pCER270-strain (Fig. 3A and3B). The presence of pCER270 in the B.th HD73 strain also resulted in a higher expression of acetoin metabolism genes in HCT medium. Interestingly, pCER270 seems to induce the reverse regulation of acetoin consumption genes in the B.we KBAB6 pCER270+ strain since the global expression level of this group of genes is significantly lowered in this strain (Fig. 3C). According to Deseq2 results for the B.we KBAB6 strains, all individual genes for this group are downregulated. However, according to Deseq2 analysis, none of the aco genes seems to be differentially expressed in LB medium (Fig. S2).

The expression of genes implicated in the initiation of the sporulation is affected by pCER270

In Bacilli, Spo0A is a master regulator controlling various transition phase functions and sporulation initiation [START_REF] Sonenshein | Control of sporulation initiation in Bacillus subtilis[END_REF]. Its transcription is tightly regulated and Spo0A activity depends on a phosphorelay regulated by the quorum sensing system Rap-Phr [START_REF] Perego | Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis[END_REF]. Deseq2 analysis of B.ce F4810-72 and B.th HD73 strains indicate that spo0A expression is significantly upregulated in presence of pCER270 (padj<0.05 in both cases). However, this is not the case for J o u r n a l P r e -p r o o f the B.we KBAB6 strain, despite the slight increase visible on IGV profile. These IGV profiles illustrate the distribution of reads at the genomic loci but do not provide information on the statistical strength of these observations, as they are only average profiles of the biological replicates. For the B.we KBAB6 strain, the heterogeneity between biological replicates had an impact on the p-value (padj>0.05) for the spo0A gene, therefore the fold increase cannot be considered as significant (Fig. 4A).

Interestingly, the expression of SigF forespore specific factor expression is also statistically up-regulated in B.ce F4810-72 and B.th HD73 pCER270+ strains but not in B.we KBAB6 pCER270+ strain. The expression of the SigE mother-cell specific sporulation factor is also increased in B.th HD73 pCER270+ strain but remains stable in the two other hosts, with or without the plasmid. On the other hand, the expression of Yttp transcriptional regulator ortholog, involved in sporulation septum formation, is up-regulated in the B.ce F4810-72 and B.th HD73 strains but not in the B.we KBAB6 strain.

When generalizing to all direct Spo0A targets involved in the initiation of sporulation [START_REF] Molle | The Spo0A regulon of Bacillus subtilis: The Spo0A regulon[END_REF], we can see that pCER270 has a variable impact on the expression of these genes depending on the host. The B.ce F4810-72 natural host strain is again the most impacted by the megaplasmid (Fig. 4B), with 9 genes out of 13 being significantly up-regulated in the pCER270+ strain. This proportion is reduced to 5 out of 13 and 3 out of 13 for B.th HD73 and B.we KBAB6 strains, respectively (Fig. 4C).

The only sporulation-related regulation induced by pCER270 that is conserved across all pCER270 hosts, is the increased expression of sinR and sinI (Fig. 4D), which likely relates to the transcriptional up-regulation of Spo0A.

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The expression of pCER270 genes is impacted by the host genomic background

According to Genbank (CP001179.1), the pCER270 plasmid contains 277 defined transcriptional features, of which only 112 have annotated functions, including the virulence ces operon. The pCER270 plasmid also contains 17 transcriptional regulator genes.

To determine whether the host genomic context could influence pCER270 expression and describe the locus-specific regulation of the pCER270 genes in relation to its hosts, global expression levels of the megaplasmid were compared in all pCER270+ strains. Then, a Deseq2 normalization and analysis restricted to pCER270 transcriptional features was performed.

The results show that the overall expression level of pCER270 genes was equivalent in all media, albeit slightly lower in the B.we KBAB6 strain and LB medium. Thus, global pCER270 expression does not appear to be regulated by the host (Fig. 5A). However, some specific genes were differentially regulated depending on the genomic context. An analysis was done with the pCER270-restricted Deseq2 normalization, to compare the hosts for a same global expression of pCER270. This pCER270-centered analysis allowed us to identify the number of genes that are down-regulated or up-regulated in B.th HD73 or B.we KBAB6 strains when compared to the B.ce F4810-72 natural host strain (Fig. 5B, Table .S3). The number of genes showing a clear up-or down-regulated transcription in the B.th HD73 compared to the B.ce F4810-72 was higher in HCT than in LB but this was not the case in the B.we KBAB6 strain. 12% and 8% of pCER270 genes shared the same regulation in both B.th HD73 and B.we KBAB6 strains as compared to the B.ce F4810-72 strain in LB and HCT media, respectively.

We evaluated the impact of the genomic background on ces transcription, and found that the expression of the pCER270 cereulide synthetase locus was sustained in all the genomic backgrounds (Fig. 5C). The expression of ces locus was similar in the three strains grown in HCT medium. Surprisingly, in LB medium, compared to the two other strains, this expression J o u r n a l P r e -p r o o f was increased during the early stationary phase in the B.th HD73 strain. This suggests that the impact of the host on the expression of pCER270 virulence genes is dependent on the growth conditions. Cereulide was synthesized in the HD73 and the KBAB6 genetic backgrounds. In line with the RNA-seq data corresponding to LB medium, higher levels of cereulide were found in HD73 than in KBAB6 (unpublished).

pCER270 transcriptional regulator expression varies in remote hosts

To gain insights on the regulation of pCER270 gene expression, depending on the genomic context, we focused on the expression of putative transcriptional factors in the different contexts. Indeed, genes encoding DNA-binding protein are most likely involved in the local regulation of their adjacent genes present on the megaplasmid. Among the 17 genes encoding transcriptional factors, only four are showing an important variation in their hostspecific expression when the pCER270 hosts are growing in LB medium (Fig. 6 & Table 1).

Nevertheless, 13 transcriptional regulators out of 17 are differentially expressed in remote hosts compared to B.ce F4810-72 natural host strain in HCT medium (Table 1). The genes BCAH187_C0191 and BCAH187_C0192, coding for a helix-turn-helix (HTH) domain protein, and for a protein belonging to the LacI regulatory family, respectively, are strongly downregulated in both B.th HD73 and B.we KBAB6 strains, compared to the B.ce F4810-72 strain in LB, but not in HCT medium. Gene BCAH187_C0240, which also encodes an HTH-domain protein, is down-regulated in pCER270 remote hosts in both LB and HCT (Fig. 6, Table 1).

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Discussion

In order to determine the role of pCER270 in strain adaptation, virulence and ecological niche specificity, we artificially transferred the pCER270 into different strains of the B. cereus sl group. These included the pCER270 natural host-strain B. cereus F4810-72, the B.

thuringiensis serovar kurstaki HD73 and the B. weihenstephanensis KBAB6 strains, that are known for their emetic, entomopathogenic and psychrotrophic properties, respectively. This study provides the first analysis of the consequences, at the transcriptional level, of the transfer of the pCER270 megaplasmid into new hosts.

The pCER270 plasmid has an effect on B. cereus sl gene expression, especially in sporulating conditions (HCT medium). Indeed, pCER270 has an impact on carbohydrate metabolism and on sporulation in all its hosts (Fig. 7). As sporulation is an energy-costly process, a strict control of the energetic metabolism and choice of carbon sources is crucial for the bacterial self-maintenance and development in the environment [START_REF] Pisithkul | Metabolic Remodeling during Biofilm Development of Bacillus subtilis[END_REF]. TCA cycle and acetoin metabolism genes induction has been described previously in B. subtilis starved cells [START_REF] Koburger | Genome-wide mRNA profiling in glucose starved Bacillus subtilis cells[END_REF].

During starvation, when cells activate the sporulation genetic program, the pCER270 hoststrain B.ce F4810-72 was the most responsive to gene regulation induced by the megaplasmid.

Indeed, in the B.ce F4810-72 strain, pCER270 appears to accentuate the transcriptional response to a nutritional starvation by increasing the induction magnitude of the TCA cycle and acetoin catabolism genes. Transcriptional regulations are less impacted by pCER270 in its artificial hosts suggesting that gene regulation in response to the environmental conditions is partly host-specific.

We show here that after sporulation induction, pCER270 influences the expression of sporulation initiation genes by affecting the transcription rate of spo0A. The spo0A transcription has been described as an essential step in the control of sporulation during developmental J o u r n a l P r e -p r o o f transition [START_REF] Mirouze | Fluctuations in spo0A Transcription Control Rare Developmental Transitions in Bacillus subtilis[END_REF]. The up-regulation of spo0A mediated by pCER270 is more pronounced in its natural B.ce F4810-72 host-strain. This partly explains the higher number of differentially regulated genes identified in this strain and highlights the physiological implications of hosting the pCER270 megaplasmid at the regulatory level.

On the other hand, our transcriptome study provides evidence that the host genomic background also has a regulatory role on the expression of the pCER270 genes (Fig. 7). This regulation of pCER270 genes is locus-specific rather than global, highly dependent on growth conditions, and may have an impact on the expression of the virulence ces locus. Indeed, the expression of the ces locus genes and of putative transcriptional regulators is specifically and differentially regulated in response to growth conditions and in different genetic host backgrounds. This suggests that pCER270 plays a role in virulence and adaptive properties, which may differ significantly according to its hosts.

The condition-specific impact of the megaplasmid on chromosomal gene expression may represent compensatory regulations to alleviate the burden of plasmid carriage in pCER270+ strains [START_REF] Vial | Plasmid-chromosome cross-talks[END_REF]. Nevertheless, the pCER270-induced regulation may also help the host to cope with new environmental conditions, providing an adaptive advantage to the host carrying the plasmid. Artificial hosts of pCER270, which have very different ecological niches, may be less sensitive at the molecular level to the plasmid's regulatory potential. However, plasmid-chromosome incompatibility is expected to decrease after a longer co-evolution of the megaplasmid with the host genome. The domestication of the plasmid (43) could then subsequently increase the potential prevalence of the artificial hosts in new environments, such as food, and would be a concern in terms of new pathogen emergence. Furthermore, the new genetic background could in turn affect the expression of virulence and regulatory factors encoded on pCER270 depending on the conditions, and possibly lead to increased pathogenicity of the new strains harboring the pCER270.

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In conclusion, our study shows that the pCER270 megaplasmid can be transferred to new hosts, with the help of pXO16 conjugative plasmid, and expressed at the transcriptional level. Depending on the environmental conditions, the megaplasmid impacts its host transcriptome, and the genetic background of pCER270 can in turn specifically impact plasmid gene expression (Fig. 7). However, the pCER270 interaction with its host genomes is greater with its natural host, the F4810-72 strain, than with remote hosts, suggesting a co-evolution between the plasmid and its genetic background. J o u r n a l P r e -p r o o f

Legends to figures

Fig. 1 .Fig. 2 .

 12 Fig.1. B.ce F4810-72 is more sensitive to condition-specific gene regulations induced by

Fig. 3 .Fig. 4 .

 34 Fig.3. pCER270 increases acetoin catabolism genes expression in B.ce F4810 and B.th

Fig. 5 .

 5 Fig.5. pCER270 gene expression is impacted by the host genomic background (A) Boxplots

Fig. 6 .Fig. 7 .

 67 Fig.6. The expression of pCER270 transcriptional regulator genes varies in remote hosts.

  pCER270 to phylogenetically distant B. cereus sl hosts. The B. cereus group forms about five major phylogenetic clades, in which different B. cereus group species are intermingled (2).

	2. Materials and methods
	2.1. Strains construction and culture conditions
	Spontaneous streptomycin resistant mutants were selected from spores preparation
	plated on 300 µg/ml of streptomycin for B. cereus F4810-72 emetic strain (synonymous to B.
	cereus AH187) cured for pCER270 plasmid (B.ce F4810-72), B. thuringiensis serovar kurstaki
	HD73 (B.th HD73) and B. weihenstephanensis KBAB6 strains (B.we KBAB6). The B.we
	KBAB6 strain is closely related to the more generally used B.we KBAB4 strain, but competent
	Thus, we aimed to (i) choose host-strains that are only distantly related to the emetic B. cereus lineage and (ii) represent non-B. cereus sensu stricto species of economic importance, such as B. thuringiensis (commercially used biopesticide) and B. weihenstephanensis (known for its food spoilage potential). According to the panC gene sequence (https://toolcereusid.shinyapps.io/Bcereus, (3)), the B. weihenstephanensis KBAB6 strain belongs to the same phylogenetic group as the previously described B. weihenstephanensis emetic strains (group VI). In particular, we questioned the interaction of pCER270 with the chromosome, by identifying differentially expressed genes in presence of the plasmid, and J o u r n a l P r e -p r o o f
	analyzing pCER270 gene regulation in the different genetic backgrounds.

  pCER270+ is distinctly increased in HCT. The Venn diagram representations, corresponding to LB medium (Fig.1B), show that 17, 2 and 2 genes are significantly up-regulated in B.ce F4810-72, B.th HD73 and B.we KBAB6, respectively, while 52, 8 and 3 genes are downregulated, respectively (Table.S1). Under sporulation inducing conditions, the transcriptional impact of pCER270 was more pronounced, with 1507, 83 and 123 up-regulated genes and 1569, 64 and 495 down-regulated genes in B.ce F4810-72, B.th HD73 and B.we KBAB6 strains, respectively (Fig.1B, Table.S2). The identified genes are not markedly shared between the two conditions, with calculated overlaps ranging from 0.2% to 7.5%.

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Table 1 .

 1 Summary of the expression variations of pCER270-encoded transcriptional regulators in remote hosts, compared with the natural host of the megaplasmid B.ce F4810-72 strain.

Acknowledgements

This work was supported by the Agence Nationale de la Recherche (ANR AAPG2019 CEREMET PRCI; ANR-19-CE35-0012-01) and by the Austrian Science Fund (FWF) through the project I 4497-B to MES. We acknowledge the sequencing and bioinformatics expertise of the I2BC High-throughput sequencing facility, supported by France Génomique (funded by the French National Program "Investissement d'Avenir'' ANR-10-INBS-09).

We are grateful to Cyril Denby Wilkes for critical reading of the manuscript.

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Supplementary material

Table S1. Differential expressed genes in pCER270+ versus pCER270-for all strains grown in LB medium. Table S2. Differential expressed genes in pCER270+ versus pCER270-for all strains grown in HCT medium. Table S3. Differential expressed genes in remote versus natural hosts of pCER270, pCER270-centered Deseq2 analysis in LB and HCT media.

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