Laura C Bohorquez

Joana De Sousa

Transito Garcia-Garcia

Gaurav Dugar

Biwen Wang

Martijs J Jonker

Marie-Françoise Noirot-Gros

Michael Lalk

Leendert W Hamoen
email: l.w.hamoen@uva.nl.

Jing Han

Metabolic and chromosomal changes in a Bacillus subtilis whiA mutant

Keywords: WhiA, Bacillus subtilis, cell division, branched-chain fatty acids, chromo some compaction

The conserved protein WhiA is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motif and is a transcription regulator of the key cell division gene ftsZ in actinomycetes. In Bacillus subtilis, the absence of WhiA influences both cell division and chromosome segregation; however, the protein does not regulate any gene involved in these processes. In this study, we addressed three alternative mechanisms by which WhiA might exert its activity in B. subtilis and examined whether WhiA influences either (i) central carbon metabolism, (ii) fatty acid composition of the cell membrane, or (iii) chromosome organization. Mutations in glycolytic enzymes have been shown to influence both cell division and DNA replication. To measure the effect of WhiA on carbon metabolism, we tested different carbon sources and measured exometabolome fluxes. This revealed that the absence of WhiA does not affect glycolysis but does influence the pool of branchedchain fatty acid precursors. Due to the effect of WhiA on chromosome segregation, we examine chromosome organization in a ∆whiA mutant using chromosome conforma tion capture (Hi-C) analysis. This revealed a local reduction in short-range chromosome interactions. Together, these findings provide new avenues for future research into how this protein works in the non-actinomycete firmicutes.

IMPORTANCE WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mecha nisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

the N-terminal homing endonuclease-like domain of WhiA interacts with the protein WhiB, forming a complex that stimulates transcriptional activation [START_REF] Lilic | Structural basis of dual activation of cell division by the actinobacterial transcription factors WhiA and WhiB[END_REF]. In Bacillus subtilis, inactivation of WhiA reduces the growth rate in rich medium and affects the expression of various genes but not that of ftsZ or other cell division-related genes [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. WhiB is unique to the actinomycetes, and B. subtilis lacks a WhiB homolog. There is also no correlation between WhiA-binding sites on the chromosome and genes that show a different expression when whiA is inactivated, suggesting that WhiA does not function as a classic transcription factor in this organism [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. Nevertheless, WhiA is important for cell division in B. subtilis, and the absence of WhiA is lethal when cell division proteins are inactivated, which regulates the formation of the Z-ring, including the MinCD proteins that prevent aberrant division leading to minicell formation, or Noc, responsible for the nucleoid occlusion of FtsZ polymerization, or the FtsZ polymer crosslinker ZapA [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. Later, it was found that WhiA is also important for proper chromosome segregation in B. subtilis, and a ∆whiA mutant displays increased nucleoid spacing and is synthet ically lethal when either the DNA replication regulator ParB or the DNA replication inhibitor YabA is absent [START_REF] Bohorquez | The conserved DNA binding protein WhiA influences chromosome segregation in Bacillus subtilis[END_REF]. It is still unclear how WhiA operates in B. subtilis and other Gram-positive non-actinomycetes. Many different processes influence cell division and chromosome replication, including carbon metabolism, the lipid composition of the cell membrane, and chromosome organization. In this study, we have explored whether the activity of WhiA is related to one of these processes.

RESULTS AND DISCUSSION

Growth on different carbon sources

The earliest event in bacterial cell division is the polymerization of the tubulin-like protein FtsZ at midcell. Much of our understanding of bacterial cell division has been derived from heat-sensitive FtsZ mutants. Interestingly, inactivation of the glycolytic enzyme pyruvate kinase can rescue some of these FtsZ mutants [START_REF] Monahan | Coordinating bacterial cell division with nutrient availability: a role for glycolysis[END_REF]. Deletion of phosphoglucomutase, which catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate, also suppresses certain heat-sensitive FtsZ mutants, both in B. subtilis and Escherichia coli [START_REF] Weart | A metabolic sensor governing cell size in bacteria[END_REF]. Mutations in different glycolytic enzymes, among which pyruvate kinase, can also suppress temperature-sensitive mutants in DNA initiation and replication proteins in different bacteria [START_REF] Horemans | Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication[END_REF]. How these glycolytic mutants rescue certain cell division and chromosome replication mutants is not yet clear. Since the inactivation of WhiA affects both cell division and DNA segregation, it is tempting to speculate that WhiA influences cell division and chromosome replication by playing a role in central carbon metabolism. This hypothesis is further supported by the fact that in many bacteria whiA is located adjacent to the gene glmR and crh [START_REF] Szklarczyk | STRING V11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimen tal datasets[END_REF]. GlmR has been shown to be essential for growth and normal cell shape under gluconeogenic growth conditions [START_REF] Görke | YvcK of Bacillus subtilis is required for a normal cell shape and for growth on krebs cycle intermediates and substrates of the pentose phosphate pathway[END_REF]. This protein regulates carbon partitioning between central carbon metabolism and peptidoglycan biosynthesis [START_REF] Patel | A metabolic checkpoint protein GlmR is important for diverting carbon into peptidoglycan biosynthesis in Bacillus subtilis[END_REF]. crh codes for the carbon-flux regulator HPr that regulates the methylglyoxal synthase MgsA, which provides a bypass of glycolysis when phosphorylated glycolytic intermediates accumulate in the cell [START_REF] Dickmanns | Structural basis for the regulatory interaction of the methylglyoxal synthase MgsA with the carbon flux regulator Crh in Bacillus subtilis[END_REF]. Interestingly, elevated methylglyoxal levels affect cell division, possibly by reducing ftsZ expression [START_REF] Shin | Methylglyoxal synthase regulates cell elongation via alterations of cellular methyl glyoxal and spermidine content in Bacillus subtilis[END_REF]. In B. subtilis, the whiA gene forms an operon together with glmR and crh, located upstream and downstream of whiA, respectively, and this conserved organization might be related to a shared functional activity, which could mean that the cell division and possible DNA segregation phenotype of a whiA mutant is related to a change in carbon metabolism.

Inactivation of glmR blocks growth on citrate and results in very poor growth on either fumarate or malate as sole carbon sources [START_REF] Foulquier | The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis[END_REF]. To examine whether the absence of WhiA also affects growth on these carbon sources, we grew a ∆whiA mutant in Spizizen minimal salt medium (SMM) using either malate, fumarate, or citrate as a carbon source. To prevent any downstream effects, a marker-less ∆whiA mutant was used, containing a stop codon at the beginning of the gene [strain KS696 [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]]. As shown in Fig. 1, the ∆whiA mutant was able to grow in these different media with growth rates similar to that of the wild-type strain, indicating that WhiA and GlmR work in different pathways.

Cell division and chromosome segregation phenotype in minimal medium

As shown in Fig. 1, and reported before, the ∆whiA mutant grows slower in rich Luria-Bertani (LB) medium [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. The reason that this reduction in growth rate was not observed in the minimal medium (Fig. 1) could be related to the lower doubling time in the minimal medium compared to LB (~53 min versus ~21 min), which can mitigate chromosome segregation and cell division defects [START_REF] Arjes | Failsafe mechanisms couple division and DNA replication in bacteria[END_REF][START_REF] Galli | Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli[END_REF]. Previously, we have shown that removing WhiA in a strain lacking the FtsZ crosslinker ZapA interferes with the formation of Z-rings and blocks cell division [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. To examine whether this synthetic phenotype could be abrogated by growing cells in a minimal medium, we first tried to obtain a whiA zapA double deletion mutant by using minimal medium plates to select transformants, but this did not yield viable colonies. Subsequently, we tested the effect of WhiA depletion in a ∆zapA strain by placing whiA under the control of the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter. This strain was grown in a minimal medium without IPTG and although no clear effect on growth was observed over the measured period (Fig. 2A), as has been observed before in a depletion experiment in a rich medium [START_REF] Bohorquez | The conserved DNA binding protein WhiA influences chromosome segregation in Bacillus subtilis[END_REF], over time cells became elongated, indicative of a defect in cell division and occasionally generated aberrant nucleoids (Fig. 2A). Of note, these cell division defects are not caused by downregulation of the downstream located crh gene [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. Also, the inter-nucleoid spacing in a ∆whiA mutant grown in a minimal medium was still larger than the wild-type cells (Fig. 2B), thus slower growth in a minimal medium does not overcome the cell division and DNA segregation phenotypes of a ∆whiA mutant.

Utilization of carbon sources during growth

As outlined above, changes in glycolysis can affect both cell division and chromosome replication. To examine whether inactivation of WhiA affects glycolysis, we measured the carbon consumption by means of exometabolomics, using proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) [START_REF] Meyer | A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis[END_REF]. This required a minimal chemically defined medium, for which M9 medium is often used. However, the M9 medium has been optimized for E. coli and not for B. subtilis, and the latter easily lyses in this medium in the stationary phase [START_REF] Meyer | A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis[END_REF]. Therefore, we composed an alternative chemically defined medium based on different minimal media used for B. subtilis, as listed in Table S1.

In essence, the resulting medium, named Amber medium, uses a phosphate buffer, and ammonium salt and glutamate as nitrogen sources. The concentration of different carbon sources tested was 22 mM. To determine the exometabolome, 2 mL of culture was collected at regular time intervals and rapidly filtered for 1 H-NMR spectroscopic analysis. Identification of metabolites was based on NMR spectra alignment of pure standard compounds, and the quantification was done based on the integration, and comparison of the designated peaks to an internal standard peak (see the Materials and Methods section for details). The final results were based on three independent biological replicates, and the quality of the replicates was confirmed using a principal component analysis (PCA) (Fig. S1). First, we tested the consumption of different carbon sources in the presence of glucose to check for possible effects on catabolite repression. Cells were grown in Amber medium containing glucose alone, glucose and citrate, glucose and fumarate, and glucose and malate. Both the wildtype and the marker-less ∆whiA mutant grew fine in these media (Fig. S3). Malate was incorporated in this analysis since it is the second preferred carbon source of B. subtilis, and its utilization is not subjected to carbon catabolite repression in this organism [START_REF] Doan | The Bacillus subtilis YwkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM twocomponent system in response to malate[END_REF]. As shown in Fig. 3, citrate and fumarate utilization was initiated when most glucose was exhausted, confirming that fumarate and citrate were subjected to glucose-dependent catabolite repression in both strains. Malate was consumed faster than glucose as has been shown before [START_REF] Meyer | A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis[END_REF].

There was no apparent difference between the wild-type and ∆whiA strains, indicating that WhiA does not influence catabolite repression.

Exometabolome analysis with different carbon sources

Aside from the supplied carbon sources (glucose, citrate, fumarate, and malate), we were able to detect 18 other metabolites in the medium. To facilitate the interpretation of the exometabolome data, the time-resolved extracellular metabolite concentrations were plotted onto the relevant pathways. As shown in Fig. 4 and5, the levels of pyruvate, although differing substantially between growth conditions, were comparable for the wild-type and the ∆whiA strains, indicating that, at least under the conditions tested here, WhiA does not change the efficiency of glycolysis. After approximately 3 h of growth, when glucose levels started to go down, ∆whiA mutant cells started to show a reduced level of the branched-chain fatty acid precursors, isovalerate, isobutyrate, and 2-methylbutyrate, and an increase in acetate and 2-oxoglu tarate. We were not able to identify isoleucine, leucine, and oxaloacetate due to the detection limits of the method [START_REF] Meyer | A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis[END_REF]. Citrate and isocitrate were only measurable when the medium contained the trichloroacetic acid (TCA) intermediate citrate or fumarate (Fig. 4, lower panel, and Fig. 5, upper panel). The reason for the latter could be that the expression of citrate synthase and aconitase is induced when citrate is present in the medium or when fumarate becomes the sole carbon source after glucose levels have fallen [START_REF] Meyer | A time resolved metabolomics study: the influence of different carbon sources during growth and starvation of Bacillus subtilis[END_REF][START_REF] Sonenshein | Control of key metabolic intersections in Bacillus subtilis[END_REF]. S3).

Transcriptome analysis with glucose and malate as carbon sources

To examine whether the changes in metabolism were related to changes in gene expression, we compared the transcriptomes of wild-type and ∆whiA mutant cells grown in Amber medium supplemented with glucose and malate as carbon sources, when the differences in branched-chain fatty acid precursors became noticeable, i.e., around an OD 500 of 0.5. The medium with glucose and malate was chosen since this combination showed the clearest difference in branched-chain fatty acid precursors. The experiment was repeated to provide a biological replicate. The volcano plot in Fig. 6 depicts the distribution of expression differences against adjusted P-values. A total of 57 genes were upregulated and 40 downregulated more than threefold when an adjusted P-value < 0.05 was used as a threshold value (Table 1, data for all genes are listed in Table S6). The most highly upregulated genes, ydcF, ydcG, and pamR, form an operon. PamR is a transcription factor that affects the expression of prophages and certain metabolic genes [START_REF] De San Eustaquio-Campillo | PamR, a new MarR-like regulator affecting prophages and metabolic genes expression in Bacillus subtilis[END_REF]. Another strongly upregulated operon is the bmrB operon, coding for a multidrug ABC transporter [START_REF] Torres | The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter[END_REF]. This transporter is also involved in the activation of KinA, one of the key regulators of sporulation [START_REF] Fukushima | The putative ABC transporter YheH/YheI is involved in the signalling pathway that activates kina during sporulation initiation[END_REF]. Of note, a ∆whiA mutant displays only a very mild defect in sporulation [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. The upregulated tapA operon is required for the synthesis of the major extracellular matrix [START_REF] Branda | Fruiting body formation by Bacillus subtilis[END_REF]. Other upregulated genes were the epeX operon encoding proteins controlling the activity of the LiaRS cell envelope stress-response system (29), the fatR operon involved in lipid degradation [START_REF] Palmer | Adaptive responses to fatty acids are mediated by the regulated expression of cytochromes P450[END_REF], and the sunA and nupN operons involved in the biosynthesis of an antimicrobial peptide and the uptake of guanosine, respectively [START_REF] May | The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic sidero phore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin[END_REF][START_REF] Paik | Identification and characteriza tion of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168[END_REF][START_REF] Molle | Additional targets of the Bacillus subtilis global regulator cody identified by chromatin immunoprecipitation and genome-wide transcript analysis[END_REF]. Several genes involved in amino acid biosynthesis were downregulated, including the mtnA operon involved in methionine salvage [START_REF] Sekowska | MtnK, methylthioribose kinase, is a starvation-induced protein in Bacillus subtilis[END_REF], the tdh operon involved in threonine utilization [START_REF] Zhang | An alkylpyrazine synthesis mechanism involving L-threonine-3-dehydrogenase describes the production of 2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine by Bacillus subtilis[END_REF][START_REF] Plata | Global probabilistic annotation of metabolic networks enables enzyme discovery[END_REF], and proHJ necessary for the production of proline [START_REF] Belitsky | Multiple genes for the last step of proline biosynthesis in Bacillus subtilis[END_REF]. Other strongly downregulated genes are the wapA operon, expressing one of the main cell surface proteins in B. subtilis [START_REF] Koskiniemi | Rhs proteins from diverse bacteria mediate intercellular competition[END_REF], the fadN operon involved in fatty acid degradation [START_REF] Matsuoka | Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation[END_REF], and the frlB operon coding for an amino sugar uptake system [START_REF] Deppe | Genetic control of amadori product degradation in Bacillus subtilis via regulation of frlBONMD expression by FrlR[END_REF]. Finally, the expression of the major citrate synthase encoded by citZ was also significantly downregulated [START_REF] Jin | Identification of two distinct Bacillus subtilis citrate synthase genes[END_REF]. Several of the up-and downregulated genes in Table 1 were found in a previous transcriptome study of a ∆whiA mutant grown in a rich LB medium [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. A graphical comparison of both studies is depicted in Fig. S4.

In Table 1, the main genes involved in branched-chain amino acids metabolism and fatty acid synthesis are lacking, since their differential expression was lower than a Cells were grown in a defined minimal Amber medium with glucose and malate and harvested for RNA isolation during exponential growth (OD600 ~0.5). A marker-less ∆whiA mutant was used (strain KS696). Genes with an adjusted P-value < 0.05 and fold change (FC) > 3 are listed. The fold change was calculated by ∆whiA/wt. Genes found in a previous ∆whiA transcriptome analysis performed in LB rich medium are indicated by * (9) (see also Fig. S4). Genes located in the same operon are listed together in one row. In this case, the maximum and minimum FC values are indicated. b Genes found in a previous whiA transcriptome analysis performed in LB rich medium are indicated by * threefold. The fold change difference and P-values of these genes are listed in Table S2.

The most upregulated genes, with fold differences varying between 1.9-and 1.5-fold, were the branched-chain amino acid transporters bcaP and braB and the genes ilvE and ilvD involved in branched-chain fatty acid precursors' synthesis. Possibly, this is a response to low substrate levels. However, yvbW, encoding a putative leucine permease, was downregulated 1.7-fold, and the leuA operon involved in leucine biosynthesis was also downregulated significantly but only by approximately 1.4-fold, and there was no significant difference in expression of either valine or isoleucine biosynthesis genes (Table S2). In conclusion, the transcriptome data did not provide a clear explanation for the observed decrease in branched-chain fatty acid precursors in the ∆whiA mutant, and also no clues for why cell division and chromosome segregation are affected in such mutant.

Fatty acid analysis of a ∆whiA mutant

Like most Gram-positive bacteria, B. subtilis contains primarily branched-chain fatty acids. Synthesis of anteiso-fatty acids requires 2-methylbutyrate, and the iso-C15 and -C17 and iso-C14 and -C16 fatty acids require isovalerate and isobutyrate, respectively (Fig. S5) [START_REF] Dd | Biosynthesis and function of membrane lipids[END_REF]. These branched-chain fatty acids are important for the regulation of membrane fluidity in Gram-positive bacteria [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. The isoform contains a methyl side chain at the second terminal C atom, whereas the anteiso form contains a methyl side chain at the third terminal C atom. Both increase the fluidity of the membrane, but the anteiso more so than the isoform. The reduced cellular concentration of branched-chain fatty acid precursors in a ∆whiA mutant might lead to a change in the fatty acid composition of the cell membrane. In theory, this could affect chromosome replication and cell division, since DNA replication involves the recruitment of essential components to the cell membrane, such as the DNA replication initiator protein DnaA in E. coli [START_REF] Hou | The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in E. coli chromosomal replication[END_REF] and the DNA helicase loader DnaB in B. subtilis [START_REF] Rokop | Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis[END_REF]. Naturally, cell division is also closely associated with the cell membrane. In fact, the association of the E. coli cell division regulator MinD with the cell membrane requires unsaturated acyl chains [START_REF] Mileykovskaya | Effects of phospholipid composition on mind-membrane interactions in vitro and in vivo[END_REF], presumably because this facilitated the insertion of the C-terminal amphipathic helix of MinD, which functions as a membrane anchor. Insertion of such a relatively bulky helix in between phospholipid molecules will cost less energy when lipids are less densely packed, and the membrane is in a more fluid state [START_REF] Antonny | Mechanisms of membrane curvature sensing[END_REF]. The B. subtilis FtsZ membrane anchors, SepF and FtsA, also use an amphipathic helix to bind to the cell membrane [START_REF] Duman | Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring[END_REF][START_REF] Pichoff | Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA[END_REF]. Changes in fatty acid composition can, therefore, influence cell division. To examine whether the fatty acid composition of ∆whiA cells differs from wild-type cells, we performed a gas chromatogra phy-based analysis (Table S3). For this, cells were harvested when the cultures reached an OD 500 of approximately 0.5, when cell division and chromosome segregation defects became visible. The majority of fatty acids, 93.9% in the wild-type strain, are branchedchain fatty acids, and this fraction hardly changes in the ∆whiA mutant (93.1%). The absence of WhiA results in the reduction of iso-fatty acids from 53.8% to 45.8 %, whereas the fraction of anteiso-fatty acids increases from 40.1% to 47.3 % (Fig. 7). The reduction in isovalerate and isobutyrate derived fatty acids is in line with the metabolome data, but the increased contribution of the 2-methylbutyrate-derived fatty acids is not. Anteisofatty acids disturb the lipid packing more than iso-fatty acids and will therefore increase membrane fluidity, which is an important way B. subtilis regulates its membrane fluidity [START_REF] Diomandé | Role of fatty acids in Bacillus environmental adaptation[END_REF]. This might explain why the ∆whiA mutant contains 2.5% less short fatty acid species (C13, C14, and C15) and 3.6% more long fatty acid species (C16, C17, and C18) (Fig. 7) so that membrane fluidity homeostasis is maintained. Indeed, a membrane fluidity assay, using the membrane fluidity-sensitive dye Laurdan [START_REF] Parasassi | Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence[END_REF][START_REF] Strahl | The actin homologue MreB organizes the bacterial cell membrane[END_REF], did not detect significant differences in membrane fluidity between both strains when grown in either minimal or LB medium (Table S4). We also checked whether the addition of branched-chain fatty acid precursors (isovalerate, isobutyrate, and 2-methyl butyrate) to LB growth medium restored the growth defect of a ∆whiA mutant, but this was not the case (data not included). It seems therefore unlikely that the phenotypes of a ∆whiA mutant can be explained by changes in the fatty acid composition of the cell membrane.

Changes in chromosome conformation in a ∆whiA mutant

Polymerization of FtsZ over the area occupied by the nucleoid is inhibited by the protein Noc in B. subtilis. Noc binds to multiple sites on the chromosome but also associates with the cell membrane. It is believed that the nucleoprotein complexes formed at the membrane periphery by Noc physically interfere with efficient FtsZ polymerization [START_REF] Adams | Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane[END_REF]; in this way, nucleoid occlusion links cell division to chromosome segregation. Noc is not essential, however, when both Noc and WhiA are absent, cells become very filamentous and grow extremely poorly [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF]. Since a ∆whiA mutant shows increased spacing between nucleoids, it is conceivable that the protein is somehow involved in altering the chromosome organization. One of the main nucleoid organizing complexes is the DNA clamp-forming SMC complex [START_REF] Wang | Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus[END_REF][START_REF] Anchimiuk | A low Smc flux avoids collisions and facilitates chromosome organization in Bacillus subtilis[END_REF]. In many bacteria, the SMC complex moves along the DNA from the origin to the terminus and thereby tethers the left and the right arms of the chromosome [START_REF] Wang | Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus[END_REF]. Interestingly, a previous ChIP-on-chip study with WhiA revealed a genome binding profile that showed some overlap with the genome binding profile of SMC [START_REF] Surdova | The conserved DNA-binding protein WhiA is involved in cell division in Bacillus subtilis[END_REF][START_REF] Gruber | Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis[END_REF]. To examine whether the activity of SMC is disturbed in a ∆whiA mutant, we performed a Hi-C (chromosome conformation capture) analysis of wild-type and ∆whiA cells. As shown in the contact map of Fig. 8A, it is clear that the absence of WhiA does not affect the juxtaposition of chromosome arms by the SMC condensin complex [START_REF] Wang | Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis[END_REF]. However, a closer inspection of the difference in chromosome contacts indicated that short-range interactions (< 50 kb) are reduced in the absence of WhiA (Fig. 8B), possibly suggesting that WhiA has a DNA organization role and is involved in altering local chromosomal interactions.

Conclusion

Despite the conserved nature of WhiA and its documented role as a transcriptional activator in actinomycetes, it is unclear how this protein functions in B. subtilis and other Gram-positive non-actinomycetes. In this study, we were able to eliminate three possible mechanisms, including an effect on central carbon metabolism, changes in the fatty acid composition of the cell membrane, and organization of the chromosome by the SMC condensin complex. Interestingly, we found that the absence of WhiA affects the compaction of the chromosome. The resolution of Hi-C is currently insufficient to correlate the differences in DNA packing with either WhiA binding sites on the chromo some or transcriptome data. It will be interesting to see whether this new phenotype of S3.

a ∆whiA mutant can be linked to transcription effects, and more importantly, cell division and/or chromosome segregation.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Luria-Bertani medium was used for routine selection and maintenance of B. subtilis and E. coli strains. S1). When indicated, the medium was supplemented with 22 mM final concentration of malate, fumarate, or citrate. All strains were grown at 37°C at 250 rpm. Growth in AMBER and LB medium was followed by measuring the optical density using 500 and 600 nm, respectively. B. subtilis strains used in this study are listed in Table S5. When indicated, LB medium was supplemented with a mixture of three branched-chain fatty acid precursors (100 µM of 2-methyl-butyrate, isobutyrate, and isovalerate, Sigma-Aldrich) or straight fatty acid precursors (100 µM of methyl-butyrate, methyl-propionate, and methyl-valer ate, Sigma-Aldrich).

WhiA depletion strain (LB45) [START_REF] Bohorquez | The conserved DNA binding protein WhiA influences chromosome segregation in Bacillus subtilis[END_REF] was always grown in the presence of erythromycin, due to the Campbell-type integration of the Pspac-whiA construct into the whiA locus. Depletion of WhiA was accomplished by inoculating a single colony into LB medium with 0.1 mM IPTG and growth at 37°C to an OD 600 of ~1. Subsequently, cells were harvested, washed in pre-warmed LB medium, resuspended to an OD 600 of 0.01, and grown in the absence of IPTG.

Microscopy

Exponentially growing cells were stained with the fluorescent membrane dye FM-95 (0.5 µg/mL final concentration), and the DNA was stained with DAPI (1 µg/mL final concentration), and after 5-min incubation mounted on microscope slides covered with a thin film of 1% agarose. Microscopy was performed on an inverted fluorescence Nikon Eclipse Ti microscope equipped with a CFI Plan Apochromat DM 100× oil objective, an Intensilight HG 130bW lamp, and a C11440-22CU Hamamatsu ORCA camera. The digital images were acquired and analyzed with ImageJ v.1.48d5 (National Institutes of Health). Cell lengths were measured manually using ImageJ software.

Metabolome analysis

The main culture (20 mL) was inoculated with an exponentially growing overnight culture to an initial OD 500 of 0.05. The optical density was monitored and 2 mL cell suspension was sampled. Three experiments were carried out to provide the necessary biological replicates. During cultivation, the pH value was determined at each sampling time point by using HI 2211 pH/mV/uC bench meter (Hanna Instruments Deutschland GmbH, Kehl, Germany). A total of 2 mL of cell culture medium was taken at 60, 120, 180, 240, 300, 360, 420, and 480 min by sterile filtration, using a 0.45 mm pore size filter (Sarstedt AG, Nuernberg, Germany), to get sterile extracellular metabolite samples of the bacterial culture and directly frozen until measurement. 1 H-NMR analysis was carried out as described previously [START_REF] Dörries | Metabolic footprint analysis uncovers strain specific overflow metabolism and D-Isoleucine production of Staphylococcus aureus COL and HG001[END_REF]. In brief, 400 µL of the sample was mixed with 200 µL of sodium hydrogen phosphate buffer (0.2 M, pH 7.0) to avoid chemical shifts due to pH, which was made up with 50% D 2 O. The buffer also contained 1 mM trimethylsilyl propanoic acid-d 4 (TSP), which was used for the quantification and also as a reference signal at 0.0 ppm. To obtain NMR spectra, a 1D-NOESY pulse sequence was used with a presaturation on the residual partly deuterated water (HDO) signal. A total of 64 FID scans were performed at 600.27 MHz and at a temperature of 310 K using a Bruker AVANCE-II 600 NMR spectrometer operated by TOPSPIN 3.1 software (both from Bruker Biospin). For qualitative and quantitative data analysis, we used AMIX (Bruker Biospin, version 3.9.14). We used the AMIX Underground Removal Tool on obtained NMR spectra to correct the baseline, thereby using the following parameters: left border region 20 ppm, right border region -20 ppm, and a filter width of 10 Hz. Absolute quantification was performed as previously described [START_REF] Dörries | Metabolic footprint analysis uncovers strain specific overflow metabolism and D-Isoleucine production of Staphylococcus aureus COL and HG001[END_REF]. In brief, a signal of the metabolite, either a complete signal or a proportion, was chosen manually and integrated. The area was further normalized on the area of the internal standard TSP and on the corresponding number of protons and the sample volume. For statistical comparison of extracellular metabolite data and growth, bar charts, and XY plots, we used Prism (version 6.01; GraphPad Software). The time-resolved extracellular metabolite concentrations were log 2 (x + 1) transformed for the separation via PCA. The PCA was done using PAST v3.16 with auto-scaled data [START_REF] Van Den Berg | Centering, scaling, and transformations: improving the biological information content of metabolomics data[END_REF].

Transcriptome analysis

Cells (2 mL cultures) were spun down (30 s Eppendorf centrifuge, 14,000 rpm, 4°C), resuspended in 0.4 mL ice-cold growth medium, and added to a screw cap Eppen dorf tube containing 1.5 g glass beads (0.1 mm), 500 µL phenol:chloroform:isoamyl alcohol (25:24:1), 50 µL 10% SDS, and 50 µL RNase-free water [START_REF] Hamoen | Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach[END_REF]. All solutions were prepared with diethylpyrocarbonate-treated water. After vortexing, tubes were frozen in liquid nitrogen and stored at -80°C. Cells were broken using a bead-beater for 4 min at room temperature. After centrifugation, the water phase was transferred to a clean tube containing 400 µL chloroform; after vortexing and centrifugation, the water phase was used for RNA isolation with High Pure RNA Isolation Kit (Roche Diagnostics GmbH, Mannheim, Germany), yielding >3 µg total RNA per sample. TapeStation System (Agilent) was used for checking the integrity of the RNA, and integrity number (RNA) values of 8.3-9.2 were obtained. For next-generation sequencing, a ribosomal RNA depletion was performed on the total RNA using the Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria, Illumina). Bar-coded RNA libraries were generated according to the manufacturer's protocols using the Ion Total RNA-Seq Kit v2 and the Ion Xpress RNA-Seq barcoding kit (Thermo Fisher Scientific). The size distribution and yield of the barcoded libraries were assessed using the 2200 TapeStation System with Agilent D1000 ScreenTapes (Agilent Technologies). Sequencing templates were prepared on the Ion Chef System using the Ion PI Hi-Q Chef Kit (Thermo Fisher Scientific). Sequencing was performed on an Ion Proton System using an Ion PI v3 chip (Thermo Fisher Scientific) according to the instructions of the manufacturer. After quality control and trimming, the sequence reads were mapped onto the genome (genome-build-accession NCBI Assembly: GCA_000009045.1) using the Torrent Mapping Alignment Program. The Ion Proton system generates sequence reads of variable lengths, and this program combines a short read algorithm [START_REF] Li | Fast and accurate short read alignment with burrows-wheeler transform[END_REF] and long read algorithms in a multistage mapping approach. The gene expression levels were quantified using HTseq [START_REF] Anders | HTSeq--a python framework to work with high-throughput sequencing data[END_REF]. The data were normalized and analyzed for differential expression using R statistical software and the DESeq2 package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2[END_REF]. The RNA-seq data have been submitted to and are accessible in the Gene Expression Omnibus using accession number GSE121479.

Lipid analysis

The fatty acid composition was determined from cells growing in Amber medium when the cultures reached an OD 600 of approximately 0.5. Cells were harvested by centrifuga tion at 10,000 × rcf for 5 min at 4°C, washed once with 0.9% ice-cold NaCl, and submitted to flash freeze in liquid N 2 . Fatty acids were analyzed as fatty acid methyl esters using gas chromatography. All analyses were carried out in triplicates at the Laboratory Genetic Metabolic Disease, Amsterdam UMC.

Laurdan GP spectroscopy

Membrane fluidity was measured as described before using Laurdan generalized polarization (GP) [START_REF] Strahl | The actin homologue MreB organizes the bacterial cell membrane[END_REF]. Cells were grown in either LB or SMM to an OD of approximately 0.5, followed by 5-min incubation with 10 mM Laurdan. Subsequently, cells were washed three times with pre-warmed buffer containing 50 mM Na 2 HPO 4 /NaH 2 PO 4 pH 7.4, 0.1% glucose, and 150 mM NaCl with and without the membrane fluidizer benzyl alcohol (30 mM). The Laurdan fluorescence intensities were measured at 435 ± 5 nm and 490 ± 5 nm upon excitation at 350 ± 10 nm, using a Tecan Infinite 200M fluorometer. The Laurdan GP was calculated using the formula GP = (I 435 -I 490 )/(I 435 + I 490 ).

Chromosome capture by Hi-C

Cultures were grown in LB media with shaking, and samples for Hi-C were collected at the exponential growth phase (OD 600 : 0.6). Hi-C was carried out exactly as described before with digestion, using HindIII [START_REF] Dugar | A chromosomal loop anchor mediates bacterial genome organization[END_REF]. Hi-C matrices were constructed using the Galaxy HiCExplorer webserver [START_REF] Wolff | Galaxy HiCExplorer: a web server for reproducible Hi-C data analysis, quality control and visualization[END_REF]. Briefly, paired-end reads were mapped separately to the B. subtilis genome (NCBI Reference Sequence: NC_000964.3) using very sensitive local setting mode in Bowtie2 (Galaxy v.2.3.4.2). The mapped files were used to build the contact matrix using the tool hicBuildMatrix (Galaxy v.2.1.2.0) using a bin size of 10 kb, and HindIII restriction site (AAGCTT) and AGCT as the dangling sequence. The contact matrix was then used for further analysis and visualization using the interactive browserbased visualization tool "Bekvaem" essentially as described before [START_REF] Dugar | A chromosomal loop anchor mediates bacterial genome organization[END_REF].

FIG 1

 1 FIG 1 Growth of a ∆whiA strain on different carbon sources. Growth of the wild-type strain (strain 168) and the whiA marker-less mutant (strain KS696) in rich Luria-Bertani (LB) medium and in (SMM supplemented with 22 mM of either citrate, fumarate, or malate. Data are shown as mean values and standard deviation of triplicate samples.

FIG 2

 2 FIG 2 Cell division and chromosome segregation defects of ∆whiA cells in minimal medium. (A) Growth curves of strain LB45 containing a ΔzapA mutation and an IPTG-inducible whiA allele. The strain was grown in minimal SMM in the presence of 0.1 mM IPTG to an OD 600 of 0.8. After that, the culture was diluted into fresh medium in the presence or absence of 0.1 mM IPTG (arrow at t 0 ). Middle panel shows fluorescence and phase contrast microscopy composite pictures of the cells after 270 min (t 1 ). Nucleoids were stained with 4′,6-diamidino-2-phenylindole (cyan), and the phase contrast images were pseudocol ored red to enhance the contrast. Scale bar is 2 µm. Arrows indicate cells with aberrant nucleoids and a-nucleate cells. Lower panels show the cell length distribution (n = 307 and 385 for +IPTG and no IPTG, respectively). (B) Fluorescence and phase contrast microscopy composite picture of exponentially growing wild-type cells and ∆whiA cells (strain KS696) cultivated in minimal SMM at 37°C and stained with DAPI (cyan) to mark nucleoids. The phase contrast images are pseudocolored red to enhance the contrast. Scale bar is 2 µm. Right panels show the inter-nucleoid distances (n = 607 and 835 for wild-type and ∆whiA cells, respectively).

FIG 3

 3 FIG 3 Carbon utilization over time. Carbon source utilization (concentration in mM) of wild-type (strain 168) and whiA marker-less mutant cells (strain KS696) during growth in defined minimal Amber medium supplemented with either glucose, glucose and citrate, glucose and fumarate, or glucose and malate. Data are shown as mean values and standard deviation of triplicate samples. The dashed lines mark the time point when the glucose culture enters the stationary phase (360 min) (see Fig.S3).

FIG 4

 4 FIG 4 Exometabolome of cells grown with either glucose or glucose and citrate. Time-resolved extracellular metabolite concentrations (in mM) of wild-type and ∆whiA marker-less mutant cells (strain KS696) grown in chemically defined minimal Amber medium with either glucose alone (upper panel) or glucose and citrate (lower panel) as carbon sources. Dashed lines indicate entry into the stationary phase (360 min). The compounds are arranged according to the main metabolic pathways: glycolysis, TCA cycle, overflow metabolites, branched-chain amino acids, and branched-chain fatty acids precursors. Data are shown as mean values and standard deviation of triplicate samples.

FIG 5

 5 FIG 5 Exometabolome in cells grown with either glucose and fumarate or glucose and malate. Time-resolved extracellular metabolite concentrations (in mM) of wild-type and ∆whiA marker-less mutant cells (strain KS696) grown in chemically defined minimal Amber medium with either glucose and fumarate (upper panel) or glucose and malate (lower panel) as carbon sources. Dashed lines indicate entry into the stationary phase (360 min). The compounds are arranged according to the main metabolic pathways: glycolysis, TCA cycle, overflow metabolites, branched-chain amino acids, and branched-chain fatty acid precursors. Data are shown as mean values and standard deviation of triplicate samples.

FIG 6

 6 FIG 6 Volcano plot of transcriptome data. Volcano plot depicting the transcriptome data as a relation between adjusted P-values and log 2 fold expression change. Wild-type and whiA marker-less mutant cells (strain KS696) were grown in a defined minimal Amber medium with glucose and malate and sampled during exponential growth. Main downregulated and upregulated genes in the whiA mutant are shown in cyan and magenta, respectively.

  I*-yxzC*-G*-J*-I*-yxiG*-H*-I*-J*-K*-M* -6 Cell wall-associated WapA protein toxin & unknown yonN-J-B-yomW-

FIG 7

 7 FIG 7 Fatty acid analysis of a ∆whiA mutant. (A) Comparison of the total iso-and anteiso-fatty acids and fatty acid chain length between wild-type B. subtilis and ∆whiA cells. (B) Details of the different fatty acids. Concentrations of individual fatty acids are listed in TableS3.

FIG 8

 8 FIG 8 Chromosome conformation capture (Hi-C) analysis. (A) Normalized Hi-C contact maps of wild-type (top) and ΔwhiA strains (below) at the exponential phase. SMC-dependent juxtaposition of the chromosome arms is observed in both strains as the secondary (vertical) diagonal. (B) Difference plot of wild-type and ΔwhiA strains. The magnified view of the difference in short-range contacts (between 10 and 100 kb) is shown below.

TABLE 1

 1 Transcriptome comparison of wild-type and ∆whiA cells a

  Spizizen minimal medium (58) consisted of 2 g/L (NH 4 ) 2 SO 4 , 14 g/L K 2 HPO 4 , 6 g/L KH 2 PO 4 , 1 g/L sodium citrate, 2 g/L MgSO 4 , 5 g/L glucose, 2 g/L tryptophan, 0.2 g/L casamino acids, and 2.2 g/L ammonium ferric citrate. The defined minimal Amber medium consisted of 70 mM K 2 HPO 4 , 30 mM KH 2 PO 4 (adjusted to pH 7.4), 15 mM sodium chloride, 10 mM (NH 4 ) 2 SO 4 , 0.002 mM of trace elements (ZnCl 2 , MnSO 4 , CuCl 2 , CoCl 2 , and Na 2 MoO 4 ), 22 mM glucose, 0.25 mM tryptophan, 10 mM glutamate, 1 mM MgSO 4 , 0.1 mM calcium chloride, and 0.01 mM ammonium ferric citrate (Table

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ACKNOWLEDGMENTS

We would like to thank Henrik Strahl (Newcastle University) for scientific assistance with the lipid analysis and for insightful discussions, Johan Westerhuis (University of Amsterdam) for his insights about the analysis of the exometabolome, Selina van Leeuwen (MAD, UvA) for providing excellent sequencing services, and members of the group, and the Advanced Multidisciplinary Training in Molecular Bacteriology (AMBER)

EU Marie Curie Initial Training Network (ITN), for insightful discussions, and Gertjan Kramer and Stanley Brul for critical reading of the manuscript.

The research was funded by EU Marie Curie ITN grant AMBER (317338), Marie Curie CIG grant DIVANTI (618452), European Commission MCSA-IF 749510, and STW Vici grant 12128.

AUTHOR AFFILIATIONS

1 Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supplemental material (Spectrum01795-23-s0001.pdf). Tables S1 to S5 and Fig. S1 to S5. Table S6 (Spectrum01795-23-s0002.xlsx). Transcriptome differences between whiA mutant and wild-type cells.

Open Peer Review PEER REVIEW HISTORY (review-history.pdf). An accounting of the reviewer comments and feedback.


Iuliia Zhurakovskaia
email: iuliia.zhurakovskaia@gmail.com

Jeanne Vezien
email: jeanne.vezien@limsi.fr

Patrick Bourdot
email: patrick.bourdot@limsi.fr

Teaching with a companion: the case of gravity

Keywords: virtual reality, companion, science education

Virtual Reality (VR) has repeatedly proven its effectiveness in student learning. However, despite its benefits, the student equipped with a personal headset remains isolated from the real world while immersed in a virtual space and the classic student-teacher model of learning is difficult to transpose in such a situation. This study aims to bring the teacher back into the learning process when students use a VR headset. We describe the benefits of using a companion for educational purposes, taking as a test case the concept of gravity. We present an experimental setup designed to compare three different teaching contexts: with a physically present real teacher, using a live video of the teacher, and with a VR avatar of the teacher. We designed and evaluated three scenarios to teach the concept of gravity: an introduction to the concept of free fall, a parabolic trajectory workshop and a final exercise combining both approaches. Due to sanitary conditions, only pre-tests are reported. The results showed that the effectiveness of using the VR simulations for learning and the self-confidence level of the students increased as well. The interviews show that the students ranked the teaching modes in this order: VR companion mode, video communication and real teacher.

I. INTRODUCTION: COMPANIONS IN THE DIGITAL ERA

Humans are social animals and usually prefer to avoid being alone whenever possible. During our studies we are usually surrounded by other people, students, and professors.

On the other hand, VR has been democratised recently on the advent of affordable, personal devices -head-mounted displays (HMDs). These systems are very immersive, but they considerably reduce classical social interactions, which poses a problem when using them for an educational purpose. For example, students often need to ask questions during their educational process. Questions arise due to a lack of attention, difficulties in understanding certain concepts, or even misconceptions on the subject. While wearing an HMD, it can be complicated to switch from VR to the real world to get assistance, then to go back to work in VR. The availability of virtual help -a companion, can solve the problem [START_REF] Chou | Redefining the learning companion: The past, present, and future of educational agents[END_REF]. This is the rationale for the present work. Thus, our research question is "Given the isolation problem of VR headsets, how can we best bring back the teacher in the learning process?".

Companions are an evolution of Embodied Conversational Agents (ECA). They are endowed with emotional abilities that make them capable of establishing social and affective relationships with people, caring for them and providing them with companionship. The latter is recognised as a key factor in children's acquisition and development of social skills [START_REF] Roa-Seïler | Designing Interaction Strategies for Companions Interacting with Children[END_REF].

The use of pedagogical agents in 3D simulations can be traced to Rickel and Johnson [START_REF] Rietzler | Breaking the Tracking: Enabling Weight Perception Using Perceivable Tracking Offsets[END_REF] creating the STEVE (Soar Training Expert for Virtual Environments) agent. STEVE was used as a teaching assistant during procedural learning, providing verbal or direct cues to students upon requests.

This type of pedagogical agent, represented by an animated character, is now described as ABLEs (Agent-Based Learning Environments) [START_REF] Harley | Toward a feature-driven understanding of students' emotions during interactions with agent-based learning environments: A selective review[END_REF]. Research has found that the visual presence of such personal assistance does not distract students from learning [START_REF] Moreno | The case for social agency in computer-based teaching: Do students learn more deeply when they interact with animated pedagogical agents?[END_REF]. The presence of a digital companion has been found to have a positive effect on learners' perception of their learning experience as well as their performance outcomes with these environments [START_REF] Lester | The persona effect: Affective impact of animated pedagogical agents[END_REF].

These works show that companions can be quite effective, but their appearance and behaviour can affect this effectiveness in the teaching process. Other works do not discuss how the companion is rendered, and instead focus on the AI (Artificial Intelligence) aspect. Since these companions are designed to replace the physical teacher in a VR-based serious game, it made sense to investigate how to include them in existing gaming education framework. Our research is complementary to these "automated" approaches.

Companions have been used in a few studies related to education. Firstly, it has been suggested by Lea Pillette et al. [START_REF] Pillette | A physical learning companion for mental-imagery BCI user training[END_REF] that users may experience difficulties or be nervous during training sessions using Mental-Imagery Brain-Computer Interfaces (MI-BCI) and that this may be partly the result of a lack of social presence and emotional support, which had been paid little attention to. One way of providing this social and emotional context is using a learning companion. PEANUT (Personalised Emotional Agent for Neurotechnology User Training) provided social presence and emotional support depending on the performance and progress of the user through interventions combining both spoken sentences and facial expressions. Pillette et al. [START_REF] Pillette | A physical learning companion for mental-imagery BCI user training[END_REF] found desirable characteristics of their learning companion in terms of verbal content and appearance (e.g., eyebrows can increase the expressiveness of cartoon faces). They found that «nonautonomous people (those who are more inclined to work in a group), who are usually at a disadvantage when using MI-BCI, benefited compared to autonomous people with PEANUT with a 3.9% increase in peak performance. In addition, in terms of user experience, PEANUT seems to have improved by 7.4% people's attitude towards their ability to learn and remember how to use MI-BCI, which is one aspect of the user experience we evaluated».

N. Roa-Seïler conducted a study regarding the presentation of companions [START_REF] Roa-Seïler | Designing Interaction Strategies for Companions Interacting with Children[END_REF]. In this study, they compared which companion the 4th-grade children preferred in maths class: Samuela is a 3D screen-based character, Nao is an actual robot, and the third embodiment was Ari, an actual person made to look like a cartoon character and projected on the screen. The study used a Wizard of Oz setup (WoZ), where the teacher controlled the companions using a panel with predefined sets of action. The children marked Ari as the most affectionate because of her behaviour ("She smiled", "She talked nice"). Nao, not having a mouth, couldn't smile, which generated some confusion; nevertheless, children found him fascinating probably because none of them had met a robot before, whereas they had already encountered screen-based characters in video games.

As a support of our work on companions, we decided to focus on the topic of gravity, as it is one of the subjects in physics that students often have misconceptions about.

II. TEACHING GRAVITY WITH COMPANIONS

A. Why free-fall resist teaching

What do an apple and a planet have in common? They are both subject to the same force that describes their movement: gravity. Gravity is the fundamental force that we experience first, as we see it in action everywhere.

In the French education system, the official teaching of free-fall is based on a progression that consists of establishing the Cartesian equation of a projectile trajectory, deducing the motion's nature, and then demonstrating why a projectile moving in a homogeneous field of gravity is a conservative system. But this approach to parabolic motion is insufficient for a complete and consistent understanding of free-fall, especially regarding students' representations of projectile trajectories in a gravitational field, launched under different initial conditions (initial velocity vector) [START_REF] Anago | Explication du mouvement parabolique par les élèves de terminale scientifique[END_REF]. This suspicion is confirmed by A. Prescott [START_REF] Prescott | Teaching projectile motion to eliminate misconceptions[END_REF], who showed that the parabola is not the preferred curve for students to represent free-fall motion (even after learning).

Halloun and Hestenes [START_REF] Halloun | The initial knowledge state of college physics students[END_REF] note that "students hold the prescientific belief that every movement has a cause". Also, A. Prescott [START_REF] Prescott | Teaching projectile motion to eliminate misconceptions[END_REF] showed that students have "misconceptions when faced with a projectile movement situation". Most students do not consider the initial velocity and draw a straight line when the trajectory of motion crosses the equilibrium position (the result is not a parabola but two upper lines of a triangle) [START_REF] Hecht | Understanding projectile acceleration[END_REF]. Thus, it is clearly necessary to dispel students' misconceptions on free-fall while teaching them parabolic motion.

Where do these errors come from? It is generally accepted that we think the way we have been taught to think. Nevertheless, regarding physics phenomena, we all start with "intuitive physics": a kind of general explanatory scheme representing a common and self-consistent set of concepts and resisting attempts to change or modify it, no matter how wrong it may be. This is not taught but comes unconsciously from our already existing knowledge and experience. These intuitive patterns are a strong obstacle to actual scientific teaching. It is precisely this "intuitive physics" that prevents us from gaining new knowledge and makes teaching less effective [START_REF] Viennot | Spontaneous reasoning in elementary dynamics[END_REF]. Spontaneous reasoning is very stable and not susceptible to learning that contradicts it. Just like delusions, they must first be destroyed before being replaced [START_REF] Strike | A conceptual change view of learning and understanding[END_REF]. It is not just "a few mistakes" by students, it is a way of thinking found even in everyday conversations [START_REF] Viennot | Spontaneous reasoning in elementary dynamics[END_REF]. Thus, one must first show students the limitations of their current reasoning and only then, teach new material. Learning becomes a process in which new concepts have to displace or rearrange stable concepts that the students have been building up for a long time.

When studying free-fall, the most common misunderstanding is the relationship between force and acceleration (force and motion) [START_REF] Clement | Students' preconceptions in introductory mechanics[END_REF] and between position, velocity and acceleration in one dimension [START_REF] Mcdermott | Students' conception and problem solving in Mechanics[END_REF]. These Newtonian concepts are often distorted or misinterpreted by students to fit their ingrained misconceptions. Students tend to memorise them separately, as formulas, with little or no connection to fundamental qualitative concepts. Put simply, students cannot apply their theoretical knowledge to practical tasks. This is aggravated by the fact that the modern education system often puts an emphasis on quantitative calculations to measure learning outcomes, instead of assessing true qualitative understanding.

B. Teaching gravity

L. McDermott [START_REF] Mcdermott | Students' conception and problem solving in Mechanics[END_REF] found that results obtained with students at the university level, including prospective and practising teachers, are equally applicable to high school students and, in some cases, to primary school children. Thus, we can conclude that students are pulling their delusions regarding physics from their earliest years. In a survey of 6000 high school, college and university students who took the Force Concept Inventory test (designed to assess students' understanding of Newtonian dynamics [START_REF] Halloun | Common sense concepts about motion[END_REF] [START_REF] Hestenes | Force concept inventory[END_REF]) before and after learning mechanics, it was found that the largest gains in scores occurred in those students who were involved in interactive activities that provided immediate feedback through discussion with peers or instructors [15] [12].

This raises the question of how to teach students more effectively. When students have misunderstandings about the differences between instantaneous velocity and constant acceleration, a common teaching strategy to help students overcome some conceptual difficulties is to use Microcomputer-Based Laboratory work (MBL) [START_REF] Mcdermott | Students' conception and problem solving in Mechanics[END_REF] [START_REF] Thornton | Learning motion concepts using realtime microcomputer-based laboratory tools[END_REF]. In kinematics, students plot in real time the relationship between position, velocity and acceleration versus time for motions, including their own. Instantaneous feedback helps to explain the links between movements and their graphical representations. An introductory course should be based entirely on such laboratory work [START_REF] Mcdermott | Students' conception and problem solving in Mechanics[END_REF]. Evaluation of the syllabus by means of pre-and post-tests shows that achievement and memorisation are then significantly higher than in courses taught by traditional methods. In another type of lab approach, students perform simple experiments that are designed to form the basis of Socratic dialogue [START_REF] Hake | Promoting student crossover to the newtonian world[END_REF] [START_REF] Hake | Socratic pedagogy in the introductory physics laboratory[END_REF].

Thus, questions that require qualitative reasoning and verbal explanation are very important. Mathematical formalism should be set aside until students have had some practice in qualitative reasoning about the phenomena being studied. Moreover, students should be asked to synthesise concepts and mathematical deductions and to formulate relationships in their own words. Likewise, learners should engage in the process of constructing qualitative models that will help them understand the connections and differences between concepts. Persistent conceptual difficulties should be explicitly overcome by repeated challenges in different contexts. Based on these findings, teaching by telling is an ineffective teaching method for most learners. Students need to be intellectually active in order to develop functional understanding. In conclusion, while teaching the concept of free fall in a gravity field, interaction on the one hand, and dialogue (questions and answers) on the other hand, seem to play a key role in the learning mechanism and in dispelling conceptual errors.

III. EXPERIMENTS

A. Research questions

VR naturally responds to the interaction aspects by creating arbitrary experimental conditions. On the other hand, when a student is in an immersive situation via a HMD, they are cut off from the outside world, particularly from the teacher, which tends to diminish or even interrupt the crucial questioning and challenging mechanisms. As the process of learning without a teacher can be difficult, this study's main interrogation concerns the isolation problem created using VR systems in an educative framework. Several objectives can be formulated: [START_REF] Zhurakovskaia | Immersive Serious Games for Learning Physics Concepts: The Case of Density[END_REF] The need to bring the teacher back into the virtual educational process and how best to do so; (2) help students understand and overcome their mistakes and misconceptions while learning free-fall; (3) convey to students the key idea of parabolic motion and how it can change in the presence or absence of gravitational fields.

B. Experimental conditions

We tested three playing conditions in a VR setting: Condition 1: The student is alone in the virtual world. The participant is equipped with a VR headset, two 3D controllers and a set of headphones. To talk to the teacher, they must remove the headset and address the teacher in person.

Condition 2: Student plays in VR but stays in contact with the teacher via an audio-visual link (videoconferencing window inside the simulation) (Fig. 1). This way the student can see and hear the teacher in real time, without breaking immersion.

Condition 3: The student experiences the VR together with a companion, a robot named Kylie (Fig. 3). Kylie will follow him everywhere. The teacher can manage this companion as WoZ, using a set of predefined actions (for example, making the robot point to the object that the student should pay attention to). The teacher can address the student with a microphone, and the student perceives the voice as coming from the robot. We chose a robot model because it is human-like [START_REF] Clark | E-learning and the Science of Instruction: Proven guidelines for consumers and designers of multimedia learning[END_REF], and more relatable than, say, a bird or a dragon. We chose to make it gender-neutral and did not try to replicate the teacher's appearance, because a teacher maybe be unwilling or unable to create a resembling avatar in an actual classroom condition.

C. Interviews with teachers

Prior to conducting the experiments, three interviews were conducted with teachers: two primary school teachers and a middle school English teacher. Each of them had more than 30 years of work experience. During the interview they got descriptions of the three game conditions and asked to choose what they would prefer and why. They were also asked how they generally felt about this method of teaching with VR.

Two teachers (one primary school teacher and an English teacher) preferred the second condition. They justified this by saying that in this case the pupil does the whole experience by himself/herself and if he/she needs help, face-to-face communication will be more effective for getting all the information. They also pointed out that face-to-face communication plays a big role in teaching and pupils should not be left to their own devices. Indeed, children in primary school may have comprehension problems due to inattention, and they are more likely to listen to the teacher (and take his face as authority) than to follow the robot's instructions. To the second question, the primary school teacher answered that in her practice this method of teaching would not be useful, again because the children were too young and did not have the necessary autonomy. The English teacher answered positively that she would use such a method in her teaching practice (close to distance learning, which the Covid-19 crisis has made commonplace). Another primary school teacher chose the first condition. She pointed out that it is necessary to combine modern technology and face-to-face communication and not to detach from social communication in general. She added that she would not use such technology in mainstream classes, but perhaps in supplementary courses. She suggested that this method could be applied to older students (middle and high school). In addition, she and her children are already using 2D simulations on tablets. According to her, today's children love technology, but human interaction must remain at the forefront.

From these interviews, we can hypothesise that there will be a strong preference towards "video" or "real teacher" conditions during actual experiments. But it also points out that, should the VR technology be spread in classrooms, teachers could be reluctant to use it for fear of losing crucial social interactions with their pupils.

D. Experimental setup

As mentioned, this study involves three exercises. The exercises are proposed one after the other in this order:

1

) Exercise 1 -Gravitation

The goal of this exercise is to show how gravity makes objects fall. This exercise is specifically designed to address the most common misconception in this regard, i.e., that heavy objects fall faster than light ones.

In this exercise, the student has 9 objects of different weights, sizes and densities at their disposal, as well as a Roberval balance scale [START_REF] Zhurakovskaia | Immersive Serious Games for Learning Physics Concepts: The Case of Density[END_REF]. The experience goes as follows: the student grabs any two objects from the table, compares their weights on the scale, places these two objects on the two predefined target locations and presses the "Start" button. The objects fall down, and a timer displays the time they each take to fall to the floor (Fig. 1).

Three game locations are proposed in succession: Earth (g=9.807m/s²), Moon (g=1.62 m/s²), outer space (no gravity). The visuals of the background change accordingly.

2) Exercise 2 -Parabolic trajectory

The purpose of this exercise is to show that in the presence of gravity, the fly trajectory of the projectile will always be parabolic in a gravity field -strongly bent, bell-shaped under Earth's gravity, less so on the Moon, and a straight line in the absence of gravity, contrary to what many students will spontaneously express.

In this game the student can choose between two types of projectiles (1 kg and 2 kg) to feed a cannon and shoot targets. When the cannon is loaded, the trajectory corresponding to the current cannon orientation is displayed in the form of a string of small beads. The student can observe the flight path in 3D live view and on a 2D orthographic monitor (Fig. 2).

The student can take up to 8 shots in each gravity condition. For each attempt, the target is randomly placed in one out of 8 positions at different distances and elevations.

3) Exercise 3 -Parabolic trajectory & Gravity

In this exercise, the student can observe how the trajectory of the projectile changes depending on the presence or absence of a gravitational field. Two kinds of cubic areas, "G -gravity" (normal earth gravity) and "NG -no gravity", called boxes, are horizontally stacked in a sandwich manner: on one platform "G-NG-G" and on another "NG-G-NG" (Fig. 3).

Again, the main task consists in shooting a cannonball. The student is asked to focus on the shape of the trajectory as the ball traverses the "gravity sandwich" (no targets). Before each shot, the student sees only the beginning of the predicted trajectory before the gravity sandwich. There are eight 1 kg projectiles on each platform. The student can watch the full trajectory when the shot is fired on a 2D orthographic video monitor.

E. Questionnaire

In order to evaluate the students' comprehension of the subject of gravity and free-falling, we developed our own questionnaire consisting of nine questions accompanied by a self-assessment of the level of confidence in answering the question. This questionnaire focuses on misconceptions and qualitative knowledge (link to the questionnaire: https://figshare.com/s/0902f3b748f76b3fb2ed).

F. Experimental procedure

Proper sanitary procedures to prevent Covid-19 contamination were designed and applied [START_REF] Posselt | Protocols of use for immersive platforms under Covid19[END_REF] during the experiments, described below.

Users passed a pre-test questionnaire. Players received basic practical instructions on how to interact with objects in VR. Then the player starts with Exercise 1. In each mode and for each exercise, instructions regarding the course of actions were given by the teacher by voice, but the general task description was also duplicated on a virtual whiteboard inside the simulation.

Following the VR experiment, the participants passed a post-test (the same as the pre-test). Then, participants were then asked to undergo a 15-minutes semi-directed interview, with the objective to collect some data regarding the subjective perception of the overall quality of the environment and interaction, to justify some answers in the post-test (if misconceptions remained). We were especially interested in feedback regarding the presence of the helper. Finally, students were given a theoretical description of each of the three game modes and asked to choose which one they would ideally prefer, and why.

IV. PRELIMINARY RESULTS

Due to the sanitary situation caused by the Covid-19 crisis, a complete round of experiments could not be undertaken at the time of writing this document. However, a preliminary study was conducted.

There were six participants (Mean age = 27, SD = 2; 2 females and 4 males; 2 per condition). All participants were right-handed, without visual problems, all have experience of using VR. All had a level equivalent to Masters in a sciencerelated field. Four students said that they had last studied the concept of gravity in secondary school, two others in high school.

During the game we recorded the time of completion for each condition and for each exercise. Because the aim was to study knowledge acquisition, success rate (target hitting skills) was not recorded. We consequently focused on test results and personal interviews. Table 1 compiles the total time results for each mode, for each state, and the total playing time. The average playing time was 26.52 minutes. However, for the real time mode students spent 19.74 minutes, for the video mode it took longer at 27.36 minutes, and the longest time was obtained for the companion mode at 32.45 minutes. On average students spent the most time on the 1st exercise with 12.95 minutes, followed by the second exercise with 8.54 minutes and the shortest time was for the third exercise with 5.03 minutes. The first and second exercise had 3 states, so there was a familiarity factor: the first exercise took the longest time, the second was shorter, and the third the shortest (for 5 out of 6 participants). For Exercise 3, the same amount of time was spent on average for each platform (2.12 minutes and 2.01 minutes).

Based on the Pre-and Post-test questionnaire success rate, all participants improved their performance, with average success rising from 77.56% to 87.82%. The self-confidence score increased by 13.42% as well. Looking at success rates for each mode, companion mode was the most effective, with an increase of 17%, followed by no-help mode with an increase of 8%, and finally video mode with 6%. However, these results need to be confirmed with a more statistically significant set of participants.

For the most part, scores increased on questions that were directly in relation to the game. In contrast, answers to question related with determining the flight path of a ball if dropped from an airplane (following a parabolic trajectory, but not directly considered in the practical exercises), remained consistently wrong in most cases. It seems the notion of initial speed is not invariant depending on how it is acquired (cannonball vs. dropping from a moving object), so that parabolic "forward" motion is not generalised easily.

During the interview, all the students noted the simplicity of Exercise 1, but one participant did not believe at first that 2 objects with different masses could fall the same way and concluded that the program did not work correctly, then later changed his mind. In Exercise 2 half of the students commented on the usefulness of the 2D orthographic view, the other half found the exercise was obvious without it, but all enjoyed shooting at targets with a cannon. Exercise 3 was also evaluated positively, but half of the students regretted that it was not possible to shoot at specific objects. In this situation, the 2D orthographic side view was deemed useful because the transition between the gravity blocks was not visible from the participant's subjective view. When asked to rank the exercises by order of preference, half of the students put Exercise 2 in first place and the other half chose Exercise 3, but all students clearly put Exercise 1 in last place, justifying this by the over simplicity.

When asked about their preferred assisting mode, five students chose the companion mode, justifying that it was more interesting, that they preferred not to see the teacher in the real world because they would feel pressure, and that interacting with the companion was more useful as it could show things in a 3D context without ambiguity. One student chose video communication, saying he preferred to see the teacher, i.e. that one-to-one communication was important. One student noted that the choice of game mode can depend on different factors: for instance, in a short simulation in VR the teacher was not required, whereas a long tutorial lesson would certainly interacting with the teacher a necessity.

Students who tested the first mode (with a real teacher) noted that they had difficulty understanding what the teacher wanted from them. Being completely immersed in the VR world, an "outside" voice was hard to focus on. This effect was absent in the second and third mode subjects.

Obviously further experiments with a higher number of participants are necessary to confirm our findings.

V. CONCLUSION

Many students have misconceptions about what could be considered the most basic concepts of physics. Although the use of VR itself for learning has proven to be effective [START_REF] Merchant | Effectiveness of virtual reality-based instruction on students' learning outcomes in k-12 and higher education: A meta-analysis[END_REF], the fact remains that the complete teaching process cannot be realised in a virtual environment alone. Often the presence of a teacher is a key factor. Because being immersed in a virtual world tend to isolate the student from the real world, a virtual companion or live video link with the teacher can significantly counterbalance this situation. Our goal was thus to study the effectiveness of assisting techniques in serious VR games. The increased average success rate of all participants of the preliminary tests confirms that indeed such assistance complemented the natural benefits of VR learning.

Preliminary studies demonstrate the ecological validity of VR, giving access to situations that would otherwise never be experienced. Based on success rates for each mode, we found that companion mode was the most effective.

Figure 1 .

 1 Figure 1. Exercise 1 -Gravitation (Condition 2).

Figure 2 .

 2 Figure 2. Exercise 2 -Parabolic trajectory (Condition 1).

Figure 3 .

 3 Figure 3. Exercise 3 -Parabolic trajectory and Gravity (Condition 3).

TABLE I .

 I TIME SPENT IN THE GAME (IN MINUTES)

ACKNOWLEDGEMENT

This work was supported by French government funding under grant ANR-21-ESRE-0030 / CONTINUUM.