Vladimir Bidnenko

Arnaud Chastanet

Christine Péchoux

Yulia Redko-Hamel

Olivier Pellegrini

Sylvain Durand

Ciarán Condon

Marc Boudvillain

Matthieu Jules

Elena Bidnenko

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

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

Introduction

A growing understanding of the importance of transcription termination in the regulation of gene expression in bacteria has stimulated extensive analysis of the proteins that control this universal step in decoding genetic information [START_REF] Peters | Bacterial transcription terminators: the RNA 3′end chronicles[END_REF][START_REF] Ray-Soni | Mechanisms of bacterial transcription termination: all good things must end[END_REF][START_REF] Kriner | Learning from the leaders: gene regulation by the transcription termination factor Rho[END_REF][START_REF] Jr | Regulation of bacterial gene expression by transcription attenuation[END_REF]Mandell et al., 2022a). Among them is transcription termination factor Rho, an ATP-dependent RNA helicase-translocase that improves the termination efficiency of many intrinsic terminators and is essential for termination at bona-fide Rhodependent terminators [START_REF] Roberts | Termination factor for RNA synthesis[END_REF]Quirk et al., 1996;[START_REF] Peters | Rho directs widespread termination of intragenic and stable RNA transcription[END_REF][START_REF] Hao | Rho-dependent transcription termination: A revisionist view[END_REF]Mandell et al., 2022b). Over the last years, considerable advances have been made in unveiling the molecular mechanism of Rho-dependent termination (Song et Recently, we provided evidence that Rho is intricately involved in the control of cellular adaptation to stationary phase and in cell-fate decision-making, in particular, the decision to sporulate, in the Gram-positive bacterium Bacillus subtilis [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF][START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF]. The involvement of Rho in the sporulation process was also demonstrated in Clostridioides difficile and Bacillus thuringiensis [START_REF] Trzilova | Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile[END_REF][START_REF] Lin | Adaptation of Bacillus thuringiensis to Plant Colonization Affects Differentiation and Toxicity[END_REF]2021). Sporulation is a complex developmental program that transforms a vegetative bacterial cell into a highly resistant dormant spore, thus ensuring the survival of bacteria under adverse environmental conditions and their dissemination within different ecological niches (de Hoon et al., 2010; [START_REF] Swick | Surviving Between Hosts: Sporulation and Transmission[END_REF][START_REF] Galperin | Conservation and Evolution of the Sporulation Gene Set in Diverse Members of the Firmicutes[END_REF]. A hallmark of sporulation in B. subtilis is the asymmetric division of a differentiating cell in two unequal parts: a forespore, which further develops into the spore, and a mother cell, which engulfs the forespore, nourishes it, ensures the synthesis of spore protective layers, and finally lyses to release the mature spore.

Proper spore morphogenesis is essential for spore resistance to external damages and its conversion back into a growing cell under favorable conditions through sequential processes of germination and outgrowth [START_REF] Setlow | Spore germination[END_REF][START_REF] Setlow | Germination of spores of the orders Bacillales and Clostridiales[END_REF][START_REF] Setlow | New Thoughts on an Old Topic: Secrets of Bacterial Spore Resistance Slowly Being Revealed[END_REF][START_REF] Segev | Molecular kinetics of reviving bacterial spores[END_REF][START_REF] Abhyankar | The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis spores[END_REF][START_REF] Boone | Protein synthesis during germination: shedding new light on a classical question[END_REF][START_REF] Mutlu | Phenotypic memory in Bacillus subtilis links dormancy entry and exit by a spore quantity-quality tradeoff[END_REF]2020).

In B. subtilis, sporulation is primarily controlled at the level of transcription initiation by the master regulator Spo0A, whose activity depends on phosphorylation mediated by a multicomponent phosphorelay, the transition phase-specific sigma factor SigH and a cascade of sigma factors (SigF, SigE, SigG and SigK) that sequentially drive temporally-and spatiallydefined transcriptional programs of spore morphogenesis. Sporulation is initiated at a threshold level of Spo0A~P, which activates the expression of sigH and several sporulation genes, including sigF and sigE encoding the inactive forms of sigma factors. Upon completion of the asymmetric septum, activation of SigF in the forespore is required for subsequent activation of SigE in the mother cell. After a complete engulfment of the forespore, the compartment-specific programs of gene expression are further continued by SigG in the forespore and SigK in the mother cell (for comprehensives reviews see [START_REF] Errington | Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis[END_REF][START_REF] Stragier | Molecular genetics of sporulation in Bacillus subtilis[END_REF][START_REF] Piggot | Sporulation of Bacillus subtilis[END_REF][START_REF] Higgins | Recent progress in Bacillus subtilis sporulation[END_REF]. The relevant regulons of each sporulationspecific sigma factor, as well as other genes involved in sporulation, were determined using the combination of experimental and bioinformatics approaches [START_REF] Eichenberger | The σE regulon and the identification of additional sporulation genes in Bacillus subtilis[END_REF][START_REF] Eichenberger | The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis[END_REF] We have previously shown that deletion of the rho gene in B. subtilis prevents intragenic termination of the kinB transcript, which encodes the sensor kinase KinB, one of the main kinases feeding phosphate into the Spo0A phosphorelay. This leads to the increased expression of KinB and, consequently, to rapid accumulation of active Spo0A~P to a threshold level that triggers sporulation. Thus, Rho inactivation stimulates sporulation in B. subtilis indicating a regulatory role of Rho-mediated transcription termination within the cell fate decision-making network centered on Spo0A~P [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. Conversely, maintaining rho expression at a stably elevated level throughout B. subtilis exponential growth and stationary phase alters the early steps of adaptive reprogramming of cellular transcription, prevents the activation of Spo0A and, additionally, inhibits some late, yet non-identified, sporulation events, with the overall result of blocking the formation of spores [START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF].

Comparative transcriptome and proteome analyses have revealed a decrease in the levels of rho mRNA and Rho protein during the transition to stationary phase in wild-type B. subtilis cells [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF][START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]2023). Considering that Rho negatively affected sporulation in our analyses, a decrease in Rho levels appears necessary for both initiation and implementation of the sporulation program. This in turn suggests that rho expression during B. subtilis growth and stationary phase is subject to reliable and timely regulation.

The only mechanism known to date for regulating rho expression is transcriptional attenuation at the Rho-dependent terminator(s) located within the leader region of the rho transcript and consequent premature transcription termination [START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF]. Similar to B. subtilis, transcription of the rho gene was shown to be autogenously regulated in Escherichia coli, Salmonella and Caulobacter crescentus [START_REF] Barik | Autogenous regulation of transcription termination factor Rho[END_REF][START_REF] Matsumoto | Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: Localization and function of its attenuators[END_REF][START_REF] Italiani | The transcription termination factor Rho is essential and autoregulated in Caulobacter crescentus[END_REF][START_REF] Silva | SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA[END_REF]. In Salmonella, rho autoregulation is counteracted by the small noncoding RNA SraL, which prevents the Rho-mediated termination by direct binding the 5′-UTR of rho mRNA [START_REF] Silva | SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA[END_REF].

In the present study, we aimed to gain further insights into rho regulation in B. subtilis by analyzing the kinetics of rho expression at various stages of cellular growth and differentiation.

Quite unexpectedly, we found that the expression of the rho gene is specifically induced early during sporulation. This counterintuitive finding has focused our subsequent analysis on understanding the mechanism of the sporulation-specific expression of rho and its biological significance.

We describe two distinct mechanisms controlling rho expression in each compartment of the sporulating cells: read-through transcription from the upstream SigH-dependent promoter in the mother cell, and a genuine SigF-dependent rho promoter active in the forespore. The latter feature allows us to classify rho as a novel member of the SigF regulon of B. subtilis. We provide evidence that altering the spatiotemporal expression of rho affects spore resistance properties and morphology, in particular, the structure of spore coat. Moreover, spores formed in the absence of Rho are impaired in their ability to revive under favorable growth conditions, exhibiting an accelerated germination and a slow outgrowth. Finally, we show that the optimal rate of spore outgrowth depends on the synthesis of Rho during spore formation and de novo after germination.

Results

Rho expression is specifically induced during sporulation.

To analyze a real-time expression of the rho gene, we constructed a scarless reporter system, in which the luciferase gene luc of firefly Photinus pyralis was fused to the rho promoter (Prho) at the position of the rho start codon. The Prho-luc fusion was similarly located in the rho locus of the chromosome in the wild type B. subtilis strain BSB1 and the rho deletion mutant RM [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. The former strain (hereinafter WT) kept an active copy of the rho gene due to duplication of the rho locus during the construction. By comparing luciferase activity in WT and RM strains under different growth conditions, we sought to assess Rho autoregulation and possibly identify other regulatory factors.

In WT cells grown in rich medium LB, the expression of rho was mainly limited to the exponential growth phase, characterized by two peaks of luciferase activity at OD600 ~0.15 and ~0.4 (Fig 1A). In stationary phase, rho expression remained at a basal level. A low-level expression of rho and its down-regulation during the transition to stationary phase were reported previously [START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF][START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF][START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]2023). In RM cells grown in DSM, luciferase activity increased ~7-fold compared to WT cells during exponential growth, but only ~1.5-fold in stationary phase. This suggests that rho expression in stationary phase is not subject to significant autoregulation. Curiously, however, the stationary phase-specific expression of rho was activated considerably earlier in RM than in WT cells (Fig 1C). This effect was reminiscent of the accelerated expression of spo0A gene in the sporulating RM cells caused by the up-regulation of Spo0A phosphorelay [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. Therefore, we compared the expression of rho and spo0A genes in WT and RM cells grown in DSM using the Prho-luc and Pspo0A-luc fusions [START_REF] Mirouze | Spo0A~P imposes a temporal gate for the bimodal expression of competence in Bacillus subtilis[END_REF][START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. In both strains, rho expression in stationary phase timely followed spo0A and, in RM cells, the maximal expression levels of both genes was reached ~1.5 hour earlier than in WT (Fig 1D). This observation additionally argued for the sporulation-specific expression of rho as a function of Spo0A~P activity.

The inactivation of

To get more insights into the sporulation-specific expression of rho, we analyzed the Prho-luc activity in B. subtilis sporulation mutants. First, we considered spo0A and sigH mutations, which block initiation of sporulation by inactivating Spo0A and the transition phase-specific sigma factor SigH, respectively. Neither spo0A nor sigH mutations affected Prho-luc activity during exponential growth in DSM, but both specifically blocked it in stationary phase, thus confirming that rho expression at this stage is dependent on sporulation-specific factors (Fig 1E).

Next, we assessed the involvement of the alternative SigF and SigE factors controlling early gene expression in the forespore and mother cell, respectively. The kinetics of Prho-luc activity in sigF and sigE mutants was rather similar to WT cells. However, the stationary phase-specific luminescence appeared slightly lower in the sigF background and, in both mutants, remained high after reaching a maximal level (Fig 1F). These results indicated some deregulating effects of sigF and sigE mutations and suggested that rho isexpressed in both compartments of sporulating cells.

Considering that the known SigA-controlled Prho promoter is mainly active during exponential growth (Fig 1A ), we sought for additional regulatory factors of rho expression during sporulation.

The read-through transcription from the upstream SigH-controlled promoter contributes to rho expression during sporulation.

The rho gene is transcribed from the cognate Prho promoter together with the upstream noncoding S1436 element and the downstream rpmE gene in a transcript of ~1.9 kb (Quirk et al., 1991;[START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF][START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]. Additionally, the genome-wide transcriptome analyses have revealed several longer read-through transcripts at the rho locus, which initiate at the upstream spo0F and fbaA-ywjH promoters and bypass the intrinsic terminators of the ywjH and glpX genes [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]Mandell et al., 2022b;Fig 2A).

We asked whether read-through transcription could play a role in the expression of rho, in particular during sporulation, as spo0F gene is known to be transcribed from alternative SigAand SigH-dependent promoters, with the latter also being regulated by Spo0A~P [START_REF] Lewandoski | Transcriptional Regulation of the spoOF Gene of Bacillus subtilis[END_REF][START_REF] Predich | Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H[END_REF][START_REF] Strauch | A positive feedback loop controls transcription of the spo0F gene, a component of the sporulation phosphorelay In Bacillus subtilis[END_REF][START_REF] Asayama | Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis[END_REF]. To this end, we sought to enhance transcription termination upstream of the rho gene and inserted a DNA fragment containing three intrinsic transcription terminators after the stop codon of the glpX gene (hereafter referred 3Ter; Fig 2B; Vagner et al., 1998;[START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. To assess read-through transcription at the rho locus and the termination efficiency of the 3Ter insertion, we analyzed rho-specific transcripts in WT, sigH and 3TER mutant cells by Northern blot (Fig 2C). Cells were grown in DSM and sampled during exponential growth (OD600 0.5), transition phase (OD600 1.0) and in stationary phase, at OD600 1.8. In WT and sigH mutant cells, we observed several rho-specific RNA species, the smallest and most abundant of which corresponded in size to the 1.9 kb rho-rpmE mRNA, and the largest (~7 kb) to transcripts initiated at the fbaA and/or spo0F promoters. Other RNAs of ~ 4-5 kb in size cannot be assigned to any defined promoter and may result from ribonuclease processing of the larger transcripts.

During transition to stationary phase, the larger rho-specific RNAs accumulated in WT cells, but gradually disappeared in sigH mutant, in accordance with the notion that spo0F To better understand the expression of rho in the absence of read-through transcription, we analyzed the activity of Prho-luc fusion in the 3TER strain and its sporulation-deficient derivatives. In 3TER Prho-luc cells grown in DSM, luciferase activity was identical to WT Prholuc during exponential growth, but decreased ~3-fold in stationary phase, highlighting the role of read-through transcription in the rho expression at this stage (Fig 2D). Intriguingly, the residual luminescence in the stationary 3TER Prho-luc cells was blocked by spo0A and sigH (Fig 2E) and partially inhibited by sigF inactivation, while mutation of sigE had no effect (Fig 2F). All together, these results indicated that the glpX-rho intergenic region contains additional structural factor(s) of rho expression active during sporulation and likely dependent on SigF.

The sporulation-specific expression of rho is compartmentalized.

Since SigF is primarily associated with the forespore, this led us to investigate potential compartmentalization of rho expression during sporulation. To this end, we constructed a Prhogfp fusion expressing green fluorescent protein GFP at the rho chromosomal locus of WT cells preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. and analyzed its activity during sporulation at a single-cell level by fluorescence microscopy.

We observed an increase of fluorescence intensity in the forespores of the sporulating WT Prhogfp cells immediately after asymmetric division (S1 Fig and Fig 3). This observation further suggested a particular role of SigF in the sporulation-specific expression of rho. Following it, we analyzed the activity of Prho-gfp fusion in sigF and sigE mutant cells.

Absence of SigE disables the SigE-dependent inhibitors of septation, therefore provoking the formation of asymmetric septa at both poles of sporulating cells [START_REF] Illing | Genetic regulation of morphogenesis in Bacillus subtilis: roles of sigma E and sigma F in prespore engulfment[END_REF][START_REF] Setlow | Condensation of the forespore nucleoid early in sporulation of Bacillus species[END_REF][START_REF] Lewis | Sigma factors, asymmetry, and the determination of cell fate in Bacillus subtilis[END_REF][START_REF] Eichenberger | A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis[END_REF]. Because activation of SigE in the mother cell sequentially depends on SigF activity in the forespore (Londono-Vallejo & Stragier, 1995; and references therein), both sigE and sigF mutants display a similar disporic cell phenotype. Such disporic cells were readily seen in Prho-gfp sigF and Prho-gfp sigE sporulating cultures, in addition to cells containing one asymmetric septum. The mother cells of both sigF and sigE sporangia maintained GFP signal at a level similar to that of WT, indicating that rho expression is SigE-independent (Fig 3). In contrast, while the forespore-like structures showed an increased fluorescence in sigE mutant similarly to the WT forespores, their brightness was significantly diminished in sigF sporangia (Fig 3). Together with the results from the previous section, this observation indicated that, in forespores, rho is expressed from a promoter located in the glpX-rho intergenic region and dependent, directly or indirectly, on SigF.

The 5'-UTR of rho contains a genuine SigF-dependent promoter active in forespores.

Previous analyses have identified a rho promoter that matches the SigA-binding sequence consensus ( sigA Prho) 293-320 bp upstream the rho start codon [START_REF] Quirk | Identification of a putative Bacillus subtilis rho gene[END_REF][START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF]Fig 4A). Recent reassessment of B. subtilis promoters using the unsupervised sequence clustering algorithm has classified sigA Prho to the M16 cluster of SigA-dependent promoters, which are characterized by an extended 3'-terminal G-rich -10 box and a variable -35 box with the conserved TTG stretch [START_REF] Nicolas | Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis[END_REF]Fig 4B). Looking for alternative rho expression signals, we analyzed the glpX-rho intergenic region for the presence of the SigFbinding consensus -35 (GYATA) and -10 (GGnnAnAHTR) sequences, where Y is C or T; H is A or C or T; R is A or G; and n is any nucleotide (Wang et al., 2006). Such features were found within the sigA Prho sequence itself (Fig 4C). Within the -10 box of sigA Prho, we identified GGTAAAAATA sequence perfectly matching the SigF -10 consensus and, 14 bp upstream, a 5-nucleotide GAATA sequence differing from the SigF -35 consensus by one nucleotide. Of note, the same -35 sequence is present in the SigF-regulated promoters of yuiC, yabT, yjbA, ypfB and ythC genes (Wang et al., 2006). Moreover, the 14-bp spacer between -35 and -10 sequences is highly A/T-rich that is characteristic of most SigF-dependent promoters (Wang et al., 2006).

To prove that the identified sequences constitute a novel SigF-dependent rho promoter (hereafter sigF Prho), we proceeded to site-directed mutagenesis of the Prho-gfp fusion, anticipating that altering the SigF-binding sequences would specifically affect GFP expression in the Overall, our results indicate that, during growth and sporulation, the expression of rho gene relies on three distinct features: (i) a SigA-dependent rho promoter that is active during exponential growth, (ii) read-through transcription from the upstream SigH-dependent spo0F promoter during early stationary phase and in the mother cells of sporangia, and (iii) a SigFdependent rho promoter active in the forespores.

Differential expression of Rho affects spore properties and morphology.

The revealed spatiotemporal expression of rho suggested that besides modulation of Spo0A activity at the onset of sporulation [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF], Rho can be involved in the subsequent steps of spore development. To address this hypothesis, we used a set of five strains differentially expressing Rho during sporulation, namely: the wild type (WT), a strain blocked for the rho read-through transcription (3TER), their derivatives inactivated for rho expression in the forespore by mF-35T/A mutation of sigF Prho (WT-mT/A and 3TER-mT/A), and a mutant deleted for rho (RM).

Initially, we compared sporulation capacities of the five strains. To this end, we induced sporulation by the resuspension method and assessed asymmetric division of individual cells and formation of the heat-resistant spores at the initial and final stages of sporulation, respectively. Among the five strains, RM cells showed a highest rate of asymmetric division corroborating our previous data on the accelerated sporulation of Δrho mutant, most probably due to a more efficient activation of Spo0A (S3 Fig; [START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. Interestingly, the proportion of cells with asymmetric septum was also higher in 3TER and 3TER-mT Two coats were tightly attached to each other in a large majority (over 92 percent) of WT spores or occasionally separated across a limited area (Fig 6). In contrast, RM spores contained a lamellar inner coat similar to WT spores, but their outer coat remarkably lost electron-density and striated structure and appeared half thinner compared to WT spores (respectively, 36.2 +/-0.0091 nm and 74.2 +/-0.021 nm, as measured for 100 spores of each strain). Moreover, above 80 percent of RM spores showed an extensive detachment of inner and outer coats, often along the entire spore perimeter (Fig 6). Spores differentially expressing rho exhibited intermediate ranges of structural alterations of their coats. The outer coat of 3TER spores appeared less striated and low electron-dense compared to WT, therefore resembling the RM coat. Moreover, the outermost layer often peeled off the 3TER spore surface. However, we did not observe 3TER spores with the separated inner and outer coats (Fig 6). In WT-mT/A spores, the outer coat was electron-dense and striated like a WT layer, but detached from the inner coat in ~30 percent of spores, although across less extended areas than in RM spores (Fig 6). Finally, 3TER-mT/A spores contained a low structured outer coat locally separated from the inner coat in ~30 percent of spores (Fig 6). By combining these features, 3TER-mT/A spores appear most similar to RM spores, which seems in line with the absence of sporulation-specific rho expression in both mutant strains. We conclude that altering expression of rho during sporulation in the mother cell (3TER) or in the forespore (mT/A) induces specific defects of spore morphology, which are accentuated by the entire loss of the Rho activity in RM strain.

Rho mediates spore germination and outgrowth.

The ability to resume vegetative growth in favorable nutrient conditions, referred to as spore revival, is a basic spore property and spore quality indicator ( We compared the revival abilities of spores differentially expressing Rho by evaluating the optical density (OD600) of spore suspensions after the induction of germination. Rapid rehydration and structural changes of the germinating spores alter their optical properties resulting in a decrease in OD600. The OD600 of reviving spores remains stable during the postgermination phase of metabolic resumption and molecular reorganization, designated the "ripening period", before increasing later during spore outgrowth, when the emerging cells start to grow and divide [START_REF] Segev | Molecular kinetics of reviving bacterial spores[END_REF][START_REF] Rosenberg | Phosphoproteome dynamics mediate revival of bacterial spores[END_REF]. For simplicity, we consider the outgrowth period as a time needed for a spore culture to restore its initial OD600 after germination. We asked whether Rho activity is needed exclusively during spore morphogenesis for the formation of an appropriate molecular basis for spore revival in the future, or for a correct metabolic resumption during spore outgrowth. To answer this question, we placed the rho gene under the control of the IPTG-inducible promoter Pspac, which allowed us to selectively control rho expression during sporulation and/or spore outgrowth. By immunoblot analysis of Pspacrho cells using the Rho Bs -specific antiserum, we established that the induction of Pspac-rho by IPTG (100µM) determines a near-natural level of Rho protein (Materials and Methods; S8 Fig) .     Following this, we allowed Pspac-rho cells to sporulate in the presence or absence of IPTG 100µM, purified the mature spores from the IPTG-induced and non-induced cultures and compared the revival of two types of Pspac-rho spores in the conditions where rho expression was omitted or de novo induced by IPTG. We observed that spores, which were formed in the absence of Rho (Pspac-rho (-IPTG) ), exhibited an accelerated germination like RM spores, while those that expressed rho during morphogenesis (Pspac-rho (+IPTG) ), germinated as WT (Fig 8A). Addition of IPTG together with the germinant did not modify germination kinetics of either spore type (Fig 8B). The two types of spores also behaved differently during outgrowth. In the absence of IPTG, the outgrowth of Pspac-rho (-IPTG) spores was identical to RM spores, while Pspac-rho (+IPTG) spores grew out at an intermediate rate between WT and RM (Fig 8C). Interestingly, de novo induction of rho expression by IPTG had a small but reproducible stimulatory effect on the outgrowth rates, so that Pspac-rho (+IPTG) spores were able to "catch up" with the outgrowth of WT spores (Fig 8D ).

IPTG had no effect on the germination or outgrowth of WT and RM spores.

From these experiments, we conclude that normal spore revival requires expression of rho during spore morphogenesis and, although to a lesser extent, de novo during spore outgrowth.

Discussion

The main achievement of the present analysis is the revelation of spatiotemporal expression of the termination factor Rho during sporulation in B. subtilis via two distinct mechanisms: readthrough transcription initiated at a distal promoter controlled by SigH in the mother cells, and transcription from the authentic SigF-dependent rho promoter in the forespores. These regulatory elements compensate for the inactivation of SigA-dependent rho expression at the end of exponential growth and allow the "refueling" of Rho protein in both compartments of the sporangium.

Our results also show that maintaining Rho levels during sporulation is necessary for the formation of spores with normal morphology, resistance properties and the ability to return to a vegetative state under favorable environmental conditions.

Compartmentalized expression of rho during sporulation.

The sporulation-specific read-through transcription of rho is initiated by the activation of the For instance, the stationary phase transcriptome of RM cells does not provide an obvious explanation for the decreased UV-resistance of spores differentially expressing rho. Indeed, the observed upregulation of several ssp genes in the stationary RM cells, in particular sspC, is contrary to what one could expect if spores become sensitive to UV. It is therefore likely that the effect of Rho in stationary phase and in sporulating cells is different for some genes. and the SigK-controlled gerPA-gerPB-gerPC-gerPD-gerPE-gerPF operon, whose products facilitate the passage of nutrient germinants across the spore coat [START_REF] Behravan | Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores[END_REF].

Although these features suggest that Rho controls both operons during sporulation, they seem insufficient to explain the rapid germination of RM spores in response to L-alanine or L-valine.

Among the gerAA-gerAB-gerAC genes encoding the L-alanine germinant receptor GerA (Feavers et To conclude, this and our previous analyses trace the activity of Rho through each stage of the complex program of B. subtilis sporulation, from the regulation of Spo0A~P activation at the onset of sporulation, through the subsequent morphogenesis of spores and up to the modulation of spore revival. This highlights the existence of specific targets for Rho-mediated transcription termination within the regulon of each alternative Sigma factor, which certainly include, alongside the known coding genes, a particular set of non-coding and anti-sense transcripts.

Appropriate regulation of these targets requires in turn an accurate time-and spatial-specific regulation of the rho expression, through mechanisms we describe. Overall, our results strengthen our vision of Rho as a built-in regulatory module of B. subtilis cell differentiation.

Materials and Methods

Bacterial strains and growth conditions

All strains used in the analysis originate from B. subtilis 168 trp + strain BSB1 (Nicolas et al., Spectrophotometer, Pharmacia Biotech. When required, antibiotics were added at following concentrations: 0.5 μg per ml of erythromycin, 3 µg per ml of phleomycin, 100 μg per ml of spectinomycin, and 5 μg per ml of chloramphenicol to B. subtilis cells; and 100 μg per ml of ampicillin or 20 µg per ml of kanamycin to the plasmid-containing E. coli cells. For the induction of Pspac-rho fusion, IPTG (isopropyl-β-D-1-thiogalactopyranoside) inducer was added to cells and spores to final concentration 100µM. (WT) and BRL1 (Δrho) cells using selection for chloramphenicol-resistance. By this event, the luc gene was placed under all natural regulatory signals of the rho expression. In the WT background, the replaced intact copy of rho gene remained active under the control of own promoter.

2012) and are listed in S2

Strains and plasmids construction

Insertion of three intrinsic transcription terminators (3TER) upstream the chromosomal Prho-luc fusion was performed as follows. The 3TER DNA fragment amplified from pMutin4 plasmid using oligonucleotides veb734 and veb735 was end-joined with the DNA fragments amplified from pBRL862 using the pairs of oligonucleotides glpXBam/veb798 (5'-complementary to veb734) and lucintrev/veb797 (5'-complementary to veb735). The primers glpXBam and lucintrev were used in the joining reaction. The PCR product was cut by EcoRV and BstBI endonucleases and cloned at a similarly cut and gel-purified pBRL862 plasmid. The resulting plasmid pBRL1107 was integrated at the rho locus of BSB1 chromosome as above. The created 3TER-Prho-luc transcriptional fusion contains intact 5'-UTR of rho and the 3TER insertion immediately after the glpX stop codon.

Transcriptional fusion between the gfp gene, encoding Green Fluorescent Protein, and the rho promoter (Prho-gfp) was constructed similarly to Prho-luc. The oligonucleotides glpXBam and veb742 were used for amplification of the rho moiety from BSB1 chromosome, and the gfp gene was amplified from the plasmid pCVO119 using the primers veb741 and veb740 complementary to veb742. The primers glpXBam and veb741 were used for joining PCR, and the resulting DNA fragment was cloned in pCVO119 plasmid using BamHI and NcoI endonucleases and T4 DNA ligase. The resulting plasmid pBRL893 was integrated by single crossover at the rho locus of the BSB1 chromosome using selection for spectinomycin resistance.

To construct the 3TER-Prho-gfp transcriptional fusion, the DNA fragment containing 3TER and the rho 5'-UTR was amplified from pBRL1107 plasmid using the primers glpXBam and veb742 and fused to the gfp gene as described above for pBRL893. The product of joining PCR was cut by EcoRV and NcoI endonucleases and cloned at a similarly cut and gel-purified pBRL893. The resulting pBRL1150 plasmid was inserted in the BSB1 chromosome as above. Structural modifications of the rho expression unit at natural locus were performed by the method for allelic replacement using a shuttle vector pMAD (Arnaud et al., 2004).

Site-directed mutagenesis of the

To insert 3TER transcription terminators at the rho locus, two DNA fragments amplified from the BSB1 chromosome with the pairs of oligonucleotides veb795/veb796 and veb797/veb798

were end-joined to the 3TER DNA fragment (see above) by PCR using the primers veb795 and veb796. The product of joining PCR was cut with BamHI and EcoRI endonucleases and cloned in the pMAD vector (Arnaud et al., 2004). The resulting plasmid pBRL1108 was integrated into the rho locus of the BSB1 chromosome with selection of the erythromycin-resistant transformants at 37°C non-permissive for plasmid replication. Single transformants were propagated in LB without antibiotic at 30°C to induce the loss of the vector and platted at LBplates at 37°C. The erythromycin-sensitive clones were selected among the grown colonies and tested by PCR for the presence of the 3TER insertion using oligonucleotides veb734 and veb735, and the selected clones were controlled for structural integrity of the glpX-3TER-rho region by sequencing.

The point mutation mF-35T/A was introduced in the rho locus of BSB1 and BRL1130 (3TER) strains as follows. Two DNA fragments were amplified from pBRL1164 plasmid and BSB1

chromosome by PCR with the respective pairs of oligonucleotides veb796/veb808 and veb806/veb807, complementary to veb808. The fragments were joined by PCR using veb796 and veb806 as primers, and the resulting product was cloned at pMAD vector using BamHI and EcoRI endonucleases and T4 DNA ligase. The resulting plasmid was transformed into BSB1 cells and the transformants were processed as described above to select the point mutant WT-mT/A. The mutant strain 3TER-mT/A was constructed in a similar way using the plasmid pBRL1162 as a template in the first PCR and WT-mT/A strain as a recipient for transformation.

To construct the IPTG-controlled system for rho expression, the DNA fragment amplified from BSB1 chromosome by PCR with the oligonucleotides veb806 and veb880 was digested by EcoRI and BamHI endonucleases and clones at pMUTIN4 vector (Vagner et al., 1998). The resulting plasmid was transformed in BSB1 cells with selection to erythromycin resistance.

For the purification of the B. subtilis Rho protein, rho gene was amplified by PCR with oligonucleotides veb596 and veb599, treated by NdeI and SalI endonucleases and cloned at the expression vector pET28a (Novagen) allowing expression of Rho with N-terminal hexahistidine tag [START_REF] Ingham | Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis[END_REF]. The resulting plasmid pETRho was transferred to E. coli strain BL21-CodonPlus (DE3)-RIL (Stratagene).

Luciferase assay

Analysis of promoters' activity using luciferase fusions was performed as described previously [START_REF] Mirouze | Fluctuations in spo0A transcription control rare developmental transitions in Bacillus subtilis[END_REF] with minor modifications detailed in [START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF]. Cells were grown in LB medium to mid-exponential phase (OD600 0,4-0,5), cultures were centrifuged and resuspended to OD 1.0 in fresh LB or DSM, to follow expression of the fusions during growth or sporulation, respectively. The pre-cultures were next diluted in respective media to OD600 0.025. The starter cultures were distributed by 200µl in a 96-well black plate (Corning, USA)

and Xenolight D-luciferin K-salt (Perkin, USA) was added to each well to a final concentration of 1.5 mg/ml. The cultures were grown under strong agitation at 37°C and analyzed in Synergy 2 Multi-mode microplate reader (BioTek Instruments). Relative Luminescence Units (RLU) and OD600 were measured at 5 min intervals. Each fusion-containing strain was analyzed at least three times. Each experiment included four independent cultures of each strain.

Epifluorescence microscopy and image processing

For all microscopic observations, cells were mounted on a 2% agarose pad and topped with a coverslip. Bacteria were imaged with an inverted microscope (Nikon Ti-E) equipped with an iLas2 laser coupling system from Roper Scientific (150 mW, 488nm and 50 mW, 561 nm), a 100× oil immersion phase objective, an ORCA-R2 camera (Hamamatsu) and controlled by the To determine the frequency of cells entering into sporulation, cultures were sampled three hours after the induction of sporulation by the resuspension method (Harwood and Cutting,1990).

Sampled cells were mixed with the lipophilic fluorescent dye Mitotracker Red (10 µg/ml final concentration) prior to microscopic observation. Asymmetric septa were manually counted in two independent replicas (N > 450 per strain and per replica).

To assess rho expression during sporulation using GFP as a reporter, cells bearing the non- Total RNA was extracted from B. subtilis cultures at OD600 indicated in the text by the glass beads method [START_REF] Bechhofer | Analysis of mRNA decay in Bacillus subtilis[END_REF]. For Northern blotting, 5 µg of RNA were separated on 1% agarose-formaldehyde (2.2M) gel in MOPS (50mM)/EDTA (1mM) buffer (pH 7.0) and transferred to Hybond N+ membrane (GE-Healthcare) as described previously [START_REF] Redko | A minimal bacterial RNase J-based degradosome is associated with translating ribosomes[END_REF]. Membrane pre-hybridization and the rho RNA detection by hybridization with the rhospecific α 32 P -labelled riboprobe was performed as detailed in [START_REF] Gilet | Analysis of Bacillus subtilis Ribonuclease Activity In Vivo[END_REF]. The riboprobe was synthesized by T7 RNA polymerase in the presence of [α- 32 P]UTP at the template of the purified PCR fragment obtained with oligonucleotides YRH1 and YRH2 (S3 Table ). The reverse PCR primer YRH1 contained at the 5'-end the T7 RNA polymerase promoter sequence (TAATACGACTCACTATA).

Sporulation assay

Cells were induced for sporulation by the resuspension method, and the cultures were analyzed for the presence of spores starting from six hours after resuspension. The percentage of spores in a sample was calculated as proportion of viable cells after heating at 70°C for 15 min to the total number of cells as described in [START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF].

Spore purification and treatments

B. subtilis cells growing exponentially in LB were suspended in DSM at OD600 0.05 and cultured at 37°C with aeration for 24 hours. Spores were purified by sequential rounds of intensive washing in ice-cold water and centrifugation over 3 days as described by Nicholson and Setlow in (Harwood and [START_REF] O'brien | The nutritional requirements for germination and out-growth of spores and vegetative cell growth of some aerobic spore forming bacteria[END_REF]. The samples of the purified spores were controlled for the absence of cells by the heat-resistance test as described above and stored at 4°C in water at OD600 > 10. The purity of standard spore samples was above 95 percent.

For electron microscopy analysis, spores were additionally purified on Nicodenz (Axis-Shield, United Kingdom) density gradients. Spores were suspended in 1 ml of 20 % Nicodenz solution, layered on 50 % Nicodenz (15 ml) in the centrifuge tubes and centrifuged at 14,000 x g for 30 min at 10°C. The pelleted pure spores were washed 5 times in ice-cold water to remove traces of Nicodenz and kept at 4°C before analysis.

Prior the assays of spore resistance phenotypes and germination, the purified spores were suspended in 10 mM Tris HCl (pH 8.0) at OD600 1.0 and activated by heating at 70°C for 30 min and subsequent cooling in ice for 20 min. The activated spores were used in the assays within 1 hour.

Chemical removal of spore coats was performed according to [START_REF] Riesenman | Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation[END_REF]. Spores were suspended in decoating solution (50 mM Tris base, 8 M urea, 50 mM dithiothreitol, 1% sodium dodecyl sulfate; pH 10.0) at OD600 5.0 and incubated at 60°C for 90 min with vortexing. After the treatment, spores were intensively washed in STE buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA; pH 8.0) as described.

Transmission electron microscopy

Purified spores were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2)

for 1 h at room temperature. Samples were contrasted with 0.5% Oolong tea extract in cacodylate buffer and postfixed with 1% osmium tetroxide containing 1.5% potassium cyanoferrate. The samples were dehydrated and embedded in Epon (Delta Microscopies, France), as described [START_REF] Theodorou | A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis[END_REF]. Thin sections (70 nm) were collected onto 200mesh copper grids and counterstained with lead citrate. Grids were examined with a Hitachi HT7700 electron microscope operated at 80 kV, and images were acquired with a chargecoupled device camera (Advanced Microcopy Techniques; facilities were located on the MIMA2 platform [INRAE, Jouy-en-Josas, France;

https://doi.org/10.15454/1.5572348210007727E12]). The post-acquisition treatment of the images and measurement of the thickness of the spore coat were performed using the Fiji software [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Spore UV resistance assay

The activated spores were suspended in Tris-HCl (pH 8.0) at OD600 0.5, pipetted in triplicates (50µl) in the wells of 48-well sterile microtiter plates (Evergreen Scientific) and irradiated by ultraviolet (254 nm) light using Stratalinker 2400 UV Crosslinker (Stratagene) at the J/m 2 doses indicated in the text (one plate per UV dose). The irradiated spore suspensions were plated in sequential dilutions at LB plates and the colonies were counted after 24 hours of incubation at 37 °C. The UV-resistance was determined as a ratio of the colony-forming units in the irradiated and non-irradiated samples of spores. Five experiments were performed with two independent sets of spores differentially expressing Rho.

To determine the effect of rho inactivation on the UV resistance of vegetative cells, WT and RM cultures in the late exponential phase (~10 8 cells/ml) were plated on LB plates and irradiated with the increasing doses of UV (254 nm) light using a Stratalinker 2400 UV Crosslinker (Stratagene). The UV-resistance of cells was estimated as above. Three independent experiments were performed.

Dipicolinic acid (DPA) assay

The DPA content of spores was analyzed according to the protocol of Nicholson and Setlow (in Harwood and Cutting, 1990).

Spore germination and outgrowth assay

Spore germination and outgrowth assays were performed in MS medium (10.8 g l -1 of K2HPO4, 6 g l -1 of KH2PO4, 1 g l -1 of C6H5Na3O7.2H2O, 0.2 g l -1 of MgSO4.7H2O, and 2 g l -1 of K2SO4) supplemented with 0.5% glucose, 0.01% L-tryptophan, 0.1% glutamate, 0.1 mM of FeCl3 citrate, 0.1 mM of CaCl2, 1 mM of MgSO4 and trace elements (0.001 g l -1 of MnCl2 4H2O, 0.0017 g l -1 of ZnCl2, 0.00043 g l -1 of CuCl2• 2H2O, 0.0006 g l -1 of CoCl2 6H2O and 0.0006 g

Western blotting

The crude cell extracts were prepared using Vibracell 72408 sonicator (Bioblock scientific).

Bradford assay was used to determine total protein concentration in each extract. Equal amounts of total proteins were separated by SDS-PAGE (10% polyacrylamide). After the run, proteins were transferred to Hybond PVDF membrane (GE Healthcare Amersham, Germany), and the transfer quality was evaluated by staining the membrane with Ponceau S (Sigma-Aldrich). The Rho protein was visualized by hybridization with antiserum against B. subtilis Rho (Eurogentec, Belgium; dilution 1:5,000) and the secondary peroxidase-coupled anti-rabbit IgG antibodies A0545 (Sigma-Aldrich; dilution 1:20,000).

  al., 2022; Molodtsov et al., 2023; Murayama et al., 2023; Rashid and Berger, 2023). Transcriptome analyses of rho knockout and conditionally deficient mutants of various bacteria have established the essential role of Rho in controlling pervasive, mainly non-coding and antisense, transcription that originates from cryptic initiation signals or from read-through of transcription terminators (Nicolas et al., 2012, Peters et al., 2012; Mäder et al., 2016; Botella et al, 2017). By controlling transcription genome-wide, Rho directly and indirectly influences various aspects of cellular physiology in bacteria living in diverse habitats (Bidnenko et el., 2017, 2023; Botella et al., 2017; Nagel et al., 2018; Trzilova et al., 2020; Lin et al.; 2021; Krypotou et al., 2023). It is notable that Rho is involved in the regulation of phenomena associated with stationary phase, including stress survival, cell fate determination, antibiotic sensitivity, host colonization and virulence in different bacteria (Lee and Helmann, 2014; Liu et al., 2016; Hafeezunnisa et al., Bidnenko et al., 2017, 2023; Nagel et al., 2018; Trzilova et al. 2020; Lin et al., 2021; Figueroa-Bossi et al., 2022).

;

  Steil et al., 2005; Wang et al., 2006; de Hoon et al., 2010; Overkamp et al., 2015; Meeske et al., 2016; Galperin et al., 2022).

  Rho in RM cells did not alter the kinetics of rho expression, but rather increased its level approximately threefold, suggesting a release from autoregulation (Fig 1A). WT cells grown in the sporulation-promoting Difco Sporulation Medium (DSM) showed a different rho expression kinetics, characterized by the additional peak of luciferase activity in stationary phase (Fig 1B). The reactivation of rho expression coincided in time with the induction of the sporulation-specific spoIIAA-AB-sigF operon, monitored in a separate strain using the PspoIIAA-luc transcriptional fusion (Fig 1B; Bidnenko et al, 2017). The similarity in timing suggested that rho expression during stationary phase in DSM might be linked to sporulation.

  transcription in stationary phase depends on SigH activity (Fig 2C). Notably, none of the larger rho-specific transcripts were detectable in 3TER cells (Fig 2C) confirming that they do indeed originate from upstream of the inserted terminators. All together, these results show an efficient read-through transcription of the rho gene, mainly initiated at the spo0F promoter. Northern analysis also provided information about Prho functioning. The levels of the 1.9 kb rho transcript decreased in stationary WT cells, in line with the down-regulation of the SigAdependent Prho at this stage (Fig 2C; Nicolas et al., 2012), and became barely detectable in the stationary sigH mutant cells (Fig 2C). However, the dramatic decrease in the abundance of this short transcript in the sigH mutant was not due to the inhibition of read-through transcription from Pspo0F promoter, as the amount of the 1.9 kb rho transcript remained high in the stationary 3TER cells (Fig 2C).

  forespores. Because of a strong overlap between the -10 sequences of sigA Prho and putative sigF Prho, we targeted the -35 GAATA sequence of the latter and replaced the invariant T nucleotide[START_REF] Amaya | Analysis of Promoter Recognition In Vivo Directed by ςF of Bacillus subtilis by Using Random-Sequence Oligonucleotides[END_REF] Wang et al., 2006) with A (mF-35T/A Prho-gfp) or C (mF-35T/C Prho-gfp) (Fig4C). Next, we compared the activity of the mutant fusions with the original one (WT Prho-gfp) and the one preceded by the transcriptional terminators (3TER Prho-gfp). Cells were set to sporulate in DSM medium, and the fluorescence levels in predivisional cells and two compartments of sporangia were quantified at the time of a maximal SigF activity. As we observed earlier (Fig3), the sporulating WT Prho-gfp cells showed a higher level of fluorescence in the forespores than in the mother cells; in the latter, gfp expression appeared similar to predivisional cells confirming our hypothesis that rho expression in the mother cell is SigEindependent (Fig 4Dand 4E). A similar pattern of the brighter forespores was observed in 3TER Prho-gfp cells, even though the fluorescence was globally lower probably due to the inhibition of the read-through transcription by the 3Ter insert (Fig 4Dand 4E).In contrast, and as expected, both mF-35T/A and mF-35T/C mutations of Prho-gfp specifically inhibited the burst of fluorescence in the forespores, similarly to sigF mutation (Fig 4Dand 4E, and Fig 3). At present, it is unclear why mF-35T/A Prho-gfp mutant strain displayed a higher basal fluorescence (Fig 4E). Nevertheless, these results clearly indicate that the foresporespecific expression of rho depends on the alternative promoter sigF Prho. This conclusion was further supported by the analysis of the mutated Prho-luc fusion: both point mutations mF-35T/A and mF-35T/C efficiently inhibited the sporulation-specific luciferase activity in 3TER cells (S2 Fig).

  Fig). This indicates that the permeability of spore coat to lysozyme was not detectably affected by altered expression of rho. Likewise, all five types of spores showed similar levels of wetheat resistance, a complex spore phenotype determined by factors expressed in both spore compartments (S4 Fig). At the same time, unlike the others, RM spores contained higher (~35%) levels of dipicolinic acid (DPA) (Fig 5A), an important factor of the heat-resistance

  Henriques and Moran, 2007; Setlow, 2013; Sinai et al, 2015; Ramírez-Guadiana et al., 2017a; Christie and Setlow, 2020).

  Fig). Other spores germinated similarly to WT with all three germinants (Fig 7A and S6 Fig). In contrast to the rapid germination, the outgrowth of RM spores in a defined minimal medium was significantly delayed compared to other spores (Fig 7B). At the same time, the viability of RM spores during germination was not decreased compared to WT spores (S7 Fig) and therefore could not explain the outgrowth delay. As for other spores, the enrichment of the growth medium by casamino acids (0.5%) increased the outgrowth rate of RM spores, which, nevertheless, remained below the WT level (Fig 7C). These results indicate that rho inactivation imposes stricter requirements on the spore outgrowth.

  SigH-dependent promoter of spo0F gene encoding the phosphotransferase Spo0F of the Spo0A phosphorelay. The spo0F expression is regulated by Spo0A~P and constitutes one of the positive feedback loops of Spo0A~P at the onset of sporulation[START_REF] Lewandoski | Transcriptional Regulation of the spoOF Gene of Bacillus subtilis[END_REF][START_REF] Predich | Bacillus subtilis early sporulation genes kinA, spo0F, and spo0A are transcribed by the RNA polymerase containing sigma H[END_REF][START_REF] Strauch | A positive feedback loop controls transcription of the spo0F gene, a component of the sporulation phosphorelay In Bacillus subtilis[END_REF][START_REF] Asayama | Dimer form of phosphorylated Spo0A, a transcriptional regulator, stimulates the spo0F transcription at the initiation of sporulation in Bacillus subtilis[END_REF] Chastanet et al., 2010).Previously, we have established that Rho negatively regulates the expression of some components of the Spo0A phosphorelay, like the sensor kinase KinB[START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF].Therefore, correct modulation of Spo0A~P activity at the initial steps of B. subtilis differentiation requires the decrease in the rho expression levels[START_REF] Bidnenko | Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis[END_REF]. As we show here, this decrease is partially due to the silencing of the SigA-dependent rho promoter by yet unknown mechanism. It appears that alongside a self-reinforcing loop involving Spo0F, Spo0A~P simultaneously activates the Rho-mediated regulatory circuit, which can be viewed as a negative feedback loop of Spo0A~P. This regulatory function of Rho could be particularly important to fine-tune Spo0A~P activity during initiation of sporulation and in the mother cells, where Spo0A~P remains functional after asymmetric division[START_REF] Fujita | The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division[END_REF]. Our data indicate that the mother cell-specific sigma factor SigE is not involved in the expression of rho. Nevertheless, inhibition of the rho read-through transcription results in the structural changes of spore coat, whose assembly depends on the activity of the SigE-and SigK-controlled genes[START_REF] Eichenberger | The σE regulon and the identification of additional sporulation genes in Bacillus subtilis[END_REF][START_REF] Henriques | Structure, assembly, and function of the spore surface layers[END_REF][START_REF] Driks | The Spore Coat. The bacterial spore: from molecules to systems[END_REF].This emphasizes an extended activity of Rho during the mother cell gene expression program.Identification of the alternative forespore-specific rho promoter matching the SigF-binding consensus allows us to classify rho as a member of the SigF regulon. Interestingly, an earlier analysis of forespore gene expression has detected the up-regulation of rho in sporulating sigF + B. subtilis cells (see Supplemental Table1 inWang et al., 2006). However, rho was not induced by the artificial expression of SigF during exponential growth in LB, a second criterion used for gene classification to the SigF regulon(Wang et al., 2006). It is possible that, in that analysis, the untimely activated SigF failed to compete with the abundant vegetative SigA for binding to the overlapping cognate rho promoters. The silencing of the SigA-dependent rho promoter might facilitate SigF binding. However, the mechanism of this silencing is unclear, and despite some discrepancies between the reports, SigA remains active in the forespores[START_REF] Lord | Replacement of vegetative ςA by sporulationspecific ςF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis[END_REF][START_REF] Fujita | Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis[END_REF][START_REF] Li | Development of a two-part transcription probe to determine the completeness of temporal and spatial compartmentalization of gene expression during bacterial development[END_REF][START_REF] Marquis | SpoIIIE strips proteins off the DNA during chromosome translocation[END_REF][START_REF] Riley | Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell-specific requirement of σH and σA[END_REF] 2021). It is also notable that the rho gene is located within a chromosomal cluster of SigF-dependent genes activated the first during the forespore formation[START_REF] Steil | Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis[END_REF] Wang et al., 2006), what pinpoints its early expression. At the same time, our data indicate the role of Rho in the resistance of spores to ultraviolet radiation, the main factors of which, the α/β-type small acidsoluble spore proteins (SASPs) and the spore photoproduct lyase SplB, are controlled by the late forespore-specific sigma factor SigG[START_REF] Mason | Essential role of small, acid-soluble spore proteins in resistance of Bacillus subtilis spores to UV light[END_REF][START_REF] Pedraza-Reyes | Temporal regulation and forespore-specific expression of the spore photoproduct lyase gene by sigma-G RNA polymerase during Bacillus subtilis sporulation[END_REF][START_REF] Setlow | Resistance of spores of Bacillus species to ultraviolet light[END_REF]. Therefore, like the read-through transcription in the mother cells, the SigFmediated rho expression appears to ensure Rho activity during the entire forespore-specific gene expression program. It can also mediate the packaging of Rho protein in mature spores, where it has been recurrently identified by mass spectrometric analysis[START_REF] Swarge | Integrative Analysis of Proteome and Transcriptome Dynamics during Bacillus subtilis Spore Revival[END_REF] Tu et al, 2020).Rho involvement in the spatiotemporal regulation of spore development.Two spore phenotypes, a decreased UV-resistance and a structurally altered outer coat, resulting from the complete inactivation of rho, or following the inhibition of rho expression in the forespores and in the mother cells, respectively, highlight the importance of the compartmentalized Rho-mediated transcription termination for spore development. Our previous transcriptome analysis of RM cells in stationary phase revealed disordered and untimely expression of many sporulation genes via their extensive sense or antisense readthrough transcription or, more rarely, unscheduled activation of promoters[START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF]. The relevant gene expression changes are compiled in S1 Table and can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/[START_REF] Bidnenko | Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase[END_REF][START_REF] Dérozier | Genoscapist: online exploration of quantitative profiles along genomes via interactively customized graphical representations[END_REF]. We posit that at least some of the sporulation genes abnormally expressed in the stationary RM cells are subject to Rho-mediated regulation during sporulation. At the same time, we are aware that most of potential sporulation-specific targets of Rho are not yet expressed in stationary phase.

  The existing transcriptome data probably provide the most straightforward mechanistic hints on the involvement of Rho in the regulation of spore coat morphogenesis. The prominent coat defects in RM and 3TER-mT/A spores resemble those induced by the inactivation of morphogenetic proteins CotO, CotH or CotG, or auxiliary proteins of coat assembly, superoxide dismutase SodA and holin YwcE (McPherson et al., 2005; Zilhão et al., 1999; Isticato et al., 2020; Freitas et al., 2020; Henriques et al., 1998; Real et al., 2005; Henriques and Moran, 2007). All of the abovementioned mutant spores exhibit a disordered, low electron-dense and thinner outer coat frequently detached from the inner layer. The protein kinase CotH and its target protein CotG were recently identified as the main morphogenetic factors determining the regular structure of spore coat (Nguyen et al, 2016; Freitas et al., 2020; Isticato et al., 2020; Di Gregorio Barletta et al., 2022). It has been shown that phosphorylated CotG assembles in the coat, while its non-phosphorylated form sequesters other coat proteins in the mother cell cytoplasm (Di Gregorio Barletta et al., 2022). Interestingly, the expression levels of cotG are strongly upregulated in RM cells, due to the transcription upshift at, or close to, the SigK-dependent cotG promoter and the read-through transcription initiated at the upstream alsR gene (Nicolas et al., 2012; Bidnenko et al., 2023; S1 Table). The same read-through transcript appears antisense to the oppositely oriented neighboring genes cotB and cotH. We suggest that Rho participates in the control of the transcription of the cotB-cotH-cotG gene cluster during sporulation and that inactivation of the rho expression in the mother cells modifies the CotG-CotH protein balance important for correct assembly of CotG, thus leading to spore coat defects. Work is in progress to test this hypothesis. Alongside the shared phenotypes linked to the inhibition of spatiotemporal expression of rho, RM spores are specifically characterized by the increased levels of DPA content. The synthesis of DPA and its transfer to the core across the spore membranes involves the protein products of both mother cell-and forespore-specific genes (Daniel and Errington, 1993; Ramírez-Guadiana et al., 2017b; Gao et al., 2022). None of the genes involved in the DPA metabolism are subject to unscheduled activation in RM cells in stationary phase. However, we detected strong induction of a defined SigA-dependent antisense promoter located within the SigGregulated spoVAA-spoVAB-spoVAC-spoVAD-spoVAEB-spoVAEA-spoVAF-lysA operon, whose products are essential for DPA uptake into the core and release during germination (Tovar-Rojo et al., 2002; Gao et al., 2022; Nicolas et al., 2012; Bidnenko et al, 2023). In the wild type B. subtilis cells, this antisense promoter is weakly activated under some stress conditions and late in sporulation (Nicolas et al., 2012). Remarkably, in RM cells, the generated ~5 kb-long antisense transcript extends across spoVAA, spoVAB, spoVAC genes and the dacF-spoIIAA-spoIIAB-sigF operon. It remains to be established whether this antisense transcript affects the expression of the genes on the opposite strand during RM sporulation. Rho involvement in spore revival. RM spores also differ from their siblings by their impaired revival capacity, showing a rapid germination followed by slow outgrowth. The rate of germination in response to different nutrient stimuli is mainly determined by the levels of corresponding germinant receptors in the spore membrane (Cabrera-Martinez et al., 2003; Ghosh et al., 2012; Chen et al., 2014). The RM stationary phase transcriptome showed unscheduled up-regulation of the SigG-controlledgerBA-gerBB-gerBC operon encoding the AGFK germinant receptor GerB[START_REF] Corfe | The gerB region of the Bacillus subtilis 168 chromosome encodes a homologue of the gerA spore germination operon[END_REF]

  All intermediate plasmids were constructed using Q5 High Fidelity DNA Polymerase and DNA modification enzymes purchased from New England Biolabs. Transcriptional fusion of the luciferase gene luc and the rho promoter (Prho-luc) was constructed as follows. The oligonucleotide pairs glpXBam/veb738 were used to amplify a ~1 Kb fragment of B. subtilis chromosome located directly upstream the rho start codon. The 5' part of the luc gene was amplified from the plasmid pUC18cm-Luc (Mirouze et al., 2012) using oligonucleotides lucintrev and veb739 complementary to veb738. Two DNA fragments were joined by PCR using the primers glpXBam and lucintrev, and the resulting DNA fragment was cloned at pUC18cm-Luc using BamHI and BstBI endonucleases and T4 DNA ligase. The resulting plasmid pBRL862 was integrated by single crossover at the rho chromosomal locus of BSB1

  Sigma F-dependent rho promoter of the Prho-luc and Prho-gfp transcriptional fusions was performed as follows. The plasmids pBRL893 and pBRL1107 were entirely amplified using the pairs of side-by-side oligonucleotides veb803/veb802, containing a 5'-terminal C nucleotide to introduce the point mutation mF-35T/C, and veb803/veb805, with a 5'-terminal A for the point mutation mF-35T/A. The PCR products were phosphorylated using T4 polynucleotide kinase, self-ligated and treated with DpnI endonuclease to remove the template DNA prior transformation in E. coli cells. The resulting mutated derivatives of the plasmids pBRL893 (pBRL1141 and pBRL1164) and pBRL1107 (pBRL1116 and pBRL1162) were controlled by sequencing and inserted into the B. subtilis BSB1 chromosome as above to produce the mutated fusions mF-35T/C Prho-gfp and mF-35T/A Prho-gfp and 3TER mF-35T/C Prho-luc and 3TER mF-35T/A Prho-luc, respectively.

  MetaMorph software package (v 7.8; Molecular Devices, LLC). The post-acquisition treatment of the images was done with the Fiji software (van Ooij et al., 2004; Schindelin et al., 2012).

  modified or the point-mutated Prho-gfp transcriptional fusions were induced for sporulation by the resuspension method and sampled as above. Sampled cells were mixed with the lipophilic fluorescent dyes Nile Red (10 µg/ml final concentration) prior to microscopic observation. To measure the fluorescence intensity in the different compartments, circular areas of a constant 0.45 µm diameter were drawn in the center of individual compartments (forespores, mother cells, or predivisional cells) using images showing membrane-labelled cells, and recorded as a list of ROI. ROIs were subsequently applied over the corresponding image displaying the GFP signal, and the average fluorescence over each ROI recorded. Background fluorescence, determined as the average fluorescence from ROIs of identical areas spread over the field (outside cells), was subtracted to give the final fluorescence intensity of individual compartments. Counting was performed in at least two fields of view and for a minimum of 800 cell compartments per strain, in two independent experiments. Plotted values are average fluorescence intensities from pooled replicas, thus standard deviations account for cell to cell variability across all fields and replicas.

l - 1

 1 of Na2MoO4• 2H2O). The stock solutions (100mM) of L-alanine and L-valine germinants were prepared in 10 mM Tris-HCl buffer (pH 8.0) containing 10mM D-glucose and 100mMKCl. The stock solution of AGFK germinant mixture contained 100 mM L-asparagine, 10mM D-glucose, 10 mM D-fructose and 100mM KCl in 10 mM Tris-HCl buffer (pH 8.0). The activated spores were diluted to OD600 0.2 in the cold MS medium and distributed by 135 µl in the 96-well plate. Spore suspensions were simultaneously induced for germination by adding 15µl of a stock germinant solution, incubated under strong agitation at 37°C and monitored for OD600 at 2 min intervals in Synergy 2 Multi-mode microplate reader (BioTek Instruments). It usually took ~1 min between germinant addition and the first OD600 reading. When needed, the stock solution of the germinant L-alanine was supplemented with 1mM IPTG or 5% casamino acids to get their final concentrations in spore suspensions 100µM and 0.5%, respectively. Three independently prepared sets of spores were used in the analysis. The assays were performed with each set of spores at least twice and included up to six replicas of each spore suspension in the same plate.Purification of the B. subtilis Rho protein for antibody preparationE. coli BL21-CodonPlus (DE3)-RIL cells containing pETRho were grown to an OD600 of 0.2 in a 400 ml culture (2YT medium) at 16°C, and Rho expression was induced by the addition of 0.5 mM IPTG with continued growth overnight. The culture was harvested, pelleted and frozen at -80°C until further use. The frozen cells were resuspended in 10 ml of lysis buffer at 4°C containing 20 mM Tris-HCl pH 9.0, 100 mM Na2HPO4, 0.3 M NaCl, 10% glycerol and 0.1% Triton X-100, to which were added 10 mg/ml DNase I and an EDTA-free anti-protease tablet (Roche). The suspension was passed twice through a French press (20 000 p.s.i.) and the lysate centrifuged for 30 min at 15 000 g. Imidazole-HCl (pH 8.0) was added to the supernatant to give a final concentration of 1 mM and the resultant suspension was applied to a 1 ml Ni 2+ -NTA column (Qiagen). The resin was then washed sequentially with 10 ml of lysis buffer (without Triton X-100), 10 ml of buffer containing 20 mM Tris-HCl pH 9.0, 0.3 M NaCl, 20 mM Imidazole and, finally, 10 ml 20 mM Tris-HCl pH 9.0, 0.3 M NaCl and 250 mM Imidazole. B. subtilis Rho was eluted with 20 mM Tris-HCl pH 9.0, 100 mM Na2HPO4, 0.3 M NaCl and 500 mM Imidazole as 1ml fractions into collection tubes pre-filled with 3 ml elution buffer lacking Imidazole, to immediately dilute the sample 4-fold and avoid precipitation. The protein peak was determined initially by measuring the protein concentration (Bio-Rad) in the different fractions. Peak fractions were pooled and dialyzed over-night in buffer containing 20 mM Tris-HCl pH 9.0, 100 mM Na2HPO4, 0.3 M NaCl, 10% glycerol. Protein purity was verified by SDS-PAGE analysis and estimated at >95%. A sample of purified B. subtilis Rho protein at 1.1 mg/ml in storage buffer was filtered through 5µm, 0.5µm and then 0.2µm Acrodisk filters before rabbit immunization to generate custom anti-Rho polyclonal antibodies through the commercial '87-day anti-antigen classical' program of Eurogentec (Belgium). The low solubility of B. subtilis Rho in buffers suitable for immobilization on affinity chromatography columns prevented further purification of the anti-Rho antibodies from the crude sera. The best anti-Rho serum aliquot was selected from the immunization program aliquots by Western blotting with purified B. subtilis Rho and B. subtilis cell extract samples.

Fig 1 .

 1 Fig 1. Rho is specifically expressed during B. subtilis sporulation. (A) Rho expression in vegetative cells is limited to the exponential growth phase and autoregulated. Kinetics of luciferase activity in B. subtilis WT Prho-luc (empty circles) and RM Prho-luc (filled-in circles) cells grown in rich medium LB. In this and other panels, the plain dotted and solid lines represent growth curves of the Rho-proficient strains and rho-deletion mutant (RM), respectively, measured by optical density OD600. (B) In the sporulation-inducing

Fig 2 .Fig 3 .

 23 Fig 2. Role of the read-through transcription in the rho expression during sporulation. (A) Schematic representation of gene organization and transcription within the rho locus of B. subtilis chromosome. Arrow-shaped and flat rectangles indicate protein-coding genes and noncoding RNA S-segments (Nicolas et al., 2012), respectively. Curved arrow-ended lines represent promoters; their regulatory sigma factors are indicated. Straight dot-ended lines represent intrinsic terminators. The underlying lines indicate the transcripts of the rho gene, which were identified in (Nicolas et al., 2012); the transcript initiated at the known rho promoter is bolded. The approximate size of the transcripts is shown. Small black rectangle schematizes the 5' rho-specific riboprobe used in the Northern blot. (B) Cartoon of the insertion of three intrinsic terminators at the end of glpX gene in the 3TER strain. (C) Northern blot analysis of the rho-specific transcripts in the wild type (WT), and the mutant sigH and 3TERstrains during sporulation. Cells were grown in DSM and sampled during exponential growth (OD600 0.5), the transition to stationary phase (OD600 1.0) and in stationary phase (OD600 1.8).

Fig 4 .Fig 5 .

 45 Fig 4. Forespore-specific expression of rho depends on the activity of a genuine SigFdependent promoter.

Fig 6 .

 6 Fig 6. The alteration of the spatiotemporal expression of rho affects the morphology of spores. Thin section transmission electron micrographs of spores produced by cells differentially expressing rho. White arrows indicate a thinner and less electron-dense outer coat in RM, 3TER and 3TER-mT/A spores, which did not express rho in the forespores during maturation. Black arrows indicate the detachment of outer and inner coats in RM, WT-mT/A and 3TER-mT/A which did not express rho in the mother cells.

Fig 7 .*

 7 Fig 7. Rho activity determines the revival properties of spores.

  Fig 1

  al, 1990; Amon et al., 2022, and references therein), only gerAA appears upregulated ~3-fold in the stationary RM cells. that, in the wild type background, Rho determines the fitness of reviving spores.Using a controlled rho expression system, we showed that the normal rate of spore outgrowth requires rho expression both during spore formation and after spore germination. During

	sporulation, Rho would ensure a proper composition of a spore molecular cargo required for
	spore revival, which also includes Rho protein itself (Mutlu et al, 2018; Rao et al., 2022; Swarge
	et al., 2020). After germination, Rho would be required in the ripening period, to control a
	proper termination of the actively resuming transcription, and later, during implementation of
	the transcription program of vegetative growth. This is consistent with the differential gene
	expression analysis of the reviving B. subtilis spores, which detected the upshifts in rho
	More generally, spore germination is a complex highly heterogeneous process influenced by
	multiple factors, including, among others, the metabolic activity of cells during sporulation and
	the duration of spore formation (Segev et al. 2012; Bressuire-Isoard et al., 2018; Mutlu et al,
	2018, 2020; Riley et al., 2021; Rao et al., 2022). From this viewpoint, a more synchronous and
	rapid sporulation in RM population (Bidnenko et al., 2017, and this study) might result in a
	more homogeneous molecular composition of spores and, consequently, in a decreased
	heterogeneity of spore germination.
	At the same time, the absence of Rho negatively affects the outgrowth of the germinated RM

Interestingly, Rudner and colleagues identified rho among the genes, whose inactivation by Mariner transposon results in a premature germination of the developing spores triggered by the GerA receptor, probably in response to some undefined defects of spore envelope

[START_REF] Meeske | High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis[END_REF] Ramírez-Guadiana et al., 2017)

. The authors report that GerA activity results in a decreased sporulation efficiency of the rho::Mariner mutant, which is opposite to our recurrent observation of a highly efficient sporulation in the rho-deleted RM cells. Nevertheless, the established genetic interaction between Rho and GerA proteins may be relevant to the germination phenotype of the mature RM spores, whose coat is severely altered. Moreover, the structure of RM spore coat suggests a lower level of coat protein cross-linking

[START_REF] Henriques | Structure, assembly, and function of the spore surface layers[END_REF]

, which can facilitate spore germination, as previously observed

[START_REF] Abhyankar | Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation[END_REF]

. The germination rate of RM spores might be also influenced by high levels of DPA, a potent non-nutrient germinant that stimulates cortex hydrolysis by CwlJ hydrolase both directly and during nutrient-induced germination

[START_REF] Paidhungat | Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis[END_REF][START_REF] Paidhungat | Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate[END_REF]

. spores even in a nutrient-enriched medium. Therefore, rho inactivation imposes more stringent requirements for spore outgrowth, which in itself is more exacting than vegetative growth (O'

Brien and Campbell, 1957)

. Taken together, a rapid germination and a slow outgrowth of RM spores suggest expression shortly after germination and at the end of outgrowth

[START_REF] Swarge | Integrative Analysis of Proteome and Transcriptome Dynamics during Bacillus subtilis Spore Revival[END_REF]

. Most probably, the resumption of rho expression during outgrowth reflects the reactivation of SigAdependent rho promoter.

  Table. The Escherichia coli TG1 strain was used for construction of intermediate plasmids. Cells were routinely grown in liquid or solidified rich LB medium at by the resuspension method (Harwood and Cutting, 1990), as detailed in (Bidnenko et al., 2023). Optical density of the bacterial cultures was measured with NovaspecII Visible

37°C. Standard protocols were used for transformation of E. coli and B. subtilis competent cells (Harwood and Cutting, 1990). Sporulation of B. subtilis cells was induced by the method of nutrient exhaustion in supplemented Difco sporulation medium (DSM; Difco) (Schaeffer et al., 1965) or

sigE::erm (spoIIGBΔ::erm) Kenney and Moran, 1987 MO1780 sigE::tet (spoIIGABΔ::tet) Guérout-Fleury et al., 1996 BRL878 BSB1 sigE::erm Prho-luc (Em R Cm R ) This study BRL879 BSB1 sigF::km Prho-luc (Km R Cm R ) This study BRL894 BSB1 Prho-gfp (Sp R ) This study BRL956 BSB1 sigE::tet Prho-gfp (Tet R Sp R ) This study BRL957 BSB1 sigF::km Prho-gfp (Km R Sp R ) This study BRL1111 3TER Prho-luc (Cm R ) This study BRL1119 3TER -35mT/C Prho-luc (Cm R ) This study BRL1165 3TER -35mT/A Prho-luc (Cm R ) This study BRL1159 3TER Prho-gfp (Sp R ) This study BRL1144 BSB1 -35mT/C Prho-gfp (Sp R ) This study BRL1172 BSB1 -35mT/A Prho-gfp (Sp R ) This study BRL1175 3TER spo0A::tet Prho-luc (Tet R Cm R ) This study BRL1176 3TER sigH::km Prho-luc (Km R Cm R ) This study BRL1177 3TER sigE::tet Prho-luc (Tet R Cm R ) This study BRL1178 3TER sigF::km Prho-luc (Km R Cm R ) This study BRL1130 3TER This study BRL1185 BSB1 -35mT/A This study BRL1194 3TER -35mT/A This study BRL1300 BSB1 rhoΩPspac-rho (Em R ) This study Sp R Amp R ; used to construct BRL894 (Prho-gfp) This study pBRL1107 Cm R , Amp R ; used to construct BRL1111 (3TER Prho-luc) This study pBRL1150 Sp R Amp R ; used to construct BRL1159 (3TER Prho-gfp) This study pBRL1116 Cm R , Amp R ; used to construct BRL1119 (3TER -35mT/C Prho-luc) This study pBRL1162 Cm R , Amp R ; used to construct BRL1165 (3TER -35mT/A Prho-luc) This study pBRL1141 Sp R Amp R ; used to construct BRL1144 (-35mT/C Prho-gfp) This study pBRL1164 Sp R Amp R ; used to construct BRL1172 (-35mT/A Prho-gfp) This study pBRL1108 Em R , Amp R ; used to construct BRL1130 (3TER) This study pBRL1174 Em R , Amp R ; used to construct BRL1185 (BSB1 -35mT/A) This study pBRL1180 Em R , Amp R ; used to construct BRL1194 This study

Spores produced by WT (blue triangles), RM (red squares), 3TER (brown crosses), WT-mT/A (orange circles) and 3TER-mT/A (violet diamonds) cells were induced by the germinant Lalanine (10mM) and analyzed for germination (A) and outgrowth in the nutrient-poor MS medium (B) as described in Materials and Methods. (C) WT (filled-in blue triangles) and RM (filled-in red squares) spores were compared for outgrowth in the nutrient-replenished MS medium containing 0.5% casamino acids. The experiments were performed at least twice with three independent sets of spores. Each experiment included up to six replicas of individual suspensions of spores. The results of the representative experiment are plotted. The bars represent standard deviation from the mean values. The mutational analysis of the SigF-dependent promoter of rho using luciferase was performed in the 3TER genetic background to limit the interference of the read-through transcription. The purified spores of the wild type strain (WT), rho-deletion mutant (RM) and strains altered for rho expression in the mother cell (3TER) or in the forespore (WT-mT/A) or in the two compartments of sporangia (3TER-mT/A) were analyzed for the resistance to lysozyme (A and B) and heat (C). (A) Spores were activated as described in Materials and Methods, suspended in TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0) containing 10 mg/ml lysozyme (Fluka) at OD600 0.2 and incubated at 37°C for 1 hour. (B) Spores were chemically treated to remove spore coats as described in Materials and Methods. Decoated spores were suspended in 10mM Tris-HCl (pH 8.0) at OD600 0.2 and treated with lysozyme (10 µg/ml, final concentration) for 10 and 20 minutes at 37°C. In (A) and (B), spore viability after lysozyme treatment was established by plating spores from the treated and untreated samples at LB agar plates. (C) The activated spores were heated at 90°C for the indicated time, platted at LB agar plates and incubated at 37°C for 24 hours. The heat resistance of spores was calculated as a proportion of viable colony-forming spores in heated and unheated samples. The same set of spores was used in all experiments; spore samples in (B) originate from two independent decoating procedures.

The experiments were reproduced four times, and the established average values are presented with the standard deviations of the mean (bars).

S5 Fig. Rho inactivation does not affect UV-resistance of vegetative cells.

The wild type (WT, blue line) and ∆rho (RM, red line) cells were grown in LB to OD600 0.

S1 Table. Differential expression analysis of genes from the "Sporulation" lifestyle category in stationary ∆rho vs. stationary WT B. subtilis cells (compiled from S3