Lysate-based cell-free systems (CFS) mimic cellular functions in a controllable environment, making them versatile synthetic biology tools used from fundamental research to protein production and synthetic circuit prototyping. Here, we establish a streamlined and robust Bacillus subtilis CFS by systematically re-evaluating the traditional extract preparation and reaction environment. We show that the conventionally required runoff incubation and labor-intensive dialysis steps can be omitted without compromising performance. A key advance is the reformulation of the reaction buffer: increasing ionic strength with potassium glutamate concentration to 400 mM and adding the osmoprotectant betaine significantly enhanced translational capacity, yielding 2-3 μM sfGFP. Benchmarking against established in vivo promoter library data sets revealed that the relative hierarchy of promoter strengths is preserved, validating the platform as a predictive proxy for bacterial physiology. We further reconstituted complex genetic regulations by demonstrating functional repression of a cre-box-containing promoter by the catabolite repressor CcpA in complex with HPr, activated through a phosphorylation cascade within the CFS. Finally, we show that this high-glutamate framework is beneficial for another Gram-positive bacterium, Lactobacillus gasseri, suggesting a broad-host compatibility. Together, the simplified preparation and optimized buffer composition provide a versatile toolkit for developing CFS in diverse, nonmodel bacterial species.

Abstract Understanding genome regulation is limited by the complexity of molecular interactions in living cells. Cell-free systems provide a simplified platform for studying gene expression, but low mRNA levels have prevented RNA-seq. To address this, we develop an active learning workflow combining Bayesian optimization with automated high-throughput experimentation to systematically explore over 1.6 million buffer compositions, experimentally testing 653. We identify a “mRNA-optimized” buffer (20-fold increase in mRNA yield) and a “trade-off” buffer (13-fold increase while maintaining protein production). Using direct RNA-seq, we profile the T7 phage transcriptome in cell-free systems and compare it with a purified T7-RNAP transcription system and phage-infected bacteria. This comparative analysis reveals distinct regulatory layers: the T7-RNAP system captures promoter-strength hierarchies but lacks RNA degradation, whereas cell-free systems provide an accurate estimation of in vivo expression and reveal mRNA maturation sites. This work establishes cell-free transcriptomics as a controlled approach to study genome regulation.

Phage genome engineering methods accelerate the study of phage biology, the discovery of new functions, and the development of innovative genetic engineering tools. Here, we present QuickPhage, a rapid, technically accessible, precise, and cost-effective method for engineering Bacillus subtilis phages. Our approach uses CRISPR-Cas9 as a counter-selection system to isolate mutants of the model lytic siphovirus phage, SPP1. Efficient genome editing was achieved using homologous repair patches as short as 40 nucleotides, enabling streamlined patch construction and parallel engineering, resulting in highly accurate genome edits within a day. We applied QuickPhage to delete both essential and nonessential phage genes and to insert reporter genes. Protein production, such as GFP, was synthetically regulated using inducible systems without significantly affecting phage fitness, achieving induction levels of up to 400-fold. Time-series coinfection experiments with fluorescent protein expressing phages also revealed a highly efficient superinfection arrest mechanism that prevents reinfection as early as 13 min after initial infection. These findings highlight the potential of phages for protein production, opening new opportunities for metabolic engineering. This work also lays the foundation for systematic phage genome refactoring workflows and the development of phage-based tools for efficient DNA delivery, thereby expanding the synthetic biology toolbox for B. subtilis.

Recent evidence indicates that the bacterial Rho helicase regulates Bacillus subtilis gene expression in a growth-dependent manner. This regulation, along with extensive in vivo trimming of Rho-dependent transcript 3'-ends, complicates the identification of Rho-dependent transcription terminators using standard transcriptomic approaches. To overcome this challenge, we applied Helicase-SELEX to precisely map Rho utilization (Rut) sites genome-wide. Using B. subtilis Rho (BsRho), we identified 600 putative Rut sites, while the more permissive Escherichia coli Rho (EcRho) revealed 4189 sites, including specimens known to regulate B. subtilis genes. Comparative analysis showed that both enzymes recognize similar pyrimidine-rich sequences, though BsRho favors short unstructured Rut motifs whereas EcRho can act on presumably more structured RNAs without requiring accessory factors. In vivo validation of selected Rut sites confirmed Rho-dependent regulation and extensive PNPase-mediated processing of Rho-terminated transcripts. Collectively, our results reveal a rich and complex Rho-dependent regulatory network in B. subtilis, encompassing the widespread control of antisense transcription and genes/operons of both primary and secondary metabolism. Although nonessential under standard laboratory conditions, Rho thus likely contributes to B. subtilis fitness and survival in more demanding environments. Our comprehensive compendium of Rut sites offers a valuable resource for exploring this adaptive regulatory landscape.

Probiotics are widely used for their health promoting effects, though a lot remain to be discovered, particularly on their mechanisms of action at the molecular level. The functional genomic approach is an appropriate method to decipher how probiotics may influence human cell fate and therefore contribute to their health benefit. In the present work, we focused on Shouchella clausii (formerly named Bacillus then Alkalihalobacillus clausii ), a spore-forming bacterium that is commercially available as a probiotic for the prevention and the treatment of intestinal dysbiosis and related gastrointestinal disorders, such as diarrhoea. Several studies have demonstrated that S. clausii treatment modulated inflammatory and immune responses, as well as gut barrier functions. A functional genomic strategy was implemented to decipher the mechanisms by which S. clausii exerts its probiotic effects on human intestinal epithelial cells. To do so, a large genomic DNA fragment library was constructed for each of the four strains: O/C, N/R, SIN and T. A high throughput in vitro screening in human epithelial cells was then conducted, using the reporter gene strategy, targeting the nuclear factor kappa B (NF-κB) pathway and interleukin-10 (IL-10) gene expression. After an exhaustive in vitro screening of approximately a thousand clones per library, several clones modulating the NF-κB pathway in the HT-29 reporter cell line were identified. Among clone lysates, 1.1% (O/C), 1.4% (N/R), 2.0% (SIN), and 1.2% (T) were identified as biologically active on immune reporter systems (NF-κB and IL-10 expression). After transposon mutagenesis and a new set of screening and sequencing, 23 coding sequences (CDS) were identified, including one encoding for the glutamine synthetase, associated with NF-κB modulation, and six CDS for IL-10 modulation. The functional genomic strategy that was applied to S. clausii was an original approach to identify gene candidates that may explain the mechanisms of action of probiotics. However, further work is needed to validate the identified leads.

With the advent of MAD7, a Cpf1-like nuclease, there has been a renewed focus on the development of CRISPR-based genome engineering tools in recent years. To improve genome engineering methodologies in B. subtilis, we revisited the potential of MAD7 for gene modification and expression interference. A key challenge in these endeavors is the limited transformation efficiency. To overcome this, we developed an efficient transformation protocol using strains overexpressing competence genes. Our results showed that although MAD7 together with a B. subtilis chromosome-targeting gRNA is lethal, enabling robust counterselection, we successfully engineered a strain carrying the MAD7-gRNA machinery in a reversibly inactivated state, marking a significant advance in the field. We demonstrated that both MAD7 and its catalytically inactive variant (dMAD7) can be conditionally regulated by inactivation at elevated temperatures. In addition, the MAD7-gRNA complex is effective for multiplex genome editing, allowing for the simultaneous deletion, mutation, or insertion of up to four loci, and enabling the combination of gene deletion, gene insertion, and point mutations. Furthermore, we established a strategy that achieves the simultaneous removal of MAD7 and the gRNA along with the desired genome edits. Altogether, this comprehensive study underscores the versatility of MAD7 for complex, scarless genome engineering and lays a strong foundation for further advancing genetic manipulation in B. subtilis.

Despite advances in transcriptomics, understanding of genome regulation remains limited by the complex interactions within living cells. To address this, we performed cell-free transcriptomics by developing a platform using an active learning workflow to explore over 1,000,000 buffer conditions. This enabled us to identify a buffer that increased mRNA yield by 20-fold, enabling cell-free transcriptomics. By employing increasingly complex conditions, our approach untangles the regulatory layers controlling genome expression. ### Competing Interest Statement The authors have declared no competing interest. ANR program, ANR-24-CE44-4467, ANR-11-IDEX-0003, ANR-22-PEBB-0008 UE HORIZON BIOS program, 101070281

In continuous culture, a population of microorganisms is propagated in a stable environment over many generations. This is particularly relevant for experimental evolution and metabolic studies. However, continuous culture protocols are difficult to implement, so they are not commonly used in microbiology laboratories. Here, we present the ModuloStat, a modular, open-source framework that facilitates continuous culture in mini-bioreactors. The ModuloStat system is grounded on digital fabrication tools easily accessible in FabLabs and programmable electronics. Maintaining a culture is divided into tasks assigned to dedicated printed circuit boards with a microcontroller connected to a Wi-Fi network. According to Internet of Things principles, each board operates a set of sensors and actuators autonomously and can receive and send information. The boards are stacked to implement complex behaviors and can be modified to accommodate new features. A thermoregulated box holds the components and can be placed on a laboratory bench or transported under a sterile hood for inoculation. Sterility is ensured by autoclaving, after assembly, all components that will come into contact with the culture medium. In-situ optical density monitoring combined with modularity and computer control enables many cultivation modes. Additionally, we present the construction of the Bacillus subtilis strain ZB designed for bioreactor culture that exhibits a zero-biofilm phenotype. To demonstrate the system's versatility, we performed several experimental cultures with this model organism, including chemostat, turbidostat, medium swap, and a cascade of bioreactors.

Polymerase errors during DNA replication are a major source of point mutations in genomes. The spontaneous mutation rate also depends on the counteracting activity of DNA repair mechanisms, with mutator phenotypes appearing constantly and allowing for periods of rapid evolution in nature and in the laboratory. Here, we use the Gram-positive model bacterium Bacillus subtilis to disentangle the contributions of DNA polymerase initial nucleotide selectivity, DNA polymerase proofreading, and mismatch repair (MMR) to the mutation rate. To achieve this, we constructed several conditional hypermutators with a proofreading-deficient allele of polC and/or a deficient allele of mutL and performed mutation accumulation experiments. These conditional hypermutators enrich the B. subtilis synthetic biology toolbox for directed evolution. Using mathematical models, we investigated how to interpret the apparent probabilities with which errors escape MMR and proofreading, highlighting the difficulties of working with counts that aggregate potentially heterogeneous mutations and with unknowns about the pathways leading to mutations in the wild-type. Aware of these difficulties, the analysis shows that proofreading prevents partial saturation of the MMR in B. subtilis and that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.

Termination factor Rho, responsible for the main factor- dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to the formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis, de-repression of pervasive transcription by inactivation of rho revealed the role of Rho in the regulation of post-exponential differentiation programs. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain (Rho+) that expresses rho at a relatively stable high level in order to compensate for its decrease in the wild-type cells entering stationary phase. The RNAseq analysis of Rho+, WT and Δrho strains (expression profiles can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/) shows that Rho over-production enhances the termination efficiency of Rho-sensitive terminators, thus reducing transcriptional read-through and antisense transcription genome-wide. Moreover, the Rho+ strain exhibits global alterations of sense transcription with the most significant changes observed for the AbrB, CodY, and stringent response regulons, forming the pathways governing the transition to stationary phase. Subsequent physiological analyses demonstrated that maintaining rho expression at a stable elevated level modifies stationary phase-specific physiology of B. subtilis cells, weakens stringent response, and thereby negatively affects the cellular adaptation to nutrient limitations and other stresses, and blocks the development of genetic competence and sporulation. These results highlight the Rho-specific termination of transcription as a novel element controlling stationary phase. The release of this control by decreasing Rho levels during the transition to stationary phase appears crucial for the functionality of complex gene networks ensuring B. subtilis survival in stationary phase.

Because they mimic cells while offering an accessible and controllable environment, lysate-based cell-free systems (CFS) have emerged as valuable biotechnology tools for synthetic biology. Historically used to uncover fundamental mechanisms of life, CFS are nowadays used for a multitude of purposes, including protein production and prototyping of synthetic circuits. Despite the conservation of fundamental functions in CFS like transcription and translation, RNAs and certain membrane-embedded or membrane-bound proteins of the host cell are lost when preparing the lysate. As a result, CFS largely lack some essential properties of living cells, such as the ability to adapt to changing conditions, to maintain homeostasis and spatial organization. Regardless of the application, shedding light on the black-box of the bacterial lysate is necessary to fully exploit the potential of CFS. Most measurements of the activity of synthetic circuits in CFS and in vivo show significant correlations because these only require processes that are preserved in CFS, like transcription and translation. However, prototyping circuits of higher complexity that require functions that are lost in CFS (cell adaptation, homeostasis, spatial organization) will not show such a good correlation with in vivo conditions. Both for prototyping circuits of higher complexity and for building artificial cells, the cell-free community has developed devices to reconstruct cellular functions. This mini-review compares bacterial CFS to living cells, focusing on functional and cellular process differences and the latest developments in restoring lost functions through complementation of the lysate or device engineering.

AbstractReprogramming of gene expression during transition from exponential growth to stationary phase is crucial for bacterial survival. In the model Gram-positive bacteriumBacillus subtilis, this process is mainly governed by the activity of the global transcription regulators AbrB, CodY and Spo0A. We recently showed that the transcription termination factor Rho, known for its ubiquitous role in the inhibition of antisense transcription, is involved in Spo0A-mediated regulation of differentiation programs specific to the stationary phase inB. subtilis. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed aB. subtilisstrain that expressesrhoat a relatively stable high level in order to circumvent its decrease occurring in the wild-type cells entering the stationary phase. We show thatB. subtiliscells stably expressing Rho fail to sporulate and to develop genetic competence, which is largely, but not exclusively, due to abnormally low expression of the master regulator Spo0A. Moreover, in addition to a global decrease of antisense transcription, these cells exhibit genome-wide alterations of sense transcription. A significant part of these alterations affects genes from global regulatory networks of cellular adaptation to the stationary phase and reflects the attenuated de-repression of the AbrB and CodY regulons and the weakened stringent response. Accordingly, stabilization of Rho level reprograms stationary phase-specific physiology ofB. subtiliscells, negatively affects cellular adaptation to nutrient limitations and alters cell-fate decision-making to such an extent that it blocks development of genetic competence and sporulation. Taken together, these results indicate that the activity of termination factor Rho constitutes a previously unknown layer of control over the stationary phase and post-exponential adaptive strategies inB. subtilis, from the adjustment of cellular metabolism to the activation of survival programs.

Multispecies microbial adherent communities are widespread in nature and organisms, although the principles of their assembly and development remain unclear. Here, we test the possibility of establishing a simplified but relevant model of multispecies biofilm in a non-invasive laboratory setup for the real-time monitoring of community development. We demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians, and Rhodocyclus sp.) form a dynamic community that deterministically reaches its equilibrium after ~30 h of growth. We reveal the emergence of complexity in this simplified community as reported by an increase in spatial heterogeneity and non-monotonic developmental kinetics. Importantly, we find interspecies interactions consisting of competition for resources-particularly oxygen-and both direct and indirect physical interactions. The simplified experimental model opens new avenues to the study of adherent bacterial communities and their behavior in the context of rapid global change.

Our study presents new insights into the molecular mechanism that regulates the activity of SppA, a widely conserved bacterial membrane protease. We show that the membrane proteins SppA and SppI form a complex in the Gram-positive model bacterium B. subtilis and that SppI inhibits SppA protease activity in vitro and in vivo. Furthermore, we demonstrate that the C-terminal domain of SppI is involved in SppA inhibition. Since SppA, through its protease activity, contributes directly to resistance to lantibiotic peptides and cationic antibacterial peptides, we propose that the conserved SppA-SppI complex could play a major role in the evasion of bactericidal peptides, including those produced as part of human innate immune defenses.

Bacteria are sophisticated systems with high capacity and flexibility to adapt to various environmental conditions. Each prokaryote however possesses a defined metabolic network, which sets its overall metabolic capacity, and therefore the maximal growth rate that can be reached. To achieve optimal growth, bacteria adopt various molecular strategies to optimally adjust gene expression and optimize resource allocation according to the nutrient availability. The resulting physiological changes are often accompanied by changes in the growth rate, and by global regulation of gene expression. The growth-rate-dependent variation of the abundances in the cellular machineries, together with condition-specific regulatory mechanisms, affect RNA metabolism and fate and pose a challenge for rational gene expression reengineering of synthetic circuits.