Alfonso Soler-Bistué

Bradyrhizobia are widely used as plant growth promoters in agriculture, yet their cell biology remains poorly understood. Here, we describe, for the first time, nucleoid segregation dynamics and the cell cycle of Bradyrhizobium at the cellular level. We show that highly asymmetric polar growth generates a short daughter cell and a longer mother cell at each division. Nucleoid compaction and positioning change across growth phases, culminating in a highly compacted, asymmetrically positioned nucleoid in stationary phase. ParB presents a subpolar localization, at the nucleoid periphery. Despite this pronounced cellular asymmetry and the presence of a single large chromosome, chromosome conformation capture reveals a global chromosome organization similar to that of bacteria undergoing conventional binary fission. Together, these findings uncover a distinctive cell cycle program in Bradyrhizobium , showing that such extreme asymmetry in growth and division, significantly different from that observed in closely related characterized α-Proteobacteriota, is compatible with conserved principles of chromosome organization.

How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (oriC)–terminus (ter) axis. The spatial arrangement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. We tested the importance of the universally conserved rplKAJL-rpoBC locus, which encodes the β/β′ subunits of the sole bacterial RNA polymerase (RNAP), by relocating it to different genomic positions in the fast-growing pathogen Vibrio cholerae. Relocation close from locus native site was neutral but relocating it near either chromosomal terminus reduced exponential growth and competitive fitness specifically in nutrient-rich media. Marker-frequency analysis showed that distal positioning lowered locus copy number from ~3 to ~1 per cell, causing a 20%–25% depletion in cellular RNAP without altering its subcellular distribution. Introducing an additional oriC-proximal copy restored wild-type phenotypes, whereas two terminus copies rescued growth solely through increased dosage. Deleting the oriC-proximal RNAP genes reproduced all defects, identifying them as the primary drivers. Selection keeps RNAP genes close to oriC to harness replication-associated dosage increment during exponential growth, ensuring adequate transcription capacity for rapid proliferation. Gene order is a key but overlooked layer of bacterial genome evolution and ecological adaptation.

Free-living soil bacteria can exist in two main states: planktonic, as motile single cells, or sessile, within biofilms. In biofilms, bacterial cells are embedded in an extracellular matrix that provides protection from environmental stresses and enhances long-term survival. The transition from planktonic to biofilm states sometimes involves surface sensing and attachment, processes commonly mediated by flagella and pili. In this study, we investigated the role of Type IVc Tad pili in surface sensing, adhesion, and biofilm formation in Bradyrhizobium diazoefficiens , a nitrogen-fixing symbiont of soybean. Bioinformatic analyses revealed that Tad pili are widely distributed and highly conserved within the Bradyrhizobium genus. While pili deletion in other model organisms typically reduces biofilm formation, we found that deletion of the most conserved genomic cluster encoding Tad pili in B. diazoefficiens led to increased adhesion to abiotic surfaces and impaired motility—indicative of a physiological shift toward a biofilm-associated state. These findings suggest that Tad pili may play a sensory or regulatory role, potentially influencing cell-cell or cell-matrix interactions. Furthermore, we identified a link between Tad pili and intracellular c-di-GMP levels. Together, these results highlight the critical role of Tad pili in the physiology of B. diazoefficiens and offer new insights into bacterial surface adaptation, with potential applications in agriculture and biotechnology. Understanding these mechanisms is essential for improving biofilm management strategies and developing new approaches to enhance bacterial survival in soil and inoculant formulations, ultimately optimizing legume symbiosis. IMPORTANCE: Biofilm formation is essential for bacterial survival in soil environments. In this study, we investigated the role of Tad pili in the biofilm-forming capacity of Bradyrhizobium diazoefficiens and their connection to the second messenger c-di-GMP, a key regulator of the transition between planktonic and sessile states. Bacteria used in agricultural inoculants are typically in the planktonic state, yet survival and persistence are optimized in the sessile state. Our findings may contribute to the development of strategies that promote the transition to the biofilm lifestyle in inoculant formulations, thereby enhancing bacterial viability in storage and soil and improving symbiotic performance with host plants.

The ability to generate genomic diversity expands opportunities for understanding and engineering biology. Here, we demonstrate on-demand generation of diversity in bacterial genome configurations and its application to probing physiology under altered genome organization. We engineered the fast-growing bacterium Vibrio natriegens to enable large-scale stochastic duplications, translocations, inversions, and deletions, producing populations with a wide range of genome arrangements. We investigated phenotypic robustness to genome reconfigurations and found that distinct genome organizations can support stable physiology, indicating that bacteria may tolerate chromosomal alterations more readily than previously appreciated. Our work provides a framework for advancing the understanding and engineering of bacterial genomes and suggests that genome reconfigurations may contribute to phenogenetic drift, allowing for evolutionary exploration while preserving phenotype.

How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin ( oriC )–terminus ( ter ) axis. The spatial arrengement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. The catalytic core of the sole bacterial RNA polymerase (RNAP) is encoded by rpoB and rpoC within the universally conserved rplKAJL-rpoBC locus. In fast growing bacteria this locus is located near oriC potentially linking genome organization to cellular physiology. Here, we investigated the functional relevance of rplKAJL-rpoBC chromosomal positioning by relocating it within Vibrio cholerae genome. While viable, strains harboring rplKAJL-rpoBC far from oriC exhibited nutrient-dependent fitness defects. These phenotypes correlated with reduced locus copy number and RNAP abundance, without changes in its subcellular distribution. Introducing a second copy of rpIKAJL-rpoBC at a distal genomic location from oriC abolished the phenotypes, demonstrating that replication-associated gene dosage effects were the main mechanism behind the observed physiological changes. Further uncoupling rpoB and rpoC from the rplKAJL-rpoBC locus revealed that fitness costs were specifically linked to RNAP gene relocation. Our results reveal that selective pressures act to maintain RNAP-encoding genes near oriC to optimize replication-associated dosage effects, ensuring efficient RNAP production during exponential growth. We discuss the relationship between gene order and ecological strategies linked to bacterial growth. Our work underscores gene order as an underestimated but critical factor shaping cellular physiology and genome evolution. Significance: Bacterial chromosomes have a highly plastic gene content but the importance of their spatial organization and gene order is started to be appreciated as a driver of cellular physiology. Growing evidence suggests that the spatial organization of bacterial genomes plays a critical role in gene expression, replication dynamics, and fitness. In this study, we demonstrate that the conserved positioning of rpoB and rpoC , encoding the catalytic core of RNA polymerase (RNAP), near the replication origin ( oriC ) is essential for optimal bacterial growth. By relocating the rplKAJL-rpoBC locus in Vibrio cholerae , we show that its chromosomal position directly impacts nutrient-dependent fitness, RNAP abundance, and replication-associated gene dosage effects. These findings suggest that selective pressures maintain genes encoding RNAP near oriC to ensure efficient transcriptional output during exponential growth. More broadly, our study highlights gene order as an underappreciated factor in bacterial genome evolution, beyond its known roles in operon structure and transcriptional regulation. Given the fundamental role of RNAP, our results have implications for understanding bacterial ecological strategies, adaptation, and synthetic genome design. By integrating positional genomics with physiological studies, we provide a theoretical and experimental framework for investigating how genome architecture shapes cellular function.

Free-living soil bacteria may exist in two states: planktonic as swimmer cells, and sessile within biofilms. In biofilms, bacterial cells are embedded into an extracellular matrix, which confers protection against environmental stresses and extends bacterial survival. The transition from planktonic to biofilm state involves surface sensing and attachment mediated by flagella and pili. Here, we explored the role of Tad pili (Type IVc) in surface sensing, adhesion, and biofilm formation in Bradyrhizobium diazoefficiens , a nitrogen-fixing symbiont of soybean. Bioinformatic analyses revealed that Tad pili are widely distributed and highly conserved in Bradyrhizobium. In other model bacteria, pili deletion leads to decrease in biofilm-forming capacity, but other functions are also affected depending on the microorganism. Surprisingly, deletion of the most conserved genomic cluster encoding Tad pili in B. diazoefficiens caused an increased adhesion to abiotic surfaces and loss of movement, indicating a physiological shift towards a biofilm state. These observations suggest a sensor or regulatory role for this apparatus that could affect cell-cell interactions or interactions with the extracellular matrix. Additionally, a connection between pili and c-di-GMP levels in the cell was established. These findings highlight the critical role of Tad pili in B. diazoefficiens physiology, providing insights into bacterial adaptability and potential applications in agriculture and biotechnology. Understanding these mechanisms is key to improving biofilm management strategies and generating novel approaches to enhance bacterial viability in soil and inoculants as well as to optimize the symbiosis.

Growth rate is a key prokaryotic trait that allows for estimating fitness and understanding cell metabolism. While it has been well studied in model organisms, there is limited data on slow-growing bacteria. In particular, there is a lack of quantitative studies on Brucella species. This genus includes important microorganisms that are causative agents of brucellosis, one of the most widespread bacterial zoonoses, affecting several species of animals, including humans. Brucella species exhibit approximately 97% genomic similarity. Despite this, Brucella species show different host preferences, zoonotic risks, and pathogenicity. After more than one hundred years of research, numerous aspects of Brucella biology, such as in vivo and in vitro growth, remain poorly characterized. In this work, we analyzed vegetative and intracellular growth of the classical Brucella species (B. abortus, B. melitensis, B. suis, B. ovis, and B. canis). Strikingly, each species displayed distinct growth parameters in culture. Doubling time (DT) ranged from 2.7 hs⁻¹ in B. suis to 18 h⁻¹ for B. ovis. In the context of intracellular infection of J774A.1 phagocytic cells, DT was longer, but it widely varied across species, closely correlating with the growth observed in vitro. Overall, and despite high genome similarity, we also found species-specific growth parameters in the intracellular cell cycle.

Growth rate is a key prokaryotic trait that allows estimating fitness and understanding cell metabolism. While in some clades it has been well studied in model organisms, there is little data on slow-growing bacteria. In particular, there is a lack of quantitative studies on the species of the Brucella genus. This is an important microorganism since it is the causative agent of brucellosis, one of the most widespread bacterial zoonoses affecting several species of animals, including humans. Brucella species exhibit approximately 97% genomic similarity. Despite this, Brucella species show different host preferences, zoonotic risks, and pathogenicity. After more than one hundred years of research, numerous aspects of Brucella biology, such as in vivo and in vitro growth remain poorly characterized. In this work, we analyzed vegetative and intracellular growth of the classical Brucella species ( B. abortus 2308, B. melitensis 16M. B. suis 1330, B. ovis , and B. canis ). Strikingly, each species displayed particular growth parameters in culture. Doubling time (DT) spanned from 2.7 hs ^-1 in B. suis to 18h ^-1 for B. ovis . In the context of intracellular infection of J774A.1 phagocytic cells, DT was higher, but it widely varied across species, closely correlating to the growth observed in vitro . Overall, despite high similarity of the genomes, we found species-specific growth parameters in the intracellular cell cycle.

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

The guanine/cytosine (GC) content of prokaryotic genomes is species‐specific, taking values from 16% to 77%. This diversity of selection for GC content remains contentious. We analyse the correlations between GC content and a range of phenotypic and genotypic data in thousands of prokaryotes. GC content integrates well with these traits into r/K selection theory when phenotypic plasticity is considered. High GC‐content prokaryotes are r‐strategists with cheaper descendants thanks to a lower average amino acid metabolic cost, colonize unstable environments thanks to flagella and a bacillus form and are generalists in terms of resource opportunism and their defence mechanisms. Low GC content prokaryotes are K‐strategists specialized for stable environments that maintain homeostasis via a high‐cost outer cell membrane and endospore formation as a response to nutrient deprivation, and attain a higher nutrient‐to‐biomass yield. The lower proteome cost of high GC content prokaryotes is driven by the association between GC‐rich codons and cheaper amino acids in the genetic code, while the correlation between GC content and genome size may be partly due to functional diversity driven by r/K selection. In all, molecular diversity in the GC content of prokaryotes may be a consequence of ecological r/K selection.