Among members of the Bacillales order, there are several species capable of forming a structure called an endospore. Endospores enable bacteria to survive under unfavourable growth conditions and germinate when environmental conditions are favourable again. Spore-coat proteins are found in a multilayered proteinaceous structure encasing the spore core and the cortex. They are involved in coat assembly, cortex synthesis and germination. Here, we aimed to determine the diversity and evolutionary processes that have influenced spore-coat genes in various spore-forming species of Bacillales using an in silico approach. For this, we used sequence similarity searching algorithms to determine the diversity of coat genes across 161 genomes of Bacillales. The results suggest that among Bacillales, there is a well-conserved core genome, composed mainly by morphogenetic coat proteins and spore-coat proteins involved in germination. However, some spore-coat proteins are taxa-specific. The best-conserved genes among different species may promote adaptation to changeable environmental conditions. Because most of the Bacillus species harbour complete or almost complete sets of spore-coat genes, we focused on this genus in greater depth. Phylogenetic reconstruction revealed eight monophyletic groups in the Bacillus genus, of which three are newly discovered. We estimated the selection pressures acting over spore-coat genes in these monophyletic groups using classical and modern approaches and detected horizontal gene transfer (HGT) events, which have been further confirmed by scanning the genomes to find traces of insertion sequences. Although most of the genes are under purifying selection, there are several cases with individual sites evolving under positive selection. Finally, the HGT results confirm that sporulation is an ancestral feature in Bacillus .

Over the years, many researchers have reported a great diversity of bacteriophages infecting members of the Ralstonia solanacearum species complex (RSSC). This diversity has driven bacterial evolution by leading the emergence and maintenance of bacterial defense systems to combat phage infection. In this work, we present an in silico study of the arsenal of defense systems that RSSC harbors and their evolutionary history. For this purpose, we used a combination of genomic, phylogenetic and associative methods. We found that in addition to the CRISPR-Cas system already reported, there are eight other antiphage defense systems including the well-known Restriction-Modification and Toxin-Antitoxin systems. Furthermore, we found a tenth defense system, which is dedicated to reducing the incidence of plasmid transformation in bacteria. We undertook an analysis of the gene gain and loss patterns of the defense systems in 15 genomes of RSSC. Results indicate that the dynamics are inclined toward the gain of defense genes as opposed to the rest of the genes that were preferably lost throughout evolution. This was confirmed by evidence on independent gene acquisition that has occurred by profuse horizontal transfer. The mutation and recombination rates were calculated as a proxy of evolutionary rates. Again, genes encoding the defense systems follow different rates of evolution respect to the rest of the genes. These results lead us to conclude that the evolution of RSSC defense systems is highly dynamic and responds to a different evolutionary regime than the rest of the genes in the genomes of RSSC.

Bile acids (BA) not only influence the gut microbiome composition but are also metabolized by gut bacteria to form various microbial BAs. Among these, 3-oxo-lithocholic acid (3-oxo-LCA) and isoallo-LCA have been reported to modulate host immunity, suppress intestinal pathogens, and provide antiaging benefits, suggesting that they could also affect intestinal epithelial cells and colorectal cancer progression. To investigate the impact of 3-oxo-LCA on intestinal tumorigenesis, we evaluated its activity in vitro on mouse and human colorectal cancer cell lines, as well as primary mouse intestinal organoids and patient-derived colorectal cancer organoids, and in vivo using a genetically engineered mouse model, cell line–derived syngeneic and xenograft tumors, and patient-derived xenografts. 3-Oxo-LCA functioned as a potent FXR agonist that restored FXR signaling both in vitro and in vivo. Activation of FXR signaling reduced the growth of colorectal cancer cell lines and suppressed the proliferation of intestinal stem cells in both mouse organoids and patient-derived colorectal cancer organoids. In the APCMin/+ genetically engineered mouse model, 3-oxo-LCA reduced BA levels, enhanced gut barrier function, decreased tumor burden, and suppressed tumor initiation. Furthermore, 3-oxo-LCA significantly inhibited tumor progression in syngeneic and xenograft mouse models and promoted apoptosis within the tumors. Together, these results underscore the function of 3-oxo-LCA as an FXR agonist with the ability to inhibit colorectal cancer tumorigenesis and progression by modulating epithelial cell growth and death. Significance: The microbial bile acid 3-oxo-LCA activates FXR signaling in intestinal epithelial cells that inhibits cancer stem cell proliferation and induces apoptosis, highlighting the potential of 3-oxo-LCA for treating intestinal tumorigenesis.

We investigate the distribution and evolution of prokaryotic cell size based on a compilation of 5,380 species. Size spans four orders of magnitude, from 100 nm (Mycoplasma) to more than 1 cm (Thiomargarita); however, most species congregate heavily around the mean. The distribution approximates but is distinct from log normality. Comparative phylogenetics suggests that size is heritable, yet the phylogenetic signal is moderate, and the degree of heritability is independent of taxonomic scale (i.e., fractal). Evolutionary modeling indicates the presence of an optimal cell size to which most species gravitate. The size is equivalent to a coccus of 0.70 μm in diameter. Analyses of 1,361 species with sequenced genomes show that genomic traits contribute to size evolution moderately and synergistically. Given our results, scaling theory, and empirical evidence, we discuss potential drivers that may expand or shrink cells around the optimum and propose a stability landscape model for prokaryotic cell size.

Viral populations are large and highly heterogeneous. Despite the evolutionary relevance of such heterogeneity, statistical approaches to quantifying the extent to which viruses maintain a high genotypic and/or phenotypic diversity have been rarely pursued. Here, we address this issue by analyzing a nucleotide-to-protein sequence map through deep sequencing of populations of the Q$\beta$ phage adapted to high temperatures. Tens of thousands of different sequences corresponding to two fragments of the gene coding for the viral replicase were recovered. A diversity analysis of two independent populations consistently revealed that about $40\%$ of the mutations identified caused changes in protein amino acids, leading to an almost complete exploration of the protein neighborhood of (non-silent) mutants at a distance of one. The functional form of the empirical distribution of phenotype abundance agreed with analytical calculations that assumed random mutations in the nucleotide sequence. Our results concur with the idea that viral populations maintain a high diversity as an efficient adaptive mechanism and support the hypothesis of universality for a lognormal distribution of phenotype abundances in biologically meaningful genotype–phenotype maps, highlighting the relevance of entropic effects in molecular evolution.

Understanding how viral mutant spectra organize and explore genotype space is essential for elucidating the mechanisms that drive molecular evolution. Here, we use deep-sequencing data of an amplicon in the A2 protein of the RNA bacteriophage Q β to reconstruct genotype networks comprising tens of thousands of haplotypes. The study of populations evolved under different temperature regimes reveals robust and reproducible patterns that arise from the interplay between fundamental geometrical motifs of sequence spaces and population dynamics. Mutant swarms exhibit a self-similar, hierarchical organization in which sequences cluster around highly connected, abundant cores that continuously regenerate diversity during evolution. The immediate neighborhood of these cores is rapidly rebuilt and extensively sampled, while a few mutations away sampling becomes dynamical and sparse. This population structure emerges from a dynamic, out-of-equilibrium balance between replication and mutational exploration and suggests that Q β populations do not rely primarily on neutral networks to navigate genotype space or to generate diversity. Combining genotype networks from populations adapted to different temperatures reveals early evolutionary divergence, with partially overlapping yet distinct populations that remain connected through short mutational paths. Even at the time scale of these experiments, evolutionary trajectories remain multiple, preventing the backward reconstruction of unique trajectories once mutations have been fixed. Together, this analysis provides a detailed view of the fine-scale processes shaping the evolution of heterogeneous viral populations and establishes genotype networks as a powerful framework for visualizing and interpreting the organization and diversification of viral quasispecies.