Bacteria are able to coordinate cell growth and genome replication in different growth conditions.The DNA-binding protein DnaA is responsible for determining initiation of replication, thereby playing a central role in this coordination. Theoretical and experimental studies have shown that stability of the cell cycle requires an ultrasensitive response, i.e., a sharp dependence of the initiation firing rate on the cell volume. However, the source of such ultrasensitivity remains elusive. In this work, we elucidate how the structure and binding affinities of the DnaA regulatory system determine its ultrasensitive response. Our theory sets precise constraints on inding parameters, that are necessary for cell cycle stability. Our findings show how the variety of regulatory mechanisms of the DnaA system are required for ultrasensitivity across growing conditions.

We evaluate whether tryptophan (W), widely thought to be the last of the 20 canonical amino acids added to the genetic code, was already present in the Last Universal Common Ancestor (LUCA). We reconstruct the evolutionary history of tryptophanyl-tRNA synthetase (WRS), the enzyme that attaches W to its tRNA, and the related tyrosyl-tRNA synthetase (YRS). We identify and exclude sequences derived from ancient recombination between archaeal and bacterial YRSs. Diverse rooting methods, including a novel approach exploiting time non-reversible evolution, all place the root between bacterial and archaeal YRS rather than between YRS and WRS. This supports post-LUCA WRS origination in Archaea, followed by its horizontal transfer to Bacteria. However, ancestral sequence reconstruction suggests that Archaea were depleted for W while Bacteria were not, and enzymes essential for W biosynthesis emerged in Bacteria. This suggests that W usage originated in Bacteria, with later WRS emergence in Archaea allowing the archaeal genetic code to converge with the bacterial code. The universality of the genetic code is usually attributed to common descent from LUCA, but the final step making the code universal was instead achieved by horizontal gene transfer. This gives credence to similar mechanisms for earlier steps in genetic code evolution.

Förster resonance energy transfer (FRET), an optical distance ruler, provides the unique ability to monitor molecular interactions and conformational changes in single biomacromolecules and multi-subunit complexes. It has proven to be a powerful tool to study enzymatic reactions, membrane transport, protein folding, but also the properties of nucleic acids, proteins, molecular motors, and many other biological systems and processes. A prerequisite for FRET is targeted (covalent) labeling of macromolecules with two distinct fluorophores. Here, we present a strategy for stochastic labeling of protein residues with the required donor and acceptor dyes via anion exchange chromatography. While this technique has been used before for this purpose, we provide a conceptual basis to systematically design a purification protocol for an arbitrary choice of fluorophores. By characterizing the interaction of hydrolyzed fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow a successful purification of donor–acceptor-labeled protein with yields up to 98%. We demonstrate the capabilities of the method for bulk and single-molecule FRET assays of various bacterial substrate-binding proteins, which are examples of soluble, folded, and well-characterized protein systems.

This study presents a mathematical framework for investigating the dynamics of coexistence and competition among heterotrophic microbes across different time scales. Focusing on metabolic interactions, we examine how three strategies: public metabolizing, private metabolizing, and cheating, shape population behavior. The framework integrates generalized Lotka-Volterra dynamics with evolutionary game theory to capture the effects of resource exchange, particularly glucose made available by public metabolizers and sucrose as a shared substrate driving population growth. Game-theoretic payoffs encode ecological costs and benefits, enabling analysis of frequency-dependent interactions among strategies. To capture evolutionary realism, we implement laboratory-inspired simulations in which strategies can switch between generations, mimicking mutation or phenotypic plasticity in microbial populations. These eco-evolutionary dynamics reveal conditions under which all three strategies coexist at interior equilibria and show how variation in growth advantages and, illustratively, phenotype-switching perturbations produce evolutionary shifts. Numerical analysis identifies ecological thresholds and fitness asymmetries that determine system robustness, long-term coexistence, and the persistence of a synthetic, cross-kingdom system linked by nutrient exchange. Together, these insights provide general principles for microbial coexistence and offer design guidelines for ecosystem engineering, biotechnological applications, and the construction of stable synthetic communities under ecological and evolutionary constraints.