Biology subjects

Lopez-Garrido, J.

Publications and source records attributed to Lopez-Garrido, J..

7 recordsLinked to original sources

bioRxiv2026

The ferredoxin/flavodoxin-NADP+ oxidoreductase YumC is essential for isoprenoid and peptidoglycan biosynthesis in Bacillus subtilis

Redox reactions mediated by ferredoxin/flavodoxin-NADP+ oxidoreductases (FNRs) and their associated electron-carrier proteins, ferredoxins and flavodoxins, are essential in biology. Although the biochemical activities of these redox proteins are conserved, their precise physiological roles can differ among organisms and cannot be easily inferred. Here we have defined an essential role for Bacillus subtilis YumC, a member of a distinct group of bacterial FNRs that resemble thioredoxin reductase. We have used targeted protein degradation, cytological profiling, metabolomics, and genetic complementation to show that YumC catalyzes the transfer of electrons from NADPH, through ferredoxin (Fer) or through the flavodoxin YkuP, to the isoprenoid biosynthesis pathway, and specifically to the redox enzyme IspG. When YumC was degraded, isoprenoid biosynthesis was compromised, and the level of undecaprenyl phosphate, the isoprenoid lipid carrier for peptidoglycan building block translocation, was diminished. Degradation of YumC or of Fer in a {Delta}ykuP strain led to defective peptidoglycan biosynthesis, activation of the {sigma}M-dependent cell-wall stress response, and lethality. The introduction into B. subtilis of an alternative pathway for isoprenoid biosynthesis that does not require input from electron-carrier proteins could complement the degradation of Fer in a {Delta}ykuP strain, but not the degradation of YumC. This finding indicates that YumC is required for other essential processes that do not necessarily involve Fer and YkuP. This work provides an explanation for why YumC is essential, reveals how reducing power is delivered to isoprenoid biosynthesis in B. subtilis, and illustrates how the varied roles of redox systems among bacteria depend upon metabolic context.

microbiology↗

bioRxiv2026

Compensatory evolution facilitates loss of prfB autoregulation in Pseudomonas fluorescens SBW25

Understanding why some traits are maintained whereas others are repeatedly lost is a central question in evolutionary biology. Here, we address this problem through an analysis of the evolutionary dynamics of autoregulation of the prfB gene, which encodes peptide-chain release factor 2 (RF2), a key factor in bacterial translation termination. RF2 recognizes UGA and UAA stop codons and catalyzes the release of the completed polypeptide. In many species, prfB contains an internal UGA stop codon, triggering premature translation termination by RF2 itself. Complete RF2 translation depends on a +1 programmed ribosomal frameshifting (PRF) event on the internal stop codon, which occurs more frequently when RF2 levels are low, resulting in autoregulation of prfB expression. While widespread, this autoregulatory mechanism has been lost in multiple bacterial lineages. We combined phylogenetics, experimental evolution and molecular genetics to investigate the evolutionary forces behind this loss. We found no significant correlation between PRF loss and stop codon usage using phylogenetically informed analyses, and PRF disruption in Pseudomonas fluorescens SBW25 had no detectable fitness effect. However, engineered mutations that reduced frameshifting caused fitness defects, which were compensated by two classes of mutation: (i) mutations that impair specific ribosomal proteins, and (ii) single-nucleotide deletions in prfB that adjust the reading frame and bypass the internal stop codon. These results suggest that compensatory mutations facilitate the loss of prfB autoregulation under RF2-limiting conditions. We discuss three potential scenarios that could account for this process.

evolutionary biology↗

bioRxiv2025

Deep-learning deconvolution and segmentation of fluorescent membranes for high-precision bacterial cell-size profiling

Evolutionary studies in bacteria have emphasized genetic and metabolic diversity, while cell-size variation has received less attention. Here we introduce MEDUSSA, a high-throughput method for precise bacterial cell-size profiling based on automatic segmentation of fluorescent membrane images, well suited to studying cell-size diversity. The approach uses deep-learning-based membrane deconvolution, segmentation models fine-tuned for fluorescent-membrane images, and error-corrected cell measurement to extract accurate sizes from individual bacterial cells regardless of shape, size, chaining, or clustering. Our method overcomes limitations of phase-contrast segmentation, yielding reliable single-cell dimensions. We applied MEDUSSA to six strains of Priestia megaterium and found over twofold differences in cell volume across strains, largely driven by differences in cell width. We identified a partially-functional PBP1 allele that underlies the reduced width of one strain. Together, these results demonstrate the power of comparative analyses in bacterial cell biology and expand the toolkit to investigate the evolution of bacterial cell size.

cell biology↗

bioRxiv2025

Single capsid mutations modulating phage adsorption, persistence, and plaque morphology shape evolutionary trajectories in {Phi}X174

The evolutionary success of lytic bacteriophages depends on key life-history traits, including adsorption, lysis time, burst size, and persistence in the environment. However, how these traits evolve to allow adaptation to different environments remains poorly understood. Here, we explored this question in {Phi}X174, combining experimental evolution and mathematical modelling. By investigating how serial transfer conditions shape evolutionary outcomes in liquid culture, we found that the time between transfers imposes divergent selection on adsorption and context-dependent directional selection on persistence. Longer transfer intervals, which allow multiple infection cycles until host depletion, favoured fast-adsorbing, highly persistent mutants that could rapidly initiate infections and remained viable in the absence of the host. In contrast, shorter transfer intervals selected for slower adsorption without substantially altering persistence. Mathematical modelling of phage population dynamics predicted that adsorption evolution during short transfers reflects a trade-off between two opposing selective forces within each transfer: an early phase in which susceptible hosts are abundant and adsorption is productive, favouring fast adsorption, and a later phase in which most hosts are already infected and adsorption primarily removes phage particles via attachment to already infected cells, favouring slower adsorption. A single-point mutation in the major capsid protein was sufficient to drive these changes in adsorption. In the case of fast-adsorbing mutants, this mutation was positively pleiotropic and also enhanced environmental persistence. Our findings show how simple changes in propagation conditions can steer phage phenotypes, providing insights relevant to evolutionary biology and phage therapy.

evolutionary biology↗

bioRxiv2020

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation

The mechanistic details of bacterial cell division are poorly understood. The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the mid cell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. We use cryo-electron tomography to visualize the architectural differences in the organization of FtsAZ filaments, the major orchestrators of bacterial cell division during these conditions. We demonstrate that during vegetative growth, FtsAZ filaments are present uniformly around the leading edge of the invaginating septum but during sporulation, they are only present on the mother cell side. Our data show that the sporulation septum is thinner than the vegetative septum during constriction, and that this correlates with half as many FtsZ filaments tracking the division plane during sporulation as compared to vegetative growth. We further find that a sporulation-specific protein, SpoIIE, regulates divisome localization and septal thickness during sporulation. Our data provide first evidence of asymmetric localization of the cell division machinery, and not just septum formation, to produce different cell types with diverse fates in bacteria.

microbiology↗

bioRxiv2018

The molecular architecture of engulfment during Bacillus subtilis sporulation

The study of cell biology is limited by the difficulty in visualizing cellular structures at high spatial resolution within their native milieu. Here, we have visualized sporulation in Bacillus subtilis using cryo-electron tomography coupled with cryo-focused ion beam milling, a technique that allows the 3D reconstruction of cellular structures in near-native state at molecular resolution. During sporulation, an asymmetrically-positioned septum divides the cell into a larger mother cell and a smaller forespore. Subsequently, the mother cell phagocytoses the forespore in a process called engulfment, which entails a dramatic rearrangement of the peptidoglycan (PG) cell wall around the forespore. By imaging wild-type sporangia, engulfment mutants, and sporangia treated with PG synthesis inhibitors, we show that the initiation of engulfment does not entail the complete dissolution of the septal PG by the mother cell SpoIIDMP complex, as was previously thought. Instead, DMP is required to maintain a flexible septum that is uniformly and only slightly thinned at the onset of engulfment. Then, the mother cell membrane migrates around the forespore by forming tiny finger-like projections, the formation of which requires both SpoIIDMP and new PG synthesized ahead of the leading edge of the engulfing membrane. We propose a molecular model for engulfment membrane migration in which a limited number of SpoIIDMP complexes tether the membrane to and degrade the new PG ahead of the leading edge, thereby generating an irregular engulfing membrane front. Our data also reveal other structures that will provide a valuable resource for future mechanistic studies of endospore formation.

microbiology↗

bioRxiv2016

Cell wall remodeling drives engulfment during Bacillus subtilis sporulation

When starved, the Gram-positive bacterium Bacillus subtilis forms durable spores for survival. Sporulation initiates with an asymmetric cell division, creating a large mother cell and a small forespore. Subsequently, the mother cell membrane engulfs the forespore in a phagocytosis-like process. However, the force generation mechanism for forward membrane movement remains unknown. Here, we show that membrane migration is driven by cell wall remodeling at the leading edge of the engulfing membrane, with peptidoglycan synthesis and degradation mediated by penicillin binding proteins in the forespore and a cell wall degradation protein complex in the mother cell. We propose a simple model for engulfment in which the junction between the septum and the lateral cell wall moves around the forespore by a mechanism resembling the template model. Hence, we establish a biophysical mechanism for the creation of a force for engulfment based on the coordination between cell wall synthesis and degradation.

biophysics↗