Biology subjects

Abia, D.

Publications and source records attributed to Abia, D..

6 recordsLinked to original sources

bioRxiv2025

EXPRESSION OF CONJUGATION GENES IS CONTROLLED BY PROCESSIVE ANTITERMINATION AND A NOVEL ZIPPER-TYPE TRANSCRIPTIONAL ATTENUATION MECHANISM

Proper expression of genes clustered in operons, particularly large operons, can be complex, often consisting of multiple regulatory switches. The conjugation operon present on the Bacillus subtilis conjugative plasmid pLS20 is over 32 kb long. This operon starts with a 456 nt leader region, which is followed by the first two genes of the operon, encoding a two-component processive antitermination system. Here, we demonstrate that the long leader region encodes a transcriptional attenuator that we named cATTpLS20. In vivo and in vitro analyses showed that the attenuator is composed of three segments: a stem-loop structure with a long imperfect stem that is preceded by a sequence that may form a weak stem-loop and followed by a sequence that may form an intrinsic terminator. Sequences of the upstream stem loop and downstream terminator are complementary, allowing further extension of the long stem, thereby generating an antiterminator conformation. Based on similarity with a zipper we coined this the zipper-type attenuator. Similar zipper-type attenuators are present upstream of the first genes of conjugation operons of all pLS20 family plasmids, as well as on many other conjugative plasmids in Gram-positive bacteria, suggesting that a common attenuation mechanism regulates expression of many conjugative operons. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/689506v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a86a7eorg.highwire.dtl.DTLVardef@e787f8org.highwire.dtl.DTLVardef@8fdb75org.highwire.dtl.DTLVardef@90f842_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. SddA is a substrate for the periplasmic protease Prc, suggesting regulation via protein degradation. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage unless envC is co-expressed. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division. AUTHOR SUMMARYBacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli. We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

microbiology↗

bioRxiv2024

Time-resolved mitochondrial-focused screening identifies regulatory components of oxidative metabolism

Defects in mitochondrial oxidative metabolism contribute to various genetic inherited disorders, termed mitochondrial diseases, with limited treatment options. Given the lack of functional annotation for numerous mitochondrial proteins, there is a necessity for an extensive gene inventory related to mitochondrial function, with special interest in Oxidative Phosphorylation (OXPHOS). To address this gap, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings at various time points to uncover novel regulators of mitochondrial function. Our study resulted in a gene catalog essential for mitochondrial oxidative metabolism, and constructed a dynamic timeline mapping a broad network of mitochondrial pathways, with a particular focus on the OXPHOS complexes. Computational analysis pinpointed RTN4IP1 and ECHS1 as key genes strongly associated with OXPHOS and whose mutations are associated with mitochondrial diseases in humans. RTN4IP1 was found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Deletion of ECHS1 leads to reduced catabolism of branched-chain amino acids (BCAAs), which impairs the activity of lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.

molecular biology↗

bioRxiv2023

PC_sim: An integrated measure of protein sequence and structure similarity for improved alignments and evolutionary inference

MotivationEvolutionary inferences depend crucially on the quality of multiple sequence alignments (MSA), which is problematic for distantly related proteins. Since protein structure is more conserved than protein sequence, it seems natural to use structure alignments for distant homologs. However, structure alignments may not be suitable for inferring evolutionary relationships at the sequence level. ResultsHere we investigate the mutual relationships between four protein similarity measures that depend on sequence and structure (fraction of aligned residues, sequence similarity, fraction of superimposed backbones and contact overlap) and the corresponding alignments. Changes in protein sequences and structures are intimately correlated, but our results suggest that no individual measure can provide a complete and unbiased picture of changes in protein sequences and structure. Therefore, we propose a new hybrid measure of protein sequence and structure similarity based on Principal Components (PC_sim). Starting from an MSA, we obtain modified pairwise alignments (PA) based on PC_sim, and from them we construct a new MSA based on the maximal cliques of the PA graph. These alignments yield larger protein similarities and agree better with the Balibase "reference" MSA and with consensus MSA than alignments that target individual similarity measures. Moreover, PC_sim is associated with a divergence measure that correlates strongest with divergences obtained from individual similarities, which suggests that it can infer more accurate evolutionary divergences for the reconstruction of phylogenetic trees with distance methods. Availabilityhttps://github.com/ugobas/Evol_div Contactubastolla@cbm.csic.es

bioinformatics↗

bioRxiv2020

Pre-TCR signaling intensity shapes the TCRβ repertoire

Signaling via pre-TCR is essential for initial T cell differentiation. How the signal is triggered and whether its alteration impacts on lymphocyte repertoires is nevertheless debated. We show that a mutation in the transmembrane domain of the CD3{zeta} chain impairs murine thymocyte development at the double negative (DN) stage coinciding with reduced levels of steady-state pre-TCR signaling. Single particle tracking and TIRF microscopy on mouse primary DN3/DN4 thymocytes shows that wild-type pre-TCR form particles with a broad range of fluorescent intensities whereas mutant complexes are less intensely labeled and move faster, suggesting reduced pre-TCR nanoclustering. Notably, the mutant early-pre-TCR-selected-DP population shows less TCR{beta} repertoire diversity. We thus propose that the pre-TCR forms stable nanoclusters, the size of which determines signaling efficacy. This signaling does not merely promote maturation of any thymocyte expressing a rearranged TCR{beta} chain, but shapes the repertoire of DP thymocytes that can audition for positive and negative selection.

immunology↗

bioRxiv2020

Recreation of an antigen-driven germinal center in vitro by providing B cells with phagocytic antigen

Successful vaccines rely on activating a functional humoral response that results from generating class-switched high affinity immunoglobulins (Igs) with a superior capacity to neutralize infection. Key to this process is the germinal center (GC) reaction, in which B cells are selected in their search for antigen and T cell help. A major hurdle to understanding the mechanisms of B cell:T cell cooperation has been the lack of an in vitro system to recreate GCs in an antigen-specific manner. Here we report the generation of functional antigen-specific high affinity Igs of different isotypes in simple 2-cell type cultures of naive B and T cells. It is crucial for this process for B cells to take up antigen by a phagocytic mechanism, which results in stronger and more sustained BCR signals compared to stimulation with a soluble antigen. We also show the applicability of the system to generate antibodies of potential clinical interest.

immunology↗