BUSCADOR RECOLECTA

Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation., pbio.3002744.s019.xlsx, Peer reviewed

(A-C) Stacked bar distribution of PFAM domains, from A to C is bacteria, eukaryota and archaea, respectively. Each bar in the distribution is colored based on contribution from each phyla. Calculations were performed on a restricted subset limiting the maximum number of proteomes from each phylum to 50, as to not skew the distribution. Only the top 15 phyla in terms of contribution is shown, except for archaea. The distinct taxonomic pattern of each bar illustrates the differing contributions from each phyla. (D-F) Most common architectures of bacteria, eukaryotes and archaea, respectively. Sizes of the proteins are representative and not to scale., Peer reviewed

Only the largest clusters are shown for A) bacteria (top 36) and B) eukaryotes (top 32). All clusters are shown for (C) archaea. Proteins inside the clusters are represented by dots colored by phyla. Total number of HPs in dataset is shown for each phyla., Peer reviewed

Peer reviewed

(A) Distribution of the proportion of the dataset for each Protein Existence level (Uniprot), either for Huge proteins (Blue) or Non-Huge proteins (Orange). (B) Inset of A, zoom of the first two levels. (C) Scatter plot comparing the number of huge proteins per proteome (top row, green, each green dot is a proteome) or Protein Length (bottom row, red, each dot is a protein) with the BUSCO Completeness score for each proteome. For archaea, bacteria and eukaryota, from left to right, respectively. (D) Box plot relating the Number of Huge proteins per Proteome with the Assembly level of the genomes. For archaea, bacteria and eukaryota, from left to right, respectively. Colored by Assembly level category., Peer reviewed

(A) Whole distribution for all proteins in Uniprot (Release 2023_01). (B) Inset of the Distribution, for protein lengths bigger than 5000 amino acids, by superkingdom. Relevant proteins are highlighted. (C) Plot of the relationship between the size of the proteome (number of proteins), at log scale, and the number of huge proteins. (D) Box plot showing the likelihood of a proteome to contain huge proteins, per phylum, in percentage (number of huge proteins divided by proteome size). Each dot is a single proteome. All plots but A are colored blue, red and green for archaea, bacteria and eukaryota, respectively., Peer reviewed

An often-overlooked aspect of biology is formed by the outliers of the protein length distribution, specifically those proteins with more than 5000 amino acids, which we refer to as huge proteins (HPs). By examining UniprotKB, we discovered more than 41 000 HPs throughout the tree of life, with the majority found in eukaryotes. Notably, the phyla with the highest propensity for HPs are Apicomplexa and Fornicata. Moreover, we observed that certain bacteria, such as Elusimicrobiota or Planctomycetota, have a higher tendency for encoding HPs, even more than the average eukaryote. To investigate if these macro-polypeptides represent “real” proteins, we explored several indirect metrics. Additionally, orthology analyses reveals thousands of clusters of homologous sequences of HPs, revealing functional groups related to key cellular processes such as cytoskeleton organization and functioning as chaperones or as E3-ubiquitin ligases in eukaryotes. In the case of bacteria, the major clusters have functions related to non-ribosomomal peptide synthesis/polyketide synthesis, followed by pathogen-host attachment or recognition surface proteins. Further exploration of the annotations for each HPs supported the previously identified functional groups. These findings underscore the need for further investigation of the cellular and ecological roles of these HPs and their potential impact on biology and biotechnology., Peer reviewed

(A-C) corresponds to molecular function (MF), biological process (BP) and cellular component (CC), respectively. Each protein can contribute more than one GOterm to each category. (TIFF), Peer reviewed

(A) Scatter plot the Disorder percentage in relation to Protein Length. On the right is the same analysis, for Non-Huge proteins (less than 5000 aa’s). From top to bottom divided between Prokaryotes and Eukaryotes, respectively. Each subplot is followed by a histogram of disorder percentages. (B) Histogram of presence (red) or absence (blue) of signal peptides in relation to Protein Length. The y-axis is in logarithmic scale. (C) Density plot of the distribution of number of Transmembrane helices (TMHs) in relation to Protein Length, with corresponding rug-plot. Colored based on number of TMHs. The x-axis is in logarithmic scale. (TIFF), Peer reviewed

All clusters found colored by phyla, (A) Archaea, (B) Bacteria and (C) Eukaryotes. (TIFF), Peer reviewed