Biology subjects

Aramayo, R.

Publications and source records attributed to Aramayo, R..

7 recordsLinked to original sources

bioRxiv2024

Diverse toxin repertoire but limited metabolic capacities inferred from the draft genome assemblies of three Spiroplasma (Citri clade) strains associated with Drosophila

Spiroplasma (Class Mollicutes) is a diverse wall-less bacterial genus whose members are strictly dependent on eukaryotic hosts (mostly arthropods and plants), with which they engage in pathogenic to mutualistic interactions. Spiroplasma are generally fastidious to culture in vitro, especially those that are vertically transmitted by their hosts, which include flies in the genus Drosophila. Drosophila has been invaded by at least three independent clades of Spiroplasma: Poulsonii (the best studied; contains reproductive manipulators and defensive mutualists associated two major clades of Drosophila; and has among the highest substitution rates within bacteria); Citri (restricted to the repleta group of Drosophila); and Ixodetis. We report the first genome drafts of Drosophila-associated Citri Clade Spiroplasma: strain sMoj from D. mojavensis; strain sAld-Tx from D. aldrichi from Texas (newly discovered; also associated with D. mulleri); and strain sHy2 from D. hydei (the only Drosophila species known to naturally also harbor a Poulsonii clade strain, thereby providing an arena for horizontal gene transfer). Compared to their Poulsonii clade counterparts, we infer that the three Citri clade strains have: (1) equal or worse DNA repair abilities; (b) more limited metabolic capacities, which may underlie their comparatively lower titers and transmission efficiency; and (c) similar content of toxin domains, including at least one ribosome inactivating protein (RIP), which are implicated in the Poulsonii-conferred defense against natural enemies. As a byproduct of our phylogenomic analyses and exhaustive search for certain toxin domains in public databases, we document the toxin repertoire in close relatives of Drosophila-associated Spiroplasma, and in a very divergent newly discovered lineage (i.e., "clade X"). Phylogenies of toxin-encoding genes or domains imply substantial exchanges between closely and distantly related strains. Surprisingly, despite encoding several toxin genes and achieving relatively high prevalences in certain natural populations (sAld-Tx in this study; sMoj in prior work), fitness assays of sMoj (this study) and sAld-Tx (prior work) in the context of wasp parasitism fail to detect a beneficial effect to their hosts. Thus, how Citri clade strains persist in their Drosophila host populations remains elusive. Data summaryAll novel sequencing data are available through National Center for Biotechnology Information (NCBI) repositories. Illumina raw reads, assemblies, and NCBI annotations are available under BioProject Nos. PRJNA506493 for sHy2, PRJNA506491 for sAld-Tx, and PRJNA355307 for sMoj. Oxford Nanopore (MinIon) reads for sHy2 are under SRA Accession Number SRR12348752. Supporting Material is available under the DOI 10.6084/m9.figshare.c.7437997 or as accompanying supporting documents in the corresponding preprint server or scientific journal. Impact statementSymbiotic associations between arthropods and inherited microbes are pervasive, taxonomically and mechanistically diverse, and strongly influential. Research into the mechanisms and processes governing such heritable interactions is hindered by our inability to culture most inherited symbionts outside of their hosts. We studied three heritable strains of Spiroplasma (Citri clade) that naturally associate with Drosophila flies, and that reach relatively high prevalence in certain host populations, but appear to lack traits that would enable them to persist in host populations (e.g. such as high vertical transmission efficiency, reproductive manipulation, or fitness benefits). We compared their genomes to those of a separate Spiroplasma clade (Poulsonii) that associates with Drosophila, which does exhibit some of the traits that contribute to persistence, including protection against natural enemies of their hosts, and also has among the highest DNA substitution rates recorded for bacteria. Compared to Poulsonii, the three Citri clade strains have smaller genomes and fewer genes, leading us to predict they have similarly high DNA substitution rates, but more limited metabolic capacities, which may explain the comparatively lower densities they achieve within individual hosts, and their frequent loss in lab colonies of their hosts. However, the toxin repertoire of Citri clade was comparatively diverse, and the result of horizontal gene exchange among close and distant strains, and within-genome shuffling. We hypothesize that Citri clade strains persist via unknown fitness benefits conferred to their hosts, possibly mediated by toxins, or by substantial horizontal transmission. Our results, which also capitalized on publicly available assemblies, expand the range of Spiroplasma lineages that encode a particular combination of toxin types, and revealed the existence of a highly divergent lineage of Spiroplasma that associates with insects.

genomics↗

bioRxiv2024

A Re-Analysis of an Existing Drosophila melanogaster Dataset Reveals a New Set of Genes Involved in Post-Mating Response

RNA sequencing (RNA-seq) is a commonly used method to identify changes in gene expression between two conditions. The analysis of RNA-seq output is complicated, with the possibility of getting different results from the same raw data. We developed and deployed four parallel pipelines to reanalyze an existing dataset of two female Drosophila melanogaster tissue types before and after mating. The Drosophila post-mating response (PMR) is a well-characterized suite of changes that occur after mating, accompanied by a flux in gene expression. In comparing our study with the previous analysis of this dataset, we find our results to be more stringent, though we do identify a number of significant genes not found before. We also found variation among our own separate experiments, with gene-to-transcript isoform number and index building playing important roles in outcome. Finally, we identified a set of genes found by our pipeline that were not identified by the previous study and proposed potential roles for these genes in post-mating biology. Together, this work presents a critique of current RNA-seq analysis techniques and proposes multiple workflow adjustments that can increase the sensitivity, specificity, and stringency of differential gene expression studies.

bioinformatics↗

bioRxiv2024

Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

Human DNA licensing initiates the process of replication fork assembly. Specifically, this reaction leads to the loading of hMCM2-7 on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the biochemical reconstitution of human DNA licensing using purified proteins, the structural and functional analysis of the process and reveal the impact of cancer-associated mutations on DNA licensing. We showed that the in vitro reaction is specific and results in the assembly of high-salt resistant hMCM2-7 double-hexamers, the final product of DNA licensing. We used ATP{gamma}S to block complex assembly at the hOrc1-5-Cdc6-Cdt1-MCM2-7 step. We observed that the assembly of this intermediate is independent of hOrc6, although hOrc6 enhances the loading of the second hMCM2-7 hexamer. The structural and mutational analysis of the hOrc1-5-Cdc6-Cdt1-MCM2-7 complex provides insights into hORC-Cdc6 dependent recruitment of hMCM2-7 via five hMcm winged-helix domains. The structure highlights how hOrc1 activates the hCdc6 ATPase, while the analysis of hOrc1 and hCdc6 ATPase mutants uncovered an unexpected role for hCdc6 ATPase in complex disassembly. The structure highlights that Cdc6 binding to Orc1-5 stabilises Orc2-DNA interactions and supports Mcm3-dependent recruitment of MCM2-7. Finally, the structure allowed us to locate cancer-associated mutations at the hCdc6-Mcm3 interface, which showed specific helicase loading defects.

biochemistry↗

bioRxiv2020

Rapid molecular evolution of Spiroplasma symbionts of Drosophila

Spiroplasma are a group of Mollicutes whose members include plant pathogens, insect pathogens, and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii, a natural symbiont of Drosophila. We analysed genomic evolution of sHy within flies, and sMel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma and likely contributes to elevated substitution rates. Further, the closely related strains sMel and sHy (>99.5% sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in sHy, a protective symbiont of Drosophila hydei. Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.

evolutionary biology↗

bioRxiv2020

Translational control of methionine and serine metabolic pathways underpin the paralog-specific phenotypes of Rpl22 ribosomal protein mutants in cell division and replicative longevity

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Some may reflect general effects due to lower growth rate and ribosome levels, but a number of diverse phenotypes cannot be explained through this mechanism. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we comprehensively queried actively dividing RP paralog mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered specific translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes, such as the serine hydroxymethyltransferase Shm2p increased the longevity of wild type cells. These results provide a molecular basis for paralog-specific phenotypes in ribosomal mutants and underscore the significance of 1C metabolic pathways in mechanisms of cell division and cellular aging. 1C pathways exist in all organisms, including humans, and targeting the relevant enzymes could represent longevity interventions.

molecular biology↗

bioRxiv2019

A conserved activity for cohesin in bridging DNA molecules

Essential processes such as accurate chromosome segregation, regulation of gene expression and DNA repair rely on protein-mediated DNA tethering. Sister chromatid cohesion requires the SMC complex cohesin to act as a protein linker that holds replicated chromatids together (1, 2). The molecular mechanism by which cohesins hold sister chromatids has remained controversial. Here, we used a single molecule approach to visualise the activity of cohesin complexes as they hold DNA molecules. We describe a DNA bridging activity that requires ATP and is conserved from yeast to human cohesin. We show that cohesin can form two distinct classes of bridges at physiological conditions, a \"permanent bridge\" able to resists high force (over 80pN) and a \"reversible bridge\" that breaks at lower forces (5-40pN). Both classes of bridges require Scc2/Scc4 in addition to ATP. We demonstrate that bridge formation requires physical proximity of the DNA segments to be tethered and show that \"permanent\" cohesin bridges can move between two DNA molecules but cannot be removed from DNA when they occur in cis. This suggests that separate physical compartments in cohesin molecules are involved in the bridge. Finally, we show that cohesin tetramers, unlike condensin, cannot compact linear DNA molecules against low force, demonstrating that the core activity of cohesin tetramers is bridging DNA rather than compacting it. Our findings carry important implications for the understanding of the basic mechanisms behind cohesin-dependent establishment of sister chromatid cohesion and chromosome architecture.

biochemistry↗

bioRxiv2019

Effect of heritable symbionts on maternally-derived embryo transcripts

Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.

genomics↗