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The DNA phosphorothioate (PT) modification existing in many prokaryotes, including bacterial pathogens and commensals, confers multiple characteristics, including restricting gene transfer, influencing the global transcriptional response, and reducing fitness during exposure to chemical mediators of inflammation. While PT-containing bacteria have been investigated in a variety of environments, they have not been studied in the human microbiome. Here, we investigated the distribution of PT-harboring strains and verified their existence in the human microbiome. We found over 2000 PT gene-containing strains distributed in different body sites, especially in the gastrointestinal tract. PT-modifying genes are preferentially distributed within several genera, including Pseudomonas, Clostridioides, and Escherichia, with phylogenic diversities. We also assessed the PT modification patterns and found six new PT-linked dinucleotides (CpsG, CpsT, ApsG, TpsG, GpsC, ApsT) in human fecal DNA. To further investigate the PT in the human gut microbiome, we analyzed the abundance of PT-modifying genes and quantified the PT-linked dinucleotides in the fecal DNA. These results confirmed that human microbiome is a rich reservoir for PT-containing microbes and contains a wide variety of PT modification patterns. 

Abstract DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3′-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3′-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage. 

ABSTRACT During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK , a gene that encodes the ribosome-associated scaffold protein, LACK ( Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACK R34D35G36 →LACK D34D35E36 ) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACK D34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome. IMPORTANCE Leishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani . The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK’s putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK’s interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK’s ribosomal interactions as a potential therapeutic target. 

Phosphorothioate (PT) DNA modifications—in which a nonbonding phosphate oxygen is replaced with sulfur—represent a widespread, horizontally transferred epigenetic system in prokaryotes and have a highly unusual property of occupying only a small fraction of available consensus sequences in a genome. UsingSalmonella entericaas a model, we asked a question of fundamental importance: How do the PT-modifying DndA-E proteins select their G_PSAAC/G_PSTTC targets? Here, we applied innovative analytical, sequencing, and computational tools to discover a novel behavior for DNA-binding proteins: The Dnd proteins are “parked” at the G^6mATC Dam methyltransferase consensus sequence instead of the expected GAAC/GTTC motif, with removal of the^6mA permitting extensive PT modification of GATC sites. This shift in modification sites further revealed a surprising constancy in the density of PT modifications across the genome. Computational analysis showed that GAAC, GTTC, and GATC share common features of DNA shape, which suggests that PT epigenetics are regulated in a density-dependent manner partly by DNA shape-driven target selection in the genome.