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The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNA^{Glu, Gln, Asp}contains a variety of xnm⁵s²U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negativeEscherichia coliK12 model. Despite the ubiquitous presence of mnm⁵s²U modification, genomic analysis shows the absence ofmnmCorthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm⁵s²U to mnm⁵s²U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm⁵s²U in bothBacillus subtilisandStreptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm⁵s²U into mnm⁵s²U inB. subtilis. Analysis of tRNA modifications of bothS. mutansandStreptococcus pneumoniaeshows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm⁵s²U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm⁵s²U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications. 

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. 

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.