By integrating a literature review with transcriptomic, proteomic, and phenotypic data from two model bacteria, Escherichia coli and Vibrio cholerae, we put forward the hypothesis that defects in tRNA modification broadly impact processes that are evolutionarily tuned to be sensitive to translation speed. These include the translation of regulatory proteins associated with motility, iron homeostasis, and leader peptide-driven attenuation mechanisms. Some of these translation speed-dependent processes are influenced by the absence of a single modification, while others are affected by the absence of multiple modifications. Although further experiments are needed to clarify the mechanisms involved in each case, this work provides a foundational framework to guide future research.

Thirty to seventy percent of proteins in any given genome have no assigned function and have been labeled as the protein "unknome." This large knowledge shortfall is one of the final frontiers of biology. Machine learning (ML) approaches are enticing, with early successes demonstrating the ability to propagate functional knowledge from experimentally characterized proteins. An open question is the ability of ML approaches to predict enzymatic functions unseen in the training sets. By integrating literature and a combination of bioinformatic approaches, we evaluated individually Enzyme Commission number predictions for over 450 Escherichia coli unknowns made using state-of-the-art ML approaches. We found that current ML methods not only mostly fail to make novel predictions but also make basic logic errors in their predictions that human annotators avoid by leveraging the available knowledge base. This underscores the need to include assessments of prediction uncertainty in model output and to test for "hallucinations" (logic failures) as a part of model evaluation. Explainable artificial intelligence analysis can be used to identify indicators of prediction errors, potentially identifying the most relevant data to include in the next generation of computational models.

Epigenetic regulation of gene expression and host defense is well established in microbial communities, with dozens of DNA modifications comprising the epigenomes of prokaryotes and bacteriophage. Phosphorothioation (PT) of DNA, in which a chemically reactive sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is catalyzed by dnd and ssp gene families widespread in bacteria and archaea. However, little is known about the role of PTs or other microbial epigenetic modifications in the human microbiome. Here we optimized and applied fecal DNA extraction, mass spectrometric, and metagenomics technologies to characterize the landscape and temporal dynamics of gut microbes possessing PT modifications. Exploiting the nuclease-resistance of PTs, mass spectrometric analysis of limit digests of PT-containing DNA reveals PT dinucleotides as part of genomic consensus sequences, with 16 possible dinucleotide combinations. Analysis of mouse fecal DNA revealed a highly uniform spectrum of 11 PT dinucleotides in all littermates, with PTs estimated to occur in 5–10 The results of our studies provide a benchmark for understanding the behavior of an abundant and chemically reactive epigenetic mark in the human gut microbiome, with implications for inflammatory conditions of the gut.

The nucleobase queuine (q) and its nucleoside queuosine (Q) are micronutrients derived from bacteria that are acquired from the gut microbiome and/or diet in humans. Following cellular uptake, Q is incorporated at the wobble base (position 34) of tRNAs that decode histidine, tyrosine, aspartate, and asparagine codons, which is important for efficient translation. Early studies suggested that cytosolic uptake of queuine is mediated by a selective transporter that is regulated by mitogenic signals, but the identity of this transporter has remained elusive. Here, through a cross-species bioinformatic search and genetic validation, we have identified the solute carrier family member SLC35F2 as a unique transporter for both queuine and queuosine in Schizosaccharomyces pombe and Trypanosoma brucei. Furthermore, gene disruption in human HeLa cells revealed that SLC35F2 is the sole transporter for queuosine (Km 174 nM) and a high-affinity transporter for the queuine nucleobase (Km 67 nM), with the additional presence of second low-affinity queuine transporter (Km 259 nM). Ectopic expression of labeled SLC35F2 reveals localization to the cell membrane and Golgi apparatus via immunofluorescence. Competition uptake studies show that SLC35F2 is not a general transporter for other canonical ribonucleobases or ribonucleosides but selectively imports q and Q. The identification of SLC35F2, an oncogene, as the transporter of both q and Q advances our understanding of how intracellular levels of queuine and queuosine are regulated and how their deficiency contributes to a variety of pathophysiological conditions, including neurological disorders and cancer.

Transfer RNA (tRNA) modifications have emerged as critical post-transcriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications—the tRNA epitranscriptome—very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here, we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue would facilitate biomarker and drug discovery and development.

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX) brx genes, which were genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd, ssp, and brx genes revealed 8 PT dinucleotide settings confirmed in 6 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.

SUMMARY Deazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life.

Among dozens of known epigenetic marks, naturally occurring phosphorothioate (PT) DNA modifications are unique in replacing a non-bridging phosphate oxygen with redox-active sulfur and function in prokaryotic restriction-modification and transcriptional regulation. Interest in PTs has grown due to the widespread distribution of the dnd, ssp, and brx genes among bacteria and archaea, as well as the discovery of PTs in 5-10% of gut microbes. Efforts to map PTs in complex microbiomes using existing next-generation and direct sequencing technologies have failed due to poor sensitivity. Here we developed PT-seq as a high-sensitivity method to quantitatively map PTs across genomes and metagenomically identify PT-containing microbes in complex genomic mixtures. Like other methods for mapping PTs in individual genomes, PT-seq exploits targeted DNA strand cleavage at PTs by iodine, followed by sequencing library construction using ligation or template switching approaches. However, PT-specific sequencing reads are dramatically increased by adding steps to heat denature the DNA, block pre-existing 3'-ends, fragment DNA after T-tailing, and enrich iodine-induced breaks using biotin-labeling and streptavidin beads capture. Iterative optimization of the sensitivity and specificity of PT-seq is demonstrated with individual bacteria and human fecal DNA.

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: 1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and 2) Queuosine Precursor Transporter (QPTR), a transporter protein that imports Q precursors. Organisms like the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ(1) to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ(1) rather than q. Intriguingly, mass spectrometry analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ(1), previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ(1), akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens-from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ(1) has fitness advantages when B. henselae is growing outside a mammalian host.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome’s peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature’s preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

tRNAs are the central adaptor molecule in translation and require a wide variety of post-transcriptional modifications to fulfill their functions. The model gram-negative Escherichia coli K-12 is one of the handfuls of bacteria where all the tRNA modifications and corresponding genes have been characterized. This work dissects epistatic relationships between tRNA modification genes in E. coli by conducting a synthetic lethal screen revealing 5 pairs of modifications that cannot be deleted in combination when cells are grown in rich media, and 15 pairs of modifications that lead to growth defects when deleted in combination. The truA gene involved in the insertion of Psi residues at positions 38 to 40 in multiple tRNAs gave the highest number of synthetic lethality phenotypes that could be complemented by the expression of the truA gene in trans and in some cases suppressed by the overexpression of target tRNAs. A pilot phenotype screen of strains lacking two tRNA modifications genes viable in rich media lead to the identification of growth conditions that exhibited poor growth. This work lays the foundation to dissect the role of modifications in tRNA quality control.

Queuosine (Q) is a modification of the wobble base of tRNA harboring GUN anticodons with roles in decoding accuracy and efficiency. Its synthesis is complex with multiple enzymatic steps, and several pathway intermediates can be salvaged. The only two transporter families known to salvage Q precursors are QPTR/COG1738 and QrtT/QueT. Analyses of the distribution of known Q synthesis and salvage genes in human gut and oral microbiota genomes have suggested that more transporter families remain to be found and that Q precursor exchanges must occur within the structured microenvironments of the mammalian host. Using physical clustering and fusion-based association with Q salvage genes, candidate genes for missing transporters were identified and five were tested experimentally by complementation assays in Escherichia coli. Three genes encoding transporters from three different Pfam families, a ureide permease (PF07168) from Acidobacteriota bacterium, a hemolysin III family protein (PF03006) from Bifidobacterium breve, and a Major Facilitator Superfamily protein (PF07690) from Bartonella henselae, were found to allow the transport of both preQ0 and preQ1 in this heterologous system. This work suggests that many transporter families can evolve to transport Q precursors, reinforcing the concept of transporter plasticity.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nm YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ(0) and preQ(1) also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ(0) and preQ(1), the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ(1) from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.

The 7-deazapurine derivatives, 2 '-deoxy-7-cyano-7-deazaguanosine (dPreQ(0)) and 2 '-deoxy-7-amido-7-deazaguanosine (dADG) are recently discovered DNA modifications encoded by the dpd cluster found in a diverse set of bacteria. Here we identify the genes required for the formation of dPreQ(0) and dADG in DNA and propose a biosynthetic pathway. The preQ(0) base is a precursor that in Salmonella Montevideo, is synthesized as an intermediate in the pathway of the tRNA modification queuosine. Of the 11 genes (dpdA - dpdK) found in the S. Montevideo dpd cluster, dpdA and dpdB are necessary and sufficient to synthesize dPreQ(0), while dpdC is additionally required for dADG synthesis. Among the rest of the dpd genes, dpdE, dpdG, dpdI, dpdK, dpdD and possibly dpdJ appear to be involved in a restriction-like phenotype. Indirect competition for preQ(0) base led to a model for dADG synthesis in which DpdA inserts preQ(0) into DNA with the help of DpdB, and then DpdC hydrolyzes dPreQ(0) to dADG. The role of DpdB is not entirely clear as it is dispensable in other dpd clusters. Our discovery of a minimal gene set for introducing 7-deazapurine derivatives in DNA provides new tools for biotechnology applications and demonstrates the interplay between the DNA and RNA modification machineries.

Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ(0)), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ(0) to 7-aminomethyl-7-deazaguanine (preQ(1)). PreQ(1) is inserted into tRNAs by tRNA guanine((34)) transglycosylase (TGT). The inserted base preQ(1) is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ(0) synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ(0), preQ(1) or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ(0)/preQ(1) transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ(0) transport.

Significance The discovery of a novel modification system that inserts 7-deazaguanine derivatives in DNA, modifications thought until now to occur only in RNA, is an excellent illustration of the power of biological evolution to alter the ultimate function not only of the distinct proteins but also of entire metabolic pathways. The extensive lateral transfer of the gene cluster responsible for this modification highlights its significance as a previously unrecognized foreign DNA defense system that bacteria and phages use to protect their genomes. The characterization of these DNA modification pathways also opens the door to novel tools to manipulate nucleic acids.