Abstract tRNA modifications are crucial for fine-tuning of protein translation. Queuosine (Q) modification of tRNAs is thought to modulate the translation rate of NAU codons, but its physiological role remains elusive. Therefore, we hypothesize that Q-tRNAs control those physiological processes involving NAU codon-enriched genes (Q-genes). Here, we report a novel bioinformatic strategy to predict Q-genes, revealing a widespread enrichment in functions, especially those related to biofilm formation and virulence in bacteria, and particularly in human pathogens. Indeed, we experimentally verified that these processes were significantly affected by altering the degree of tRNA Q-modification in different model bacteria, representing the first report of a general mechanism controlling biofilm formation and virulence in Gram-positive and Gram-negative bacteria possibly through the coordination of the expression of functionally related genes. Furthermore, we propose that changes in Q availability in a microbiome would affect its functionality. Our findings open the door to the control of bacterial infections and biofilm formation by inhibition of tRNA Q-modification.

tRNA queuosine modification is involved in biofilm formation and virulence in bacteria

SUMMARYDeazaguanine 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.

Biosynthesis and function of 7-deazaguanine derivatives in bacteria and phages.

Queuine (Que) is an essential micronutrient, and its deficiency will lead to errors in tRNA modification and protein misfold, further to diseases. Meanwhile, the content of Que in food will be further reduced due to the modern planting pattern and processing. In this study, an idea, synthetic biology of gut microbiome, was introduced to meet people's demand for Que. Further, various food can be used as a media for promoting the growth of synthetic biology of flora and the concentrations of Que. Through this research, gut bacteria (Escherichia coli K12, Bacteroides fragilis ATCC25285, and Lactobacillus reuteri DSM20058) with abilities of Que independent synthesis in vitro were selected, and higher productivity of Que was fulfilled through the above three strains cocultivation via cross‐feeding. Moreover, the effect of Que on the structure of microbiome was also studied, in order to figure out the continuous synthesis of Que by this coculture system in colonic situation. Que could enhance bacterial α‐diversity, increase the abundance of Limosilactobacillus, Pediococcus, and Lactobacillus, while decrease the abundance of Eschi‐Shigella. Three bacteria combination intervention changed intestinal microbial composition, and distribution of enzymes transglycozyme, QueA, and QueG that involved in last three steps of Que synthesis in microorganisms was significantly changed. These results provide an experimental basis for colonic synthesis of Que via food intervention to solve the deficiency Que in human body. Here, we regard gut microbes as a fermentation system in human body, providing a constant supply of tiny but important substance that humans need.

Synthesis of queuine by colonic gut microbiome via cross‐feeding

Significance A poorly understood aspect of the bacterial adaptive response to host-mediated Zn(II) restriction is the upregulation of functional paralogs to replace obligatory Zn(II)-dependent proteins. Here, we find that metal limitation disrupts the biosynthesis of queuosine-transfer RNA. We present the first structure of a “low zinc” paralog of 6-carboxy-5,6,7,8-tetrahydropterin (6-CPH4) synthase, QueD2, and highlight key structural and metallocofactor-dependent differences relative to a housekeeping QueD. A key distinction is the presence of dynamic Zn(II) binding loop, incorporating an invariant cysteine, that slows dissociation of the catalytic metal of QueD2 in what is termed a “metal retention” model that is absent in QueD. This work highlights a mechanism in the adaptive response to Zn(II) limitation in an important human pathogen.

Metal retention and replacement in QueD2 protect queuosine-tRNA biosynthesis in metal-starved Acinetobacter baumannii

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 (TGT), 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 TGT and QPTR but lack predicted enzymes for converting preQ1 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 TGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, mass spectrometry analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae TGT and QPTR not only utilize preQ1, akin to their E. 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 preQ1 has fitness advantages when B. henselae is growing outside a mammalian host. Author summary Transfer RNAs (tRNAs) are adaptors that deliver amino acids to ribosomes during translation of messenger RNAs (mRNAs) into proteins. tRNA molecules contain specially-modified nucleotides that affect many aspects of translation including regulation of translational efficiency, as modified nucleotides primarily occur near the portion of tRNA (anticodon) that directly interacts with the coding sequence (codon) of the mRNA while it is associated with a ribosome. Queuosine (Q) is a modified tRNA nucleotide located in the anticodon that can be synthesized or uniquely imported from the environment as Q or a precursor using a salvage mechanism. Free-living bacteria, e.g., E. coli, can synthesize Q or salvage precursors from the environment, but many obligate intracellular pathogens, e.g., Chlamydia trachomatis, cannot synthesize Q and must import a precursor from eukaryotic hosts. In this study, we determined that Bartonella henselae, a facultative intracellular bacterial pathogen of vascular cells, falls somewhere in the middle, as it is unable to synthesize Q but can salvage Q or certain precursors. The unusual nature of Bartonella’s system suggests different evolutionary scenarios. It could be a snapshot of the transition from Q synthesis to strict Q salvage or represent a unique adaptation to a complex multi-host lifestyle.

Queuosine Salvage in Bartonella henselae Houston 1: A Unique Evolutionary Path

Abstract 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.

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12

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The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12