Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens

Abstract

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 preQ₀ and preQ₁ also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ₀ and preQ₁, 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 preQ₁ 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.

Keywords: comparative genomics; nucleoside transport; queuosine; rSAM; sequence similarity network.

Fig. 1.

Queuosine tRNA modification biosynthesis and predicted salvage pathways. (A) Biosynthesis of the Q modification at position 34 (Q₃₄-tRNA) and preQ₀/preQ₁ salvage pathway in E. coli. (B) Predicted Q₃₄-tRNA biosynthesis and queuine salvage pathway in C. trachomatis D/UW-3/CX. Red dashed arrows represent uncharacterized reactions. Molecule abbreviations and protein names are described in the main text.

Fig. 2.

C. trachomatis salvages queuine in 2 steps. (A) Amino acid sequence alignment of select TGT proteins using PROMALS3D (74). The catalytic residues are shown in bold. The residues that accommodate the 7-substituent group of the substrate are shown in red. Dots indicate regions intentionally deleted for this figure. Dashes indicate gaps in the sequence alignment. UniProt IDs for proteins included in multiple alignment are as follows: Zymomonas mobilis (P28720), E. coli (P0A847), Shigella flexneri (Q54177), Homo sapiens QTRT1 (Q9BXR0), Caenorhabditis elegans (Q23623), C. trachomatis (A0A0E9DEF3), C. caviae (Q822U8), and C. psittaci (A0A2D2DY33). (B) Comparison of substrate-binding pockets of TGT proteins. The binding pocket of TGT_Ct is modeled and docked with q (purple), compared with that of Z. mobilis TGT (green; crystal structure with docked q) and H. sapiens QTRT1 (orange; crystal structure bound to q). The residues that accommodate the substrate’s 7-substituent moiety are shown in a stick model; S₂₃₃LG₂₃₅ in TGT_Ct, L₂₃₁AVG₂₃₄ in Z. mobilis TGT and L₂₃₀SGG₂₃₃ in H. sapiens QTRT1. Queuine is colored bright green. A steric clash in Z. mobilis TGT precludes binding of q (dashed circle). (C) Protein sequence similarity network of 6,187 YhhQ sequences that were retrieved from the PubSEED subsystem “q_salvage_in_Bacteria” and colored based on the predicted salvaged molecule in the organism from which they originate: red for preQ₀, yellow for preQ₁, and dark blue and sky blue for queuine. Red and blue arrows indicate YhhQ homologs from E. coli and C. trachomatis, respectively. (D) Scheme of Q metabolism in the E. coli derivatives used to test the function of CT140 and CT193. Dashed arrows represent reactions that are being tested. Precursors in gray are not synthesized de novo in these strains. (E and F) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC in the presence of exogenous queuine (q) while expressing CT140/yhhQ_Ct and/or CT193/tgt_Ct in different E. coli derivatives.

Fig. 3.

CD1682, CD1683, and CD1684 are required for queuosine modification in tRNA of C. difficile. (A) Predicted Q-tRNA biosynthesis pathway in C. difficile strain 630. The magnified subfigure shows a model of the ECF transporters that include 4 subunits: S, the substrate-specific transmembrane component (S component); T, the energy-coupling module consisting of a transmembrane protein (T component); and A and A′, pairs of ABC ATPases (A proteins). Dashed arrows represent uncharacterized reactions. Molecule abbreviations and protein names are described in the text. (B) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC of C. difficile 630 WT, 630 Δtgt, and 630 ΔCD1682-CD1684 strains. tRNA extracted from E. coli WT and Δtgt strains was used as control. (C) Representation of the genomic context of the radical SAM cluster. C. difficile 630 (accession: NC_009089.1), C. perfringens ATCC 13124 (accession: NC_008261.1), Clostridium botulinum E1 str. “BoNT E Beluga” (accession: NZ_ACSC00000000.1), R. gnavus ATCC 29149 (accession: NZ_AAYG02000018.1), and L. bacterium 2_1_58FAA (accession: ACTO00000000.1). Each gene is colored according to Pfam domain. Predicted promoters and rho-independent terminators are indicated by dashed arrows and dots, respectively. PreQ₁ riboswitches are indicated by stem loops.

Fig. 4.

CD1682 is a queuosine hydrolase (QueK). (A) The queuosine hydrolysis activity of purified recombinant CD1682 was assayed and analyzed at different time points by injection of the reaction mixture into an HPLC system and measurement of the absorbance at 260 nm. The assay was performed with 100 µM queuosine and 100 nM CD1682. A control incubated without enzyme is included. (B and C) The identities of the substrate and product, with corresponding retention times, were verified by extracting the ion counts for the expected masses [M⁺H⁺] = m/z 278 for queuine and 410 for queuosine, respectively.

Fig. 5.

The ECF substrate specificity component CD1683 transports preQ₁, queuine, and queuosine. (A) Scheme of Q metabolism in the E. coli derivatives used to test substrate specificity (queuine, preQ₁, and preQ₀) of the ECF transporter genes. Genes encoding C. difficile ECF core components (CD100, CD101, CD102) with those encoding different substrate-specific components (CD1683, CD2097, and CD3073) were expressed in E. coli ΔqueD ΔyhhQ pBAD24::tgt_Ct strain. Dashed arrows represent reactions being tested. (B) Q-tRNA levels were detected by the ABP assay in tRNA^Asp_GUC extracted 60 min after supplementing with different precursors. (C) Scheme of Q metabolism in the E. coli strains used to test queuosine transport and hydrolysis activity. Genes coding different transporters, including YhhQ_Ct and ECF complex with S component (CD1683, CD2097, or CD3073), were coexpressed with the predicted queuosine hydrolase (CD1682) in E. coli ΔqueD ΔyhhQ strains expressing both tgt_Ec and tgt_Ct. Red dashed arrows represent reactions being tested. Precursors in gray are not synthesized de novo in these strains. (D) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC extracted 60 min after supplementing with exogenous queuosine (Q; 10 or 500 nM).

Fig. 6.

CD1684 generates preQ₁ from queuine in vivo. (A) Scheme of Q metabolism in the E. coli derivatives used to test the activity of CD1684. Red dashed arrows represent reactions being tested. Precursors in gray are not synthesized de novo in these strains. (B) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC extracted 60 min after supplementing with different precursors. The C. difficile CD1684 gene was expressed in E. coli ΔqueD ΔqueF pBAD33::yhhQ_Ct strain and ΔqueD ΔqueF ΔqueA pBAD33::yhhQ_Ct strain.

Fig. 7.

Queuine lyase (QueL) activity and structure. (A) The queuine lyase activity of purified recombinant CD1684 was analyzed by separation of quenched reaction mixture by an HPLC system with monitoring at 260 nm absorbance. Data from a representative assay are presented from an assay performed under anaerobic conditions with 100 µM queuine, 200 µM sodium dithionite, 66.67 µM S-adenosyl-l-methionine (SAM limited to allow for visualization of preQ₁; otherwise, a high concentration of SAM was used), and 10 µM of purified CD1684. The control is a reaction lacking enzyme. (B–E) The identities of the substrate and product, with corresponding retention times, were verified by extracting the ion counts for the expected masses [M+H+] = m/z 278, 180, 399, and 252 for queuine, preQ₁, SAM, and 5′dA (5′deoxyadenosine), respectively. The UV signal and mass corresponding to queuine and SAM are reduced over time, while signal and mass corresponding to preQ₁ and 5′dA are increased, demonstrating that CD1684 is an RS enzyme with queuine lyase activity. (F) Overall view of QueL_Cs with secondary structural elements assigned numerically. The α6 extension (blue) caps the active site terminating in Asp229. SAM (light gray) and queuine (dark gray) are depicted as ball-and-stick illustrations (oxygen, red; nitrogen, blue). The RS cluster is depicted as spheres (iron, burnt orange; sulfur, yellow). (G) A top-down view of the active site showing highly conserved amino acids (depicted as ball and sticks in pink) and hydrogen bonds (dashed lines) formed between substrates (coloring as in A) and the QueL_Cs active site. The red dashed line denotes the distance (3.5 Å) between the 5′-carbon of SAM and the 5′-oxygen of queuine. (H) Mechanistic proposal for catalysis by QueL.