Environmental Control of Queuosine Levels in Streptococcus mutans tRNAs

Abstract

Queuosine (Q) is a modification of the wobble base in tRNAs that decode NA(C/U) codons. It is ubiquitous in bacteria, including many pathogens. Streptococcus mutans is an early colonizer of dental plaque biofilm and a key player in dental caries. Using a combination of genetic and physiological approaches, the predicted Q synthesis and salvage pathways were validated in this organism. These experiments confirmed that S. mutans can synthesize Q de novo through similar pathways found in Bacillus subtilis and Escherichia coli. However, S. mutans has a distinct salvage pathway compared to these model organisms, as it uses a transporter belonging to the energy coupling factor (ECF) family controlled by a preQ₁-dependent riboswitch. Furthermore, Q levels in this oral pathogen depended heavily on the media composition, suggesting that micronutrients can affect Q-mediated translation efficiency.

Keywords: biofilm; oral pathogen; preQ0; preQ1; riboswitch; tRNA modification.

Figure 1.. Predicted queuosine biosynthesis and salvage pathway of S. mutans UA159.

Locus tags of the coding sequence for each enzyme are listed next to each step in the pathway. Dashed lines indicate possible substrate of the transporter QueT. Protein names are described in the main text.

Figure 2.. Validation of predicted S. mutans Q synthesis genes.

(A) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC extracted from WT and mutant strains of S. mutans UA159 grown in FMCG medium. (B) LC-MS analysis of Queuosine (Q) and Epoxyqueuosine (oQ) detected from the queG mutant and WT strain. Absolute amounts of Q and oQ in one million ribonucleosides were quantified. Error bars represent one standard deviation. Signals below the limit of detection are indicated as n.d. (C) MS chromatograms for oQ (m/z 426> 163) and Q (m/z 410> 163) in WT strain (upper panel) and queG mutant (bottom panel).

Figure 3.. QueT is required for salvaging preQ₁ and preQ_0.

Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC extracted from Wild-Type (WT) and mutant S. mutans strains grown in FMCG medium with 5 nM of preQ₀, preQ_1, or an equivalent volume of DMSO.

Figure 4.. queT Promoter-Beta-galactosidase Fusion Activity Shows Sensitivity to preQ₁.

(A) Diagram of pPqueT-lacZ construction and annotated preQ₁ riboswitch sequence. Complementary sequences that form the secondary structures of the riboswitch are highlighted in blue, green, and pink. The sections that form secondary structures in the presence of preQ₁ are highlighted in yellow (Meyer et al., 2008). The queT start codon is shown in bold, it was the site of fusion with the lacZ gene (B) Cells were plated on TYG medium supplemented with bromochloroindoxyl galactoside (X-gal). 20μL of 10 mM solutions of 7-cyano-7-deazaguanine (preQ₁) or 7-7-aminomethyl-7-deazaguanine (preQ₀) was allowed to diffuse through the center well of each plate. (C) β-galactosidase assays. S. mutans UA159 bearing a lacZ fused to the promoter sequence of queT in the Wild-Type (WT; blue bars) and the Δtgt (light grey bars) backgrounds grown in FMCG medium with various concentrations of preQ₁. Error bars = standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 (Unpaired T-test, compared with the 0 nM treatment for each strain).

Figure 5.. Effects of media composition on Q modification levels.

(A) Detection of Q by LC-MS in bulk tRNA extracted from S. mutans cells grown in Brain Heart Infusion (BHI) or semi-defined Biofilm Medium (BM), the growth curve of the cells used for sample collection is given in Fig. S2 of (Bacusmo et al., 2018). The graph displays the normalized MS signals for Q alone, excluding signals from any Q precursors (See Method). Statistical significance (adjusted p value) is performed against paired time points, indicated as follows: * (p < 0.05), *** (p < 0.001), and **** (p < 0.0001), determined by two-way ANOVA test. (B) Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC extracted at different time points from WT and Δtgt S. mutans strains grown in different growth media. tRNA extracted from Δtgt cells grown in Brain Heart Infusion (BHI) medium was added as a Q⁻ negative control. The growth curve of the cells used for sample collection is given Fig. S4.

Figure 6.. Effects of BHI and peptone on Q levels.

Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC in Wild-Type (WT) cells grown in different media. tRNA extracted from Δtgt cells grown in Brain Heart Infusion (BHI) medium was added as a Q⁻ negative control. (A) Media composed of mixtures of BHI and FMCG, V/V% are indicated at the top of each lane. (B) FMCG medium supplemented with various concentrations of peptone. The concentration of peptone added to each media preparation is indicated as a V/V% above each lane.

Figure 7.. Component(s) of low molecular weight fraction of peptone repress Q levels.

Detection of Q-tRNA by the APB assay in tRNA^Asp_GUC prepared from WT cells grown in BHI or in FMC, supplemented with 5% peptone or with the various fractions derived from peptone (see main text for details). tRNA extracted from Δtgt cells grown in BHI medium was added as a Q⁻ negative control.