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. 2019 Sep 20;201(20):e00433-19.
doi: 10.1128/JB.00433-19. Print 2019 Oct 15.

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Charles T Lauhon  1
Affiliations

Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Charles T Lauhon. J Bacteriol. .

Abstract

In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification.IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

Keywords: Fe-S cluster protein; tRNA; tRNA modification.

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Figures

FIG 1
FIG 1
Biosynthesis of ho5U derivatives in bacterial tRNA. In E. coli and other gammaproteobacteria, CmoA and CmoB convert AdoMet to carboxy-AdoMet and transfer the carboxymethyl group to ho5U to give cmo5U. The latter is then methylated by CmoM to give the terminal modification mcmo5U. In B. subtilis and most other bacteria, ho5U is methylated directly by the recently identified TrmR (YrrM) methyltransferase (22). The genes required for ho5U synthesis have yet to be described and are the subject of this work.
FIG 2
FIG 2
(A) Organization of the two peptidase U32 genes in B. subtilis and four genes in E. coli. Numbers indicate corresponding protein amino acid length. (B) Clustal-omega amino acid alignment of peptidase U32 homologs from E. coli and B. subtilis. Putative Fe-S cluster cysteines are highlighted in yellow although there are other cysteines in the region. Amino acids that differ from cysteine in the conserved positions are shown in red (YhbU is the exception in which an adjacent cysteine has been included as a potential Fe-S ligand).
FIG 3
FIG 3
(A) Representative HPLC analysis of mo5U levels of B subtilis wt and mutant strains. The first trace shows synthetic standards of modified nucleosides relevant to this work. Total tRNA isolated from each strain (12.5 μg) is enzymatically digested to nucleosides and analyzed by HPLC with detection by absorbance at 280 nm. (B) Quantitative effect of selected B. subtilis peptidase U32 gene deletions on levels of mo5U in purified total tRNA based on the ratio of peak integration relative to pseudouridine (Ψ). ***, P < 0.001 (unpaired t test); **, P < 0.01 compared to wt strain; ns = no statistically significant difference between levels in these strains.
FIG 4
FIG 4
(A) HPLC analysis of cmo5U levels of various E. coli peptidase U32 gene deletion mutants analyzed as described for Fig. 3A. The last two panels show the ΔyegQ ΔyhbUV ΔrlhA quadruple mutant without and with coinjection of cmo5U standard. The cmo5U peak was also isolated from this mutant and verified for identity by high-resolution mass spectrometry. (B) Quantitative analysis of selected gene deletions and complementation of E. coli peptidase U32 gene deletions based on HPLC analyzed levels of cmo5U in purified total tRNA. ***, P < 0.001 (unpaired t test) compared to wt strain.
FIG 5
FIG 5
Effects of other genes on xo5U levels. (A) HPLC analysis of iscS, yfhL, and aroD in E. coli. The iscS mutant lacks thionucleosides s2C and mnm5s2U and cmnm5s2U in this region but still retains cmo5U, which is decreased to a similar level as the yegQ mutant (shown in panel B). The overnight iscS mutant culture shows significant increases in s2C and some increase in cmo5U, similar to increases in other Fe-S-dependent modifications. The effect on cmo5U is not statistically significant due to variability in the log-phase data. An E. coli strain lacking the ferredoxin yfhL shows a similar HPLC spectrum as that of the yegQ mutant and a similar level of reduction in cmo5U (shown in panel B). In E. coli, shikimate pathway genes, such as aroD, are required for conversion of ho5U to cmo5U and for wt levels of ho5U, as previously reported (15, 19). Quantifying the level of ho5U shows the same reduction in ho5U as for cmo5U in the yegQ and yfhL mutants. (B) Quantitative analysis of HPLC data for E. coli aro, iscS, and yfhL mutants relative to those of the wt strain. *, the aroD strain lacks the ability to synthesize cmo5U from ho5U and thus the ratio reported here is for ho5U/Ψ. ***, P < 0.001 compared to wt (unpaired t test); **, P < 0.01 compared to wt; ns = no statistical significance among these strains or with the wt strain.
FIG 6
FIG 6
HPLC profiles of enzymatic digests of individual tRNAs (ca. 6 μg each) purified from B. subtilis wt and yrrO mutant strains. The expected modifications for each tRNA are listed on the left for reference. Some residual deoxynucleosides remain after DNase treatment and gel filtration. Detection is by absorbance at 280 nm.
FIG 7
FIG 7
Native PAGE analysis shows evidence of a YrrN-YrrO complex. Increasing concentrations of His6-YrrN are incubated with 2 μM His6-YrrO in 30 mM Tris-glycine with 5 mM DTT (20 μl total volume) for 30 min at RT and then analyzed on a 10% nondenaturing polyacrylamide gel using 30 mM Tris-glycine, pH 7.5, as the buffer. Gel was stained with Coomassie blue. Lane 1, 2 μg bovine serum albumin; lane 2, 2 μg purified His6-YrrN; lane 3, 4 μg YrrN; lane 4, 2 μg purified His6-YrrO; lane 5, 2 μg YrrO plus 0.125 eq YrrN; lane 6, YrrO plus 0.25 eq YrrN; lane 7, YrrO plus 0.5 eq YrrN; lane 8, YrrO plus 1 eq YrrN; lane 9, YrrO plus 2 eq YrrN. Arrows show position of major (A) and minor (B) potential YrrNO complexes.
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