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

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.

SUBMITTER: Lauhon CT

PROVIDER: S-EPMC6755726 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Lauhon Charles T CT

Journal of bacteriology 20190920 20

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) remai ...[more]

PMID: 31358606

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Project description:Microaerobic cultivation conditions have been shown experimentally and theoretically to improve the performance of a number of bioproduction systems. However, under these conditions, the production of l-valine by Escherichia coli is decreased mainly because of a redox cofactor imbalance and a decreased l-glutamate supply. The synthesis of one mole of l-valine from one mole of glucose generates two moles of NADH via glycolysis but consumes a total of two moles of NADPH, one in the ketol-acid reductoisomerase (KARI) reaction and the other in the regeneration of l-glutamate as an amino group donor for the branched-chain amino acid aminotransferase (BCAT) reaction. The improvement of l-valine synthesis under oxygen deprivation may be due to solving these problems. Increased l-valine synthesis under oxygen deprivation conditions was previously shown in Corynebacterium glutamicum (Hasegawa et al., 2012). In this study, we have proposed the use of NADH-dependent leucine dehydrogenase (LeuDH; EC 1.4.1.9) Bcd from B. subtilis instead of the native NADPH-dependent pathway including aminotransferase encoded by ilvE to improve l-valine production in E. coli under microaerobic conditions. We have created l-valine-producing strains on the base of the aminotransferase B-deficient strain V1 (B-7 ?ilvBN ?ilvIH ?ilvGME::PL -ilvBN N17K DA) by introducing one chromosomal copy of the bcd gene or the ilvE gene. Evaluation of the l-valine production by the obtained strains under microaerobic and aerobic conditions revealed that leucine dehydrogenase Bcd had a higher potential for l-valine production under microaerobic conditions. The Bcd-possessing strain exhibited 2.2-fold higher l-valine accumulation (up to 9.1 g/L) and 2.0-fold higher yield (up to 35.3%) under microaerobic conditions than the IlvE-possessing strain. The obtained results could be interpreted as follows: an altering of redox cofactor balance in the l-valine biosynthesis pathway increased the production and yield by E. coli cells under microaerobic conditions. Thus, the effective synthesis of l-valine by means of "valine fermentation" was shown in E. coli. This methodology has the advantages of being an economical and environmentally friendly process.

Dataset Information

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

Publications

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

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