title: The tRNA thiolation pathway modulates the intracellular redox state in Escherichia coli.
abstract: We have performed a screening of hydroxyurea (HU)-sensitive mutants using a single-gene-deletion mutant collection in Escherichia coli K-12. HU inhibits ribonucleotide reductase (RNR), an enzyme that catalyzes the formation of deoxyribonucleotides. Unexpectedly, seven of the mutants lacked genes that are required for the incorporation of sulfur into a specific tRNA modification base, 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), via persulfide relay. We found that the expression of RNR in the mutants was reduced to about one-third both in the absence and presence of HU, while sufficient deoxynucleoside triphosphate (dNTP) was maintained in the mutants in the absence of HU but a shortage occurred in the presence of HU. Trans-supply of an RNR R2 subunit rescued the HU sensitivity of these mutants. The mutants showed high intracellular ATP/ADP ratios, and overexpression of Hda, which catalyzes the conversion of DnaA-ATP to DnaA-ADP, rescued the HU sensitivity of the mutants, suggesting that DnaA-ATP represses RNR expression. The high intracellular ATP/ADP ratios were due to high respiration activity in the mutants. Our data suggested that intracellular redox was inclined toward the reduced state in these mutants, which may explain a change in RNR activity by reduction of the catalytically formed disulfide bond and high respiration activity by the NADH reducing potential. The relation between persulfide relay and intracellular redox is discussed.
authors: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan. nakayashiki@bs.naist.jp
authors: Toru
authors: T
authors: Nakayashiki
authors: Natsumi
authors: N
authors: Saito
authors: Rikiya
authors: R
authors: Takeuchi
authors: Hiroshi
authors: H
authors: Kadokura
authors: Kenji
authors: K
authors: Nakahigashi
authors: Barry L
authors: BL
authors: Wanner
authors: Hirotada
authors: H
authors: Mori
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D048168:Escherichia coli K12 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D010084:Oxidation-Reduction; D012343:RNA, Transfer / Q000235:genetics / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism; D013891:Thiouridine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D013891:Thiouridine; C011916:5-methylaminomethyl-2-thiouridine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea