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.

Unraveling the Physiological Complexities of Antibiotic Lethality

“…These data nonetheless support the general notion that aminoglycosides, downstream of their drug-target interaction, induce changes to cellular metabolism and respiration that could alter the cellular redox state. The recent discovery that IscS plays a role in a tRNA thiolation pathway that modulates intracellular redox stress (131) suggests an additional interpretation for the effects of an iscS mutation in these experiments (11,37).…”

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

“…The conservation of the yrvO–mnmA gene region and lack thereof the Tus system suggests that the abbreviated s 2 U biosynthetic pathway used in B. subtilis is common to Gram-positive bacteria. In Gram-negative E. coli , which uses the housekeeping IscS for sulfur mobilization to all thiocofactors [11,53,54], the Tus proteins also participate in additional metabolic pathways [71,175,176,177]. The multi-functionality of these and other enzymes in thiocofactor pathways has been attributed to the utilization of one major cysteine desulfurase (i.e., IscS) for all thiocofactors in Gram-negative bacteria and simultaneously demonstrates the resulting complexity of sulfur trafficking in these organisms [178,179].…”

Insufficient levels of the nrdAB‐encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain

“…Moreover, they suggest that NrdAB function is altered in the absence of hda function. Mutations affecting the tRNA thiolation pathway confer HU sensitivity (Nakayashiki et al ., ). Although NrdAB levels were reduced in these mutants, the intracellular redox was shifted toward the reduced state, suggesting NrdAB activity was enhanced due to more efficient reduction of the NrdB disulfide‐bridge.…”