Endogenous hydrogen sulfide (H 2 S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H 2 S in Escherichia coli. Cellular resistance to H 2 O 2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders ΔmstA cells hypersensitive to H 2 O 2 . Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders Δfur cells fully resistant to H 2 O 2 . Furthermore, the endogenous level of H 2 S is reduced in Δfur or ΔsodA ΔsodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H 2 S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated L-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H 2 S. These findings led us to propose a model to explain the interplay between L-cysteine metabolism, H 2 S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via L-cysteine utilization and H 2 S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.hydrogen sulfide | oxidative stress | cysteine | sulfur metabolism | antibiotics
AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR inBacillus subtiliscells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism inB. subtiliswas performed to achieve overproduction of AICAR. For this purpose, the genepurH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from theB. subtilisgenome, ensuring the accumulation of AICAR. An insertion inactivating the genepurRthat encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into theB.subtilischromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence ofpur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of therpsFgene encoding the ribosomal protein S6 was designed. The heterologousEscherichia coligenepurFencoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modifiedE.coligeneprsresponsible for the synthesis of the precursor of purines — phosphoribosyl pyrophosphate (PRPP) — was cloned into this vector under the control of therpsFgene promoter. The modifiedpurFandprsgenes were inserted into the chromosome of theB. subtilisstrain.B. subtilisstrain obtained by these genetic manipulations accumulates 11–13 g/L of AICAR in the culture fluid.
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