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

The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid β-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid β-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid β-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-P(trc-ideal-4)-SD(φ10)-adhE(Glu568Lys) attB-P(trc-ideal-4)-SD(φ10)-atoB attB-P(trc-ideal-4)-SD(φ10)-fadB attB-P(trc-ideal-4)-SD(φ10)-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.

Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.

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l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2. These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.

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.