Nitrosative stress in Escherichia coli: reduction of nitric oxide

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Role of nitric oxide in the SOS response in Escherichia coli

Having one electron with unpaired spin, nitric oxide (NO) shows high reactivity and activates or inhibits free radical chain reactions. NO toxic and genotoxic effects appear to be the result of intracellular formation of peroxinitrite that can induce some cellular damages, including DNA strand breaks, DNA base oxidation, destruction of the key enzymes, etc. Taking into account the character of DNA damages being formed under NO activity, we proposed a formation of the SOS signal and induction the SOS DNA repair response in E. coli cells treated with NO physiological donors--DNIC and GSNO. The ability of NO donor compounds to induce the SOS DNA response in E. coli PQ37 with sfiA::lacZ operon fusion is reported here at the first time. So, the SOS DNA repair response induction is one of the function of nitric oxide.     

Hemoglobin-mediated nitric oxide signaling

The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the dioxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitrosothiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered.     

Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid.     

Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli

3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l⁻¹ with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.     

Heterologous synthesis of 4-ethylphenol in engineered Escherichia coli

4-Ethylphenol (4-EP) is an industrially versatile commodity chemical widely applied in the pharmaceutical and food industries. In this study, an artificial biosynthetic pathway was constructed in Escherichia coli for production of 4-ethylphenol from simple sources of carbon. The pathway consists of the tal, pad and vpr genes, which encode tyrosine ammonia lyase (TAL), phenolic acid decarboxylase (PAD) and vinylphenol reductase (VPR), respectively. Our results confirmed that the TAL from Saccharothrix espanaensis possessed higher catalytic activity than the TAL from Rhodobacter sphaeroides for biosynthesis of p-hydroxycinnamic acid. The low solubility of Lactobacillus plantarum VPR (LpVPR) in E. coli was a critical factor limiting its availability in the biosynthetic pathway. The solubility of LpVPR was improved by E. coli strain and induction condition optimization. Under the optimized conditions, the engineered E. coli TransB-TPV produced as high as 110 mg/L 4-EP at 37 ℃ in Terrific Broth (TB) medium with glycerol as carbon source after cultivation of 48 h. This study provided a new and feasible strategy for biosynthesis of 4-EP from simple sugars, which may provide a basis for future large-scale industrial application.     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

Heterologous production of epothilone C and D in Escherichia coli

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.