δ-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA

Abstract Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of δ-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis . The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.     

Hydrogenobacter thermophilus: its unusual physiological properties and phylogenic position in the microbial world

Abstract Hydrogenobacter thermophilus (IAM12695) is an obligately autotrophic, thermophilic and aerobic hydrogen-oxidizing bacterium. Chemotaxonomic studies revealed various unusual features of this microorganism. H. thermophilus operates a reductive TCA cycle, which is the only confirmed example of the operation of a non-Calvin type carbon dioxide fixation pathway among aerobic organisms. Isolation of strains similar to Hydrogenobacter from different areas suggests a possible role of these microorganisms as primary producers of organic compounds from carbon dioxide in geothermal and/or aquatic environments.     

Divergent pathways for δ-aminolevulinic acid synthesis in two species of Arthrobacter

Abstract Secretion of coproporphyrin III by suspensions of Arthrobacter photogonimos and A. globiformis facilitated analysis of the paths of synthesis of δ-aminolevulinic acid, the precursor of tetrapyrroles. Sensitivity of coproporphyrin accumulation to gabaculine and incorporation of ¹⁴C from [1-¹⁴C]glutamate indicated that suspensions of A. photogonimos synthesized δ-aminolevulinic acid from glutamate by the widespread C5 pathway. In contrast, A. globiformis cells switched from predominantly the C5 pathway for δ-aminolevulinic acid synthesis in early exponential phase cultures to δ-aminblevulinic acid synthase in stationary phase cultures.     

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K _m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t _50%) at 70° C was approximately 8 h. Received: 9 September 1996 / Accepted: 27 December 1996     

Cadmium induced oxidative stress in soybean plants also by the accumulation of δ-aminolevulinic acid

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 μM Cd²⁺ for 48 h. δ-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-l-methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd²⁺, it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd²⁺ and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd²⁺ is highly responsible for oxidative stress generation in these tissues.     

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli

Abstract The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c -type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.     

Identification of Calmodulin in the Red Alga Susabi Laver (Porphyra yezoensis)

To test whether calmodulin is present in the red alga Susabi laver (Porphyra yezoensis), materials was concentrated from this alga, using hydrophobic chromatography. In this case, 0.25 m (NH₄)₂SO₄ should be added to bind the protein to the column in the presence of 5 mm CaCl₂. Calmodulin is present in the marine red alga, although at a very low concentration.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

Synthesis of Rhodobacter sphaeroides cytochrome c2 in Escherichia coli

The cytochrome c2 structural gene, cycA, from Rhodobacter sphaeroides was expressed in Escherichia coli. CycA-specific mRNA was detected in E. coli both under aerobic and anaerobic conditions with trimethylamine-N-oxide as electron acceptor. However mature holocytochrome c2 was only detected in anaerobically-grown cells. The mature form of cytochrome c2 (Mr = 12,500) was secreted into the periplasm of E. coli suggesting that the signal polypeptide was processed. The cytochrome c2 synthesized in E. coli exhibited absorbance maxima in the reduced form at 550 nm (alpha-band) and 521 nm (beta-band) and contained covalently attached haem c. The results indicate that a foreign c-type cytochrome can be secreted and assembled in E. coli under anaerobic conditions.     

A mutant of Pseudomonas aeruginosa that lacks c-type cytochromes has a functional cyanide-insensitive oxidase

Abstract Using transposon mutagenesis and screening for the loss of the ability to oxidise the artificial electron donor N , N , N ', N '-tetrarnethyl- p -phenylenediarnine, we have isolated a mutant of Pseudomonas aeruginosa that lacks all c -type cytochromes. This mutant is unable to grow anaerobically with nitrate as a terminal electron acceptor. Analysis of its respiratory function indicates that the mutant has lost its cytochrome c oxidase-terminated respiratory pathway but the cyanide-insensitive oxidase-terminated branch remains functional. Complementation of the mutant by in vivo cloning led to recovery of the wild-type characteristics. These data are consistent with the idea that the cyanide-insensitive respiratory pathway does not contain haem c and that the pathway's terminal oxidase is a quinol oxidase.     

Gene Structure and Expression Profile of Cytochrome bc Nitric Oxide Reductase from Hydrogenobacter thermophilus TK-6

The genes for a nitric oxide reductase-like cytochrome bc complex were cloned from a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6. The structural genes norC and norB, which encode cytochrome c and cytochrome b subunits of the complex respectively, are probably transcribed as a tricistronic operon with a following gene encoding a putative membrane protein. NorC has, unusually, two hydrophobic transmembrane spans in its N-terminus. Immunoblot analysis showed that expression of NorC was induced by nitrate, nitrite, or sodium nitropurusside, suggesting that the norCB gene product is a denitrification enzyme, nitric oxide reductase. The consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide-sensing regulators of proteobacteria were not found in the norC promoter region, but consensus ?35 and ?10 sequences were found in this region. These results indicate that strain TK-6 has a nitrogen oxide-sensing regulatory system that differs from proteobacterial systems.     

Effects of 5-aminolevulinic acid on the inflammatory responses and antioxidative capacity in broiler chickens challenged with lipopolysaccharide

5-Aminolevulinic acid (5-ALA) is an intermediate in haem biosynthesis and has anti-apoptotic, anti-inflammatory, antioxidant, and other pharmacological effects. This study aimed to investigate the effect of dietary supplementation with 5-ALA on growth performance, antioxidant capacity, and inflammatory response of the lipopolysaccharide (LPS)-challenged broiler chickens. The experiment was designed as a 2 × 2 factorial arrangement with dietary 5-ALA (0 or 60 mg/kg) and LPS (injection of saline or 0.5 mg/kg BW) levels as treatments. A total of 240 one-day-old Arbor Acres broilers were distributed into four treatments consisting of six replicates of 10 birds. All the experimental broilers were intraperitoneally injected with LPS or sterile saline at 16, 18, and 20 days of age. Our results showed that dietary 5-ALA supplementation reduced (P < 0.05) the feed to gain before broilers were stimulated with LPS (days 1–15). LPS challenge decreased (P < 0.05) the catalase (CAT), total superoxide dismutase activities and increased the content of malondialdehyde (MDA) in the serum of broiler chickens. However, 5-ALA supplementation had a tendency to increase (P = 0.08) the activity of CAT and decreased (P < 0.05) the content of MDA. LPS challenge showed higher (P < 0.05) interleukin (IL)-1β, IL-6, and IL-10 concentrations in the serum, whereas dietary 5-ALA supplementation decreased (P < 0.05) the levels of IL-1β and IL-6. Additionally, dietary 5-ALA supplementation significantly attenuated (P < 0.05) the upregulation of mRNA expression levels of hepatic toll-like receptor 4 (TLR4), IL-1β, and IL-2 induced by LPS challenge. Moreover, dietary 5-ALA supplementation also enhanced the mRNA expression of 5-aminolevulinate dehydratase, ferrochelatase, and haem oxygenase-1 (HO-1) as compared to the unsupplemented groups. In conclusion, our results suggested that supplementation of 60 mg/kg 5-ALA exhibited LPS-induced anti-inflammatory and antioxidant properties by enhancing the HO-1 expression and inhibiting the TLR4/NF-κB signalling pathway.     

Enzymology and bioenergetics of respiratory nitrite ammonification

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.