Respiratory components in acidophilic bacteria that respire on iron

Abstract

Microorganisms capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Phylogenetically distinct organisms were shown to express spectrally distinct redox‐active biomolecules during autotrophic growth on soluble iron. A new iron‐oxidizing eubacterium, designated as strain Funis, was investigated. Strain Funis was judged to be different from other known iron‐oxidizing bacteria on the bases of comparative lipid analyses, 16S rRNA sequence analyses, and cytochrome composition studies. When grown autotrophically on ferrous ions, Funis produced conspicuous levels of a novel acid‐stable, acid‐soluble yellow cytochrome with a distinctive absorbance peak at 579 nm in the reduced state.

Stopped‐flow spectrophotometric kinetic studies were conducted on respiratory chain components isolated from cell‐free extracts of Thiobacillus ferrooxidans. Experimental results were consistent with a model where the primary oxidant of ferrous ions is a highly aggregated c‐type cytochrome that then reduces the periplasmic rusticyanin. The Fe(II)‐dependent, cytochrome c‐catalyzed reduction of the rusticyanin possessed three kinetic properties in common with corresponding intact cells that respire on iron: the same anion specificity, a similar dependence of the rate on the concentration of ferrous ions, and similar rates at saturating concentrations of ferrous ions     

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450(11)beta and P-450scc, steroid-induced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADPH-dependent lipid peroxidation with P-450(11)beta being much more susceptible to this process.     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).     

Oxygen reactions with bacterial oxidases and globins: binding,reduction and regulation

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins.Recent studies of the cytochromebd-type oxidases ofEscherichia coli andAzotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotrophA. vinelandii by the cytochromebd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high V_max. The poorly characterized cytochromeo-containing oxidase in this bacterium is not required for respiratory protection. InE. coli, the cytochromebd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligandbinding sites (haemsd andb ₅₉₅) in oxidases of this type suggests plausible mechanisms for this phenomenon. InE. coli, assembly of the cytochromebd-type oxidase (and of periplasmic cytochromesb andc) requires the presence of an ABC transporter, which may serve to export haem or some assembly factor' to the periplasm.There is at least one additional oxygen-consuming protein inE. coli — the flavohaemoglobin encoded by thehmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of falling (or rising) cytoplasmic oxygen concentrations.(until October 1994: Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA)     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli)

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.