Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

Effect of exogenous glucose on electron flow to photosystem I and respiration in cyanobacterial cells

The effects of exogenous glucose on the rates of alternative pathways of photosystem II (PSII)-independent electron flow to PSI and of dark respiration in Synechocystis sp. 6803 cells were studied. The presence of glucose was shown to accelerate the electron flow to P700⁺, the PSI primary electron donor oxidized with Far-red light (FRL), which excites specifically only PSI. An increase in the glucose concentration was accompanied by a further activation of electron flow to PSI, which was supported by the dark donation of reducing equivalents to the electron transport chain. An increase in the external glucose concentration resulted also in the disappearance of lag-phase in the kinetics of P700⁺ reduction, which was observed in the cells incubated without glucose after FRL switching off. A similarity of nonphotochemical processes of electron transfer to PSI in cyanobacteria and higher plants was supposed, basing on the earlier observed fact of the occurrence of such lagphase in higher plants and its dependence on the exhausting of stromal reductants in the light. Acceleration of dark electron flow to PSI in the presence of glucose, a major respiratory substrate, may indicate the coupling between nonphotochemical processes in the photosynthetic and respiratory chains of electron transport in cyanobacterial cells. A close correlation between photosynthesis and respiration in cyanobacterial cells is also confirmed by a sharp acceleration of respiration with an increase in the glucose concentration in medium.     

Tissular distribution of heteroplasmy and ultrastructural studies of mitochondria from a Drosophila subobscura mitochondrial deletion mutant

A mutant strain of Drosophila subobscura possesses two mitochondrial genome types: a minority population (20%) identical to the wild strain mtDNA (15.9 kb), and a largely predominant population (80%) of shorter genomes (10.9 kb), presenting a deletion of more than 30% of its coding region. Study of tissular distribution of heteroplasmy shows it to be identical — about 80% — in the head (nervous tissue) and thorax (muscles). On the other hand, a lower percentage (64%) is observed in the ovaries. The strain is apparently unaffected despite this massive loss of genes, coding for four tRNA and for complex I and III subunits. Contrary to observations of similar situations in man, the mutant strain shows no accumulation or structurally abnormal mitochondria. Furthermore, cytochemical studies fail to detect mitochondria devoid of cytochrome oxidase activity (COX^?). Finally, mitoribosome populations are identical in mitochondria from both strains. These results suggest that, in the mutant strain, there are no mitochondria containing deleted genomes only: heteroplasmy would thus be intramitochondrial.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

A critique of the use of inhibitors to estimate partitioning of electrons between mitochondrial respiratory pathways in plants

The contribution of individual plant mitochondrial respiratory pathways to total respiration is commonly assessed by titration with specific inhibitors of different components in the branched electron transport chain. A pathway's contribution is equal to the activity when the other branch is blocked by an inhibitor multiplied by the degree (0-1.0) to which this activity is engaged when both pathways are operating. According to Bahr and Bonner (1973. J. Biol. Chem. 218: 3441–3445) the plot of the activities of identical titrations, one performed in the absence and the other in the presence of a specific inhibitor of the other branch of the respiratory chain, yields a straight line whose slope indicates the engagement of the titrated pathway during uninhibited respiration. An initial slope of zero may occur if electron flux is diverted between pathways during titrations. However, beyond the breakpoint (representing the point of pathway saturation), a straight line is obtained with a slope representing engagement. This technique assumes that the kinetics of inhibiting a specific component of the respiratory chain are independent of the absolute rate of electron flux through the total pathway. To test this assumption, the activity of respiratory pathways in isolated soybean (Glycine max [L]. Merr. cv. Stevens) mitochondria was titrated with specific inhibitors of the cytochrome and alternative oxidases. Under these conditions, the electron flux through a given pathway was manipulated by poising the rate of succinate oxidation with the succinate dehydrogenase inhibitor malonate. Construction of activity plots in the presence versus absence of malonate failed to result in straight lines for either KCN (when titrating the cytochrome pathway) or salicylhydroxamic acid (when titrating the alternative pathway). Rather, the resultant plots were always curvilinear whenever the activity in the presence of malonate divided by the activity in the absence of malonate was less than 1.0. In no case could the real engagement of the pathway be precisely estimated from the titration data. Titrations of cytochrome pathway activity in isolated potato tuber (Solanum tuberosum L. cv. Sabago and Canabex) mitochondria (which lack the alternative oxidase) showed that as the inhibitor concentration was increased, so did the reduction status of the ubiquinone pool, to a new steady state. The dependence of inhibition kinetics on the rate of flux through the pathway, and the increase in ubiquinone pool reduction upon KCN addition, are explained in terms of the elasticity of component enzymes as outlined in the theory of metabolic control analysis. The implications of this finding for the use of titrations to estimate engagement of plant respiratory pathways are discussed.     

On the long-wavelength spectral forms of chlorophyll a in Photosystem I: Spectroscopic and immunological investigations on a greening mutant of the green alga Scenedesmus obliquus

The origin of the long-wavelength chlorophyll (Chl) absorption (_peak > 680 nm) and fluorescence emission (_peak > 685 nm) has been investigated on Scenedesmus mutants (C-2A-series, lacking the ability to synthesize chlorophyll in the dark) grown at 0.3 (LL), 10 (ML) and 240 µE s^–1 m^–2(HL). LL cells are arrested in an early greening state; consequently, Chl availability determines the phenotype. LL thylakoids are totally lacking long-wavelength Chl; nonetheless, PS I and PS II are fully functional. Gel electrophoresis and Western blots indicate that four out of seven resolved LHC polypeptides seem to require a high Chl availability for assembly of functional chlorophyll-protein complexes. The PS I core-complex of ML and HL thylakoids contains long-wavelength chlorophylls, but in the PS I core-complex of LL thylakoids these pigments are lacking. We conclude that long-wavelength pigments are only present in the PS I core in the case of high Chl availability. The following hypothesis is discussed: Chl availability determines not only the LHC polypeptide pattern, but also the number of bound Chl molecules per individual pigment-protein complex. Chl-binding at non-obligatory, peripheral sites of the pigment-protein complex results in long-wavelength Chl. In the case of low Chl availability, these sites are not occupied and, therefore, the long-wavelength Chl is absent.     

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.     

Coordination of photosynthetic and respiratory metabolism in Chlorella vulgaris UAM 101 in the light

In Chlorella vulgaris UAM 101, the presence of glucose altered the photosynthetic and respiratory metabolism in the light. When glucose was added to the growth medium, an increase in the cellular level of enzymes involved in glucose oxidation, namely glucose-6-P dehydrogenase (EC 1.1.1.49) and NAD⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), was observed. Glucose also enhanced respiratory O₂ consumption. In addition, CO₂ released by glucose oxidation was refixed in photosynthesis. The presence of glucose also affected photosynthesis. Phosphoribulokinase (EC 2.7.1.19) and NADP⁺-dependent glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13), two regulatory enzymes of the reductive pentose phosphate cycle, were increased by glucose. However, Rubisco (EC 4.1.1.39) activity of these cells was lower than that of autotrophic cells. Despite these alterations, the photosynthetic O₂ evolution was not significantly inhibited by glucose. On the other hand, an increase in the cytosolic NADP⁺-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.9) that is involved in obtaining reducing power for anabolic processes was observed. The CO₂ levels in the growth medium did not significantly affect the cellular level of enzymes measured in this work, except those involved in biosynthetic pathways. These data suggest that the effect of glucose on photosynthesis and respiration can be explained by alteration of the cellular level of photosynthetic enzymes and respiratory substrates, respectively.     

The regulation of respiration in cell suspensions of Petunia hybrida, studied by inhibiting mitochondrial protein synthesis with chloramphenicol

When Petunia hybrida L. cv. Rosy Morn Fertile suspension cells were inoculated in fresh medium with chloramphenicol (CAP), the activity of cytochrome C oxidase (EC 1. 9. 3. 1), and the respiration via the cytochrome pathway of isolated mitochondria decreased, while in untreated cells these parameters more than doubled in 2–3 days. However, the in vivo respiratory activity of the cytochrome pathway of CAP-treated cells showed a similar course in time as that of untreated cells, even in the presence of an uncoupler, a large rise during the first 2–3 days followed by a decline. This leads to the conclusion that the respiration via the cytochrome pathway, even when measured in the presence of an uncoupler, is not the capacity of this pathway. Furthermore, the results suggest that, although new-synthesis of proteins occurs directly after in-oculation, a large overcapacity must be present of cytochrome pathway elements (at least of those that are mitochondrial encoded). CAP had little effect on the uninhibited respiration and the cyanide-resistant, alternative pathway of the Petunia cells. However, the engagement of the alternative pathway (in the presence or absence of uncoupler) was increased in CAP-treated cells, especially after day 3 of the batch cycle, possibly as an effect of higher sugar degradation in combination with substrate phosphorylation to compensate the loss of ATP-synthesizing ability of the cytochrome pathway. It will be discussed that in general one should be careful using the term 'capacity' for the respiratory pathways.     

Distribution and molecular characterization of distinct Asian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) in Japan

Bemisia tabaci (Gennadius) is considered to be the most economically important pest insect worldwide. The invasive variant, the Q biotype of B. tabaci was first identified in 2004, and has caused significant crop yield losses in Japan. The distribution and molecular characterization of the different biotypes of B. tabaci in Japan have been little investigated. In this study, B. tabaci populations were sampled from the Japanese Archipelago, the Amami Archipelago and the Ryukyu Islands between 2004 and 2008, and the nucleotide sequences of their mitochondrial cytochrome oxidase I genes were determined. Bayesian phylogenetic relationship analysis provided the first molecular evidence that the indigenous Japanese populations could be separated into four distinct genetic groups. One major native population from the Japanese Archipelago, given the genetic group name Lonicera japonica, was separated into an independent group, distinct from the other genetic groups. The second major population, the Nauru biotype in the Asia II genetic group, was identified in the Amami Archipelago and the Ryukyu Islands. Two distinct minor genetic groups, the Asia I and the China, were also identified. One invasive B‐related population belonging to the Mediterranean/Asia Minor/Africa genetic group has been identified in Honshu. All lineages generated by the phylogenetic analyses were supported by high posterior probabilities. These distinct indigenous B. tabaci populations developed in Japan under geographical and/or biological isolation, prior to recent invasions of the B and Q biotypes.     

Functional analysis of mitochondrial protein import in yeast

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild-type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.     

Partial Purification and Some Properties of ADP-glucose Phosphorylase from Potato Tubers

ADP-glucose phosphorylase [adenosine diphosphate glucose: orthophosphate adenyl- yltransferase; Dankert et ah, Biochim. Biophys. Acta, 81, 78 (1964)] was found to be widely distributed in plant tissues. The enzyme was purified 570-fold in a 24% yield from cell- free extract of growing tubers of potato (Solanum tuberosum L.). The following reaction catalyzed by the purified enzyme was found to proceed stoichiometrically. ADP-glucose +P₁→ADP+glucose-1-P

Maximal activity was observed at pH 8. The enzyme was the most stable at pH 7, showing 50% loss of its original activity after 50 min heating at 57°C. The following kinetic parameters were obtained: activation energy, 11.1 kcal/mole; Km (P₁), 2.5 mm; Km (ADP-glucose), 0.05 mm. The enzyme did not act on GDP-mannose, GDP-glucose and UDP-glucose. Neither activator nor inhibitor was found among various phosphorylated metabolites tested. The enzyme was inhibited by metal-binding reagents, EDTA and o-phenanthroline. None of the metal ions tested was found to recover the activity of chelator-treated enzyme.     

The donor side of photosystem II is impaired in a Cd-tolerant mutant strain of the unicellular green algaChlamydomonas reinhardtii

Growth of a cadmium-tolerant mutant strain of the unicellular green alga Chlamydomonas reinhardtii was found to be impaired under photoautrotrophic, but not under mixotrophic conditions. As compared to wild-type cells, oxygen evolution by the photoautotrophically grown mutant was considerably decreased and higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The capability for oxygen production was decreased in Chlamydomonas reinhardtii cells when grown in the presence of acetate without aeration. Wild-type cells grown under these conditions showed a rather low but significant oxygen evolution immediately after transfer to photoautotrophic conditions. This residual oxygen production was completely suppressed in the presence of acetate, obviously due to acetate inhibition of the water-splitting complex. In the case of our cadmium-tolerant mutant strain, however, residual oxygen production was measured even in the presence of acetate. After removal of acetate, oxygen evolution by the cadmium-tolerant mutant strain was increased to higher rates than measured for wild-type cells, but considerably higher photon fluence rates were required both for light compensation of oxygen consumption/production and maximal oxygen evolution. The conclusion that the donor side of photosystem II is affected in our cadmium-tolerant mutant strain was further corroborated by a stronger decrease of the fluorescence level caused by hydroxylamine.     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Biochemical and molecular characterization of the mitochondrial peroxiredoxin PsPrxII F from Pisum sativum.

The pea peroxiredoxin homologue PsPrxII F of the Arabidopsis thaliana mitochondrial AtPrxII F was isolated as cDNA and genomic DNA, and characterized in respect to its biochemical and molecular properties. The deduced amino acid sequence contains an N-terminal targeting address for mitochondrial import. Mitochondrial location of PsPrxII F was confirmed by immunocytochemistry. The mature enzyme, without the transit peptide, has a molecular mass of 18.75 kDa, and, at positions 59 and 84, carries the two catalytic cysteinyl residues which are characteristic for this particular Prx subgroup. Activity of site-directed mutagenized C84S-variant lacking the so-called resolving Cys dropped to about 12% of WT Prx while C59S lost its peroxidatic activity completely. Likewise, WT PsPrxII F and C84S-variant but not C59S protected plasmid DNA against strand breakage in a mixed function oxidation assay. WT PrxII F and the variant proteins aggregated to high mass oligomers not yet described for type II Prx. Upon oxidation with hydrogen peroxide PsPrxII F focussed in a series of spots of distinct pI but similar molecular masses in two-dimensional gels indicating different oxidation states of the protein. Using this technique, partial oxidation was also detected in leaf extracts and isolated mitochondria. PsPrxII F mRNA and protein accumulated in cold and heavy metals treated pea plants suggesting a particular function under stress.     

The molecular anatomy and function of thylakoid proteins

Abstract. The structure of chloroplast membrane proteins and their organization into photosynthetically-active multimeric complexes is described. Extensive use has been made of information derived from gene sequencing and other biochemical studies to predict likely protein conformations. These predictions have been assimilated into structural models of the various thylakoid complexes. The enzymatic activities of the complexes have also been described and where possible related to individual polypeptides.