Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod

The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.     

Molecular analysis of Southern Ocean skates (Bathyraja) reveals a new species of Antarctic skate

Two regions of mtDNA, cytochrome b and cytochrome c oxidase subunit 1, were sequenced in nine species of Bathyraja from the Southern Ocean and New Zealand. Based on sequence divergence, the species that has been referred to as Bathyraja eatonii from the Antarctic continental shelf and slope is a species distinct from B. eatonii from the Kerguelen Plateau (the type locality) and is a new and undescribed species Bathyraja sp. (cf. eatonii). There was no sequence divergence among samples of Bathyraja sp. (dwarf) from the Ross Sea and the South Atlantic. However, for both Bathyraja sp. (cf. eatonii) and Bathyraja maccaini in the Ross Sea and the South Atlantic Ocean, the DNA sequence divergences indicate differentiation among ocean basins and within Bathyraja sp. (cf. eatonii) divergences are similar to those among recognized species of Bathyraja in the North Pacific Ocean.     

Mitochondrial cytochrome components of Paragonimus adult worms

The cytochrome components of adult Paragonimus miyazakii mitochondria were investigated by polyacrylamide gel electrophoresis. The mitochondria were found to contain cytochromes b, c₁, c and aa₃. Two types of mitochondria, lightweight mitochondria (LWMt) and heavyweight mitochondria (HWMt), were obtained by centrifugation from the mitochondrial fraction of the adult Paragonimus ohirai. The succinate-reduced and oxidized difference spectrum of LWMt and HWMt at −196°C revealed that both mitochondria contained at least functional levels of cytochromes b, c₁, c and a low value of aa₃. Although succinate-reduced cytochromes of LWMt reoxidized in the presence of air, those of HWMt did so only minimally.     

Phylogeny of leafcutter ants in the genus Atta Fabricius (Formicidae: Attini) based on mitochondrial and nuclear DNA sequences

Leafcutting ants of the genus Atta are the most conspicuous members of the tribe Attini, the fungus-growing ants. Atta species have long attracted the attention of naturalists, and have since become a common model system for the study of complex insect societies as well as for the study of coevolutionary dynamics due to their numerous interactions with fungi and other microbes. Nevertheless, systematics and taxonomy of the 15 species in the genus Atta have proven challenging, due in part to the extreme levels of worker polymorphism these species display, leading to disagreements about the validity of as many as five different subgenera and calling into question the monophyly of the genus. Here, we use DNA sequence information from fragments of three mitochondrial genes (COI, tRNA leucine and COII) and one nuclear gene (EF1-αF1), totaling 1070 base pairs, to reconstruct the phylogenetic relationships of Atta species using maximum parsimony, maximum likelihood and Bayesian inference techniques. Our results provide support for monophyly of the genus Atta, and suggest that the genus is divided into four monophyletic groups, which correspond to four of the five previously erected Atta subgenera: Atta sensu stricto and Archeatta, each with species composition identical to earlier proposals; Neoatta and Epiatta, with major differences in species composition from earlier proposals. The current geographic ranges of these species suggest that the historical separation of South America from Central and North America has played a role in speciation within this genus.     

Structure-Guided Mutational Analysis of a Yeast DEAD-Box Protein Involved in Mitochondrial RNA Splicing

DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

The hupC gene product is a component of the electron transport system for hydrogen oxidation in Pseudomonas hydrogenovora

The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora. To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P. hydrogenovora by introducing an in-frame deletion into the hupC. The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain. Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.     

Two mitochondrial genes under episodic positive selection in subterranean octodontoid rodents

Tuco-tucos (Ctenomys) and related coruros (Spalacopus) are South American subterranean rodents. An energetically demanding lifestyle within the hypoxic, underground atmosphere may change the selective regime on oxidative phosphorylation. We examined whether weak and/or episodic positive directional selection affected the evolution of two mitochondrial genes (COX2, CytB), in a background of purifying selection in these lineages. We estimated rates of synonymous (dS) and non-synonymous (dN) substitutions and found: 1) significantly higher dN/dS ratio in subterranean groups relative to non-subterranean related species, and 2) two codons in each gene under episodic selection: 94 and 277 of COX2 and 269 and 307 of CytB.     

Use of mucus as a non-invasive sampling method for DNA barcoding of stingrays and skates (batoid elasmobranchs)

In this study we tested the use of mucus from five species of Neotropical marine batoid elasmobranchs to extract genomic DNA for barcoding and phylogenetic analysis. The DNA from all individuals sampled was successfully amplified and sequenced for molecular barcode, allowing 99–100% accuracy to the species level. This method proved to provide reliable and good-quality DNA for barcoding and phylogenetic analysis of Neotropical elasmobranchs, through rapid handling and with low disturbance to animals.     

A preliminary molecular phylogeny for New Zealand sheet-web spiders (Cambridgea) and comparison of web-building behaviour

ABSTRACT

Spider webs vary in size to meet the nutritional requirements of the resident spider with the resident’s body size strongly informing these requirements. In this way, the effect of body size on web-building behaviour should be apparent across species. To determine whether the size of analogous web structures scales with body size across closely related species, we first measured mainsheet area and adult female body size of 12 sheet-web spider species (Cambridgea). Using these species, we then generated alignments from the cytochrome c oxidase subunit I (COI) and histone 3 (H3) gene regions. These alignments were phylogenetically analysed using Bayesian inference and maximum likelihood methods. While phylogenetic trees for the COI gene suggested that Cambridgea is monophyletic relative to sampled outgroups, H3 did not. Combining our COI phylogenetic tree’s branch lengths with data on web-building behaviour, we used phylogenetic least squares to determine whether web size scales with spider size across species. While we found evidence that larger species generally build larger webs, the variation in web size across even similarly sized species suggests that environmental characteristics which influence site selection and prey type may play a role in determining the optimal web size for different species.     

Phylogenetic relationships among four species and a sub-species of Mullidae (Actinopterygii; Perciformes) based on mitochondrial cytochrome B, 12S rRNA and cytochrome oxidase II genes

DNA sequence comparisons of three mitochondrial DNA genes were used to reveal phylogenetic relationships among four species and a sub-species of Mullidae family. This is the first report using mitochondrial DNA sequence data to infer intraspecific relationship among different populations of Mullus barbatus and Mullus surmuletus; phylogenetic relationships between M. barbatus and its sub-species; M. barbatus ponticus. Cytochrome b, 12S ribosomal RNA, and cytochrome oxidase II regions of 242 individuals belonging to species M. barbatus, M. surmuletus, Upeneus moluccensis, Upeneus pori and sub-species M. barbatus ponticus were sequenced and phylogenetic trees were constructed using four different algorithms. The phylogenetic trees constructed support the existing taxonomical data of two mullid genera (Mullus, Upeneus). Molecular data shows no significant difference between same species of different geographical populations. The results suggest that the molecular difference is not large enough between M. barbatus and M. barbatus ponticus to consider them as sub-species.     

On the utility of DNA barcoding for species differentiation among brown macroalgae (Phaeophyceae) including a novel extraction protocol

The generation of a species-rich DNA barcode database in combination with rapid and affordable sequencing techniques will dramatically change specimen identification in ecological, biogeographical and taxonomic applications. Though cytochrome c oxidase 1 has been shown to be a useful tool for differentiating some groups of marine algae, its wide application in the Phaeophyceae has yet to be studied. The presence of polymerase chain reaction (PCR) inhibiting compounds in members of the Fucales, Laminariales and Tilopteridales, that are often co-extracted with DNA, has hampered the rapid processing associated with barcode projects. Polyphenolics and polysaccharides are present in concentrations such that DNA extraction methods typically include extensive series of washes, organelle extractions and/or cesium columns. In this paper we examine the utility of cytochrome c oxidase 1 for barcoding the Phaeophyceae and present a method for extracting PCR friendly DNA from brown macroalgae in about 2 h, dramatically reducing the time required from previous methods, some of which take days. This method is easily adapted to a 96 well, high-throughput format and may have applications in other organisms where the presence of similar PCR inhibiting compounds hinders molecular analyses. We extracted DNA from 106 isolates representing 29 species from 20 genera in nine families from five orders of Phaeophyceae. We were able to amplify the barcode marker (cytochrome c oxidase 1) from all samples and a nuclear marker (internal transcribed spacer region) from 54 selected samples. Cytochrome c oxidase 1 was able to differentiate clearly among species, showing within species divergence of 0.00–0.46%, with the exception of one previously studied genus, and between species divergences of greater than 3%.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii

The complete mitochondrial genome sequence of Liriomyza sativae Blanchard (15,551 bp) was determined and analyzed in this study. The circular genome contained 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A + T-rich region. The initiation codons of COI and ND1 were ‘ATCA’ and ‘GTG’, respectively. ND2 gene used the truncated termination codon ‘T’. All the tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN) gene, which was found with the absence of a DHU arm. In addition, a tRNA-like secondary structure (tRNA^Met) was found in the A + T-rich region. The great difference was that the length of L. sativae A + T-rich region was 597 bp shorter than that of Liriomyza trifolii (Burgess). Meanwhile, some minor differences such as ‘TATA’ block were also observed in L. sativae in contrast to ‘TACA’ block in L. trifolii. There were also some essential structure elements such as ‘TATA’ block, ‘G(A)_nT’ block, poly-T stretch and stem-and-loop structure in the A + T-rich region of L. sativae mitochondrial genome.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains in Escherichia coli K12

 The assembly of anaerobically induced electron transfer chains in Escherichia coli strains defective in periplasmic disulphide bond formation was investigated. Strains deficient in DsbA, DsbB or DipZ (DsbD) were unable to catalyse formate-dependent nitrite reduction (Nrf activity) or synthesize any of the known c-type cytochromes. The Nrf⁺ activity and cytochrome c content of mutants defective in DsbC, DsbE or DsbF were similar to those of the parental, wild-type strain. Neither DsbC expressed from a multicopy plasmid nor a second mutation in dipZ (dsbD) was able to compensate for a dsbA mutation by restoring nitrite reductase activity and cytochrome c synthesis. In contrast, only the dsbB and dipZ (dsbD) strains were defective in periplasmic nitrate reductase activity, suggesting that DsbB might fulfil an additional role in anaerobic electron transport. Mutants defective in dipZ (dsbD) were only slightly more sensitive to Cu⁺⁺ ions at concentrations above 5 mM than the parental strain, but strains defective in DsbA, DsbB, DsbC, DsbE or DsbF were unaffected. These results are consistent with our earlier proposals that DsbA, DsbB and DipZ (DsbD) are part of the same pathway for ensuring that haem groups are attached to the correct pairs of cysteine residues of apocytochromes c in the E. coli periplasm. However, neither DsbE nor DsbF are essential for the reduction of DipZ (DsbD). Received: 28 February 1996 / Accepted: 5 June 1996