ATP6 Gene Research Articles

AtpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples

BackgroundThe environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets.ResultsAmong the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.ConclusionsThe results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.

Investigation of nuclear and mitochondrial DNA polymorphism in closely related species of endemic Baikal sponges

In order to investigate the phylogenetic relationships among endemic Baikal sponges of the Lubomirskiidae family and to clarify their taxonomy, the sequence variation at the silicatein-encoding gene and the mtDNA intergenic region was examined. Phylogenetic analysis of the silicatein α1 gene exonic regions in six freshwater sponge species revealed considerable interspecific variability of this region. The analysis performed did not support the monophyly of the Lubomirskia and Baikalospongia genera. The mDNA region between the COX2 and ATP6 genes was examined in five species from the Lubomirskiidae family, including multiple samples for analyzing intraspecific variations. According to the data obtained, the Baikalospongia genus was monophyletic with respect to Lubomirskia, while B. bacilifera and B. recta did not form monophyletic groups. Molecular data indicate that taxonomy of Lubomirskiidae should be revised. It was demonstrated that, in endemic Baikal sponges, the accelerated evolution was accompanied by an increase in the length of the noncoding regions in both mitochondrial and nuclear genes.

Genetic divergence among disjunct populations of three Russula spp. from Africa and Madagascar

This study was a preliminary analysis of the genetic structure of the ectomycorrhizal species Russula discopus, R. pseudocarmecina and R. ochraceorivulosa with disjunct distributions in continental Africa and Madagascar. Phylogenetic analyses of the nuclear ITS1/5.8S/ITS2 region and the mitochondrial atp6 gene were performed with specimens from both locations for each species along with a suitable outgroup for each of the three taxa. Additional analyses of the ITS1/5.8S/ITS2 region using the African and Malagasy specimens and additional taxa in the genus Russula also were performed. R. pseudocarmecina and R. discopus both exhibited genetic structure as shown by a relatively high percentage difference in ITS and atp6 sequences, high bootstrap support for African or Malagasy groups and the presence of indels in the ITS sequence that are unique to either Africa or Madagascar. African and Malagasy groups of each species were more closely related to each other than to other taxa in Russula. Genetic structure also existed in populations of R. ochraceorivulosa, but bootstrap support was weaker than in the other two species. In addition, there was less sequence divergence in R. ochraceorivulosa and this species was the only one for which the same atp6 haplotype was found in both Africa and Madagascar. Reciprocal monophyly for all three species was consistent with the hypothesis that the same vicariance event may be responsible for the genetic structure observed here, although shorter branch lengths, lower bootstrap support and the presence of the same atp6 haplotype in Africa and Madagascar for R. ochraceorivulosa suggested slower evolutionary rates or geographical isolation after the other two taxa. In addition to the geological events that separated Africa and Madagascar, environmental changes during the Miocene or later might have had an effect on the distribution of these species.

Перенос митохондриальной ДНК в ядерный геном клеток пересеваемой клеточной линииDrosophila virilis

The variability of the chromosomal fragments of the atp6 mitochondrial gene, which is integrated into chromosomal DNA in the lines of flies of different geographic origins and in the passaged cell lines of D. virilis has been analyzed. We did not reveal any nucleotide variability in this DNA marker among the studied fly lines. This result is consistent with the proposition that the D. virilis species is monomorphic. The new fragments of the atp6 gene that are associated with the insertions of the Tv1 retrotransposon and are absent in the fly genome are revealed in the genome of the passaged cell line of D. virilis. This fact is evidence of the activation of the mitochondrial DNA transfer into the nuclear genome of the cells of passaged cell culture.

Vibrio toranzoniae sp. nov., a new member of the Splendidus clade in the genus Vibrio

Four motile facultative anaerobic marine isolates (Vb 10.8T [CECT 7225T, CAIM 1869T], CMJ 9.4 [CECT 8091, CAIM 1870], CMJ 9.11 and Cmf 13.9), were obtained from cultured clams (Venerupis philippinarum and Venerupis decussata) in Galicia (NW Spain). These isolates were studied by a polyphasic approach, including a phylogenetic analysis based on sequences of 16S rRNA and five housekeeping genes atpA, recA, pyrH, rpoA and rpoD, that supported their inclusion in the Splendidus clade of the genus Vibrio, forming a well-defined group separated from the others species of the clade. DNA–DNA hybridizations with the type strains of species showing more than 98.5% 16S rRNA gene sequence similarity rendered values of hybridization below 60%. These isolates could be differentiated from the closest relatives on the basis of several phenotypic and chemotaxonomic features. These results demonstr8ated that the strains constitute a novel specie of the genus for which the name Vibrio toranzoniae sp. nov. is proposed, with Vb 10.8T (=CECT 7225TCAIM 1869T) as the type strain.

Unusual Symbiotic Cyanobacteria Association in the Genetically Diverse Intertidal Marine Sponge Hymeniacidon perlevis (Demospongiae, Halichondrida)

Cyanobacteria represent one of the most common members of the sponge-associated bacterial community and are abundant symbionts of coral reef ecosystems. In this study we used Transmission Electron Microscopy (TEM) and molecular techniques (16S rRNA gene marker) to characterize the spatial distribution of cyanobionts in the widely dispersed marine intertidal sponge Hymeniacidon perlevis along the coast of Portugal (Atlantic Ocean). We described new sponge associated cyanobacterial morphotypes (Xenococcus-like) and we further observed Acaryochloris sp. as a sponge symbiont, previously only reported in association with ascidians. Besides these two unique cyanobacteria, H. perlevis predominantly harbored Synechococcus sp. and uncultured marine cyanobacteria. Our study supports the hypothesis that the community of sponge cyanobionts varies irrespective of the geographical location and is likely influenced by seasonal fluctuations. The observed multiple cyanobacterial association among sponges of the same host species over a large distance may be attributed to horizontal transfer of symbionts. This may explain the absence of a co-evolutionary pattern between the sponge host and its symbionts. Finally, in spite of the short geographic sampling distance covered, we observed an unexpected high intra-specific genetic diversity in H. perlevis using the mitochondrial genes ATP6 (π = 0.00177), COI (π = 0.00241) and intergenic spacer SP1 (π = 0.00277) relative to the levels of genetic variation of marine sponges elsewhere. Our study suggests that genotypic variation among the sponge host H. perlevis and the associated symbiotic cyanobacteria diversity may be larger than previously recognized.

Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: An ultra-large metazoan mitochondrial genome

The complete mitochondrial (mt) genome of the ark shell Scapharca broughtonii was determined using long PCR and a genome walking sequencing strategy with genus-specific primers. The S. broughtonii mt genome (GenBank accession number AB729113) contained 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 2 ribosomal RNA genes, and 42 transfer tRNA genes, in a length of 46,985 nucleotides for the size of mtDNA with only one copy of the heteroplasmic tandem repeat (HTR) unit. Moreover the S. broughtonii mt genome shows size variation; these genomes ranged in size from about 47kb to about 50kb because of variation in the number of repeat sequences in the non-coding region. The mt-genome of S. broughtonii is, to date, the longest reported metazoan mtDNA sequence. Sequence duplication in non-coding region and the formation of HTR arrays were two of the factors responsible for the ultra-large size of this mt genome. All the tRNA genes were found within the S. broughtonii mt genome, unlike the other bivalves usually lacking one or more tRNA genes. Twelve additional specimens were used to analyze the patterns of tandem repeat arrays by PCR amplification and agarose electrophoresis. Each of the 12 specimens displayed extensive heteroplasmy and had 8–10 length variants. The motifs of the HTR arrays are about 353–362bp and the number of repeats ranges from 1 to 11.

Analysis of heterologous regulatory and coding regions in algal chloroplasts

The basic photosynthetic apparatus is highly conserved across all photosynthetic organisms, and this conservation can be seen in both protein composition and amino acid sequence. Conservation of regulatory elements also seems possible in chloroplast genes, as many mRNA untranslated regions (UTRs) appear to have similar structural elements. The D1 protein of Photosystem II (psbA gene) is a highly conserved core reaction center protein that shows very similar regulation from cyanobacteria through higher plants. We engineered full and partial psbA genes from a diverse set of photosynthetic organisms into a psbA deficient strain of Chlamydomonas reinhardtii. Analysis of D1 protein accumulation and photosynthetic growth revealed that coding sequences and promoters are interchangeable even between anciently diverged species. On the other hand functional recognition of 5' UTRs is limited to closely related organisms. Furthermore transformation of heterologous promoters and 5' UTRs from the atpA, tufA and psbD genes conferred psbA mRNA accumulation but not translation. Overall, our results show that heterologous D1 proteins can be expressed and complement Photosystem II function in green algae, while RNA regulatory elements appear to be very specific and function only from closely related species. Nonetheless, there is great potential for the expression of heterologous photosynthetic coding sequences for studying and modifying photosynthesis in C. reinhardtii chloroplasts.

High-Throughput Discovery of Chloroplast and Mitochondrial DNA Polymorphisms in Brassicaceae Species by ORG-EcoTILLING

BackgroundInformation on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used.Methodology/Principal FindingsTo address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results.Conclusions/SignificanceThese results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.

Cloning and characterization of the mitochondrial genes Cox II and atpA from ramie (Boehmeria nivea (L.) Gaud.) and their possible role in cytoplasmic male sterility

Huang, H.-Q., Liu, J.-D., Duan, J.-Q., Liang, X.-N. and Liu, F.-H. 2012. Cloning and characterization of the mitochondrial genes Cox II and atpA from ramie (Boehmeria nivea (L.)Gaud.) and their possible role in cytoplasmic male sterility. Can. J. Plant Sci. 92: 1295-1304. In plants, cytoplasmic male sterility (CMS) is known to be associated with structural changes and the presence of new chimeric genes in mtDNA. In this study, fragments of the Cox II and atpA genes, cloned from mtDNA of three lines (CMS line, its maintainer and restorer lines) of ramie [Boehmeria nivea (L.) Gaud.], showed homology of 95% for Cox II and 97% for atpA compared with the reference dicotyledons in GenBank. The whole sequences of these two genes were obtained using the DNA walking strategy. Cox II showed no difference among the three lines in sequences of DNA and deduced amino acids. However, atpA from the CMS line obviously differed from that of fertile lines in DNA sequence, amino acid sequence and secondary structure of the p...

Cloning and characterization of the mitochondrial genes Cox II and atpA from ramie (Boehmeria nivea (L.) Gaud.) and their possible role in cytoplasmic male sterility

Huang, H.-Q., Liu, J.-D., Duan, J.-Q., Liang, X.-N. and Liu, F.-H. 2012. Cloning and characterization of the mitochondrial genes Cox II and atpA from ramie ( Boehmeria nivea (L.)Gaud.) and their possible role in cytoplasmic male sterility. Can. J. Plant Sci. 92: 1295–1304. In plants, cytoplasmic male sterility (CMS) is known to be associated with structural changes and the presence of new chimeric genes in mtDNA. In this study, fragments of the Cox II and atpA genes, cloned from mtDNA of three lines (CMS line, its maintainer and restorer lines) of ramie [Boehmeria nivea (L.) Gaud.], showed homology of 95% for Cox II and 97% for atpA compared with the reference dicotyledons in GenBank. The whole sequences of these two genes were obtained using the DNA walking strategy. Cox II showed no difference among the three lines in sequences of DNA and deduced amino acids. However, atpA from the CMS line obviously differed from that of fertile lines in DNA sequence, amino acid sequence and secondary structure of the predicted protein. Unusually low expression was revealed via RT-PCR for atpA in the CMS line at the budding and late-bloom stages. In conclusion, the variation in DNA sequence and the encoded product, and/or the abnormal expression of atpA in the CMS line, may cause male sterility in ramie.

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its β subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.

The plastid genome of the mycoheterotrophicCorallorhiza striata(Orchidaceae) is in the relatively early stages of degradation

Plastid genomes of nonphotosynthetic, mycoheterotrophic plants represent apt systems in which to study effects of relaxed evolutionary constraints. The few mycoheterotrophic angiosperm plastomes sequenced to date display drastic patterns of degradation/reduction relative to those of photosynthetic relatives. The goal of this study was to focus on a mycoheterotrophic orchid hypothesized to be in the "early" stages of plastome degradation, to provide perspective on this process. Short-read sequencing was used to generate a complete plastome sequence for Corallorhiza striata var. vreelandii, a mycoheterotrophic orchid, to investigate the extent of plastome degradation. Patterns of nonsynonymous/synonymous mutations were also assessed, and comparisons were made between Corallorhiza and other heterotrophic plant lineages. Corallorhiza yielded a plastome of 137505 bp, with several photosynthesis-related genes either lost or pseudogenized. Members of all major photosynthesis complexes, except ATP-synthase genes, were affected. "Housekeeping" genes were intact, despite the loss of a single tRNA. Intact photosynthesis genes (excluding atp genes) together displayed elevated nonsynonymous changes, while housekeeping genes did not. The Corallorhiza plastome is not drastically reduced in overall size (∼6% reduction relative to that of photosynthetic Oncidium), but displays a pattern congruent with a loss of photosynthetic function. Comparing Corallorhiza with other heterotrophs allows some emergent evolutionary patterns to be inferred, but these remain as hypotheses to be tested, especially at lower taxonomic levels, and in lineages illustrating transitions from autotrophy to heterotrophy. The independent, unique processes of plastome modification among mycoheterotrophic lineages illustrate the urgency of their conservation.

A unique tRNA gene family and a novel, highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae)

Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS−AGY and trnQ genes, a trnM isomerism, but it lacks trnS−CUN. We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP→trnS−AGY) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, “Oxidored_q1_N”, which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available bivalve mt tRNA and atp8 genes.

Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus tropaeoli from spontaneous cocoa bean fermentations

Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T) = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.

The Mitochondrial Atp8 Mutation Induces Mitochondrial ROS Generation, Secretory Dysfunction, and β-Cell Mass Adaptation in Conplastic B6-mtFVB Mice

Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic phenotypes. In this study, we used conplastic mouse strains to characterize the impact of a mtDNA mutation in the Atp8 gene on β-cell function, reactive oxygen species (ROS) generation, β-cell mass, and glucose metabolism in response to high-fat diet (HFD). In comparison with B6-mt(AKR) controls, the B6-mt(FVB) strain carries a point mutation of the mtDNA-coded Atp8 gene (ATP synthase), leading to a fragmentated mitochondrial phenotype. Isolated pancreatic islets from 3-month-old B6-mt(FVB) mice showed increased mitochondrial generation of ROS, reduced cellular ATP levels, reduced glucose-induced insulin secretion, higher susceptibility to palmitate stress, and pathological morphology of mitochondria. ROS generation in β-cells was not affected by changes of the ambient glucose concentrations. Feeding a HFD for 3 months resulted in impaired glucose tolerance in B6-mt(FVB) mice but not in B6-mt(AKR) controls. In B6-mt(FVB) animals, glucose intolerance positively correlated with gain of body weight. Serum insulin levels and β-cell mass significantly increased in B6-mt(FVB) mice after a 3-month HFD. The data indicate that the mutation in the Atp8 gene induces mitochondrial dysfunction in β-cells with concomitant impairment of secretory responsiveness. This mitochondrial dysfunction induced a higher susceptibility to metabolic stressors, although this effect appeared not strictly linked to nutrient-induced ROS generation. The Atp8 gene mutation caused mitochondrial dysfunction, apparently stimulating an adaptive increase of β-cell mass in response to HFD, whereas mitochondrial ROS might have had an supportive role.

Experimental Relocation of the Mitochondrial ATP9 Gene to the Nucleus Reveals Forces Underlying Mitochondrial Genome Evolution

Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.

Abstract 210: PPARγ Activation Prevents Cardiac Dysfunction in Sepsis via Stimulation of Energy Production

Septic cardiac dysfunction has been attributed to either inflammation or suppression of fatty acid oxidation (FAO). We hypothesized that energy deprivation accounts for cardiac dysfunction in lipopolysaccharide (LPS)-induced sepsis. LPS compromised cardiac function, increased cardiac inflammatory markers (interleukin (IL)-1α, IL-6 and tumor necrosis factor (TNF)α) mRNA, and reduced FAO, ATP and FAO-related gene expression: Peroxisome proliferator activating receptor (PPAR)α, cluster of differentiation 36, lipoprotein lipase (LpL), fatty acid transport protein, carnitine palmitoyl-transferase (Cpt)1, PPARγ coactivator (PGC-1)α and PGC-1β in C57BL/6 mice. LC-MS/MS analysis of cardiac lipids showed that total ceramide, which might account for defective FAO and ceramide species were not modulated by LPS. Electron microscopy and ATPase6 DNA levels showed that LPS-treated animals had fewer and smaller cardiac mitochondria. PPARα agonist, WY-14643, did not prevent cardiac dysfunction and suppression of FAO-associated gene expression in LPS-treated mice. In contrast, LPS did not affect cardiac function of mice with cardiomyocyte-specific expression of PPARγ (αMHC-PPARg), although cardiac IL-1α, IL-6 and TNFα mRNA levels were increased. Despite a 77% reduction in PPARα, mRNA levels of FAO-associated genes, such as acyl-CoA oxidase, PGC-1α, PPARδ, Cpt1 increased over 10-fold as compared to saline-treated αMHC-PPARg mice. Similarly, cardiac FAO and ATP content was 2.6 and 5-fold greater in LPS-treated αMHC-PPARγ mice compared to LPS-treated C57BL/6 mice. Moreover, treatment of C57BL/6 mice with LPS and the PPARγ agonist, rosiglitazone, prevented cardiac dysfunction, increased FAO and relevant gene expression, while IL-1α, IL-6 and TNFα mRNA levels increased. Rosiglitazone prevented LPS-mediated reduction of PGC-1α and maintained the number and size of mitochondria. Treatment of C57BL/6 mice with a lethal dose of LPS (15 mg/kg) and repeated doses of rosiglitazone (30mg/kg/day for 72 hours) improved survival, mobility and overall appearance of LPS-treated mice. Thus, PPARγ activation stabilized cardiac energy and prevented cardiac dysfunction and mortality in septic animals despite cardiac inflammation and PPARα downregulation.