Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.     

Stable isotope analyses—A method to distinguish intensively farmed from wild frogs

Consumption of frog legs is increasing worldwide, with potentially dramatic effects for ecosystems. More and more functioning frog farms are reported to exist. However, due to the lack of reliable methods to distinguish farmed from wild‐caught individuals, the origin of frogs in the international trade is often uncertain. Here, we present a new methodological approach to this problem. We investigated the isotopic composition of legally traded frog legs from suppliers in Vietnam and Indonesia. Muscle and bone tissue samples were examined for δ¹⁵N, δ¹³C, and δ¹⁸O stable isotope compositions, to elucidate the conditions under which the frogs grew up. We used DNA barcoding (16S rRNA) to verify species identities. We identified three traded species (Hoplobatrachus rugulosus, Fejervarya cancrivora and Limnonectes macrodon); species identities were partly deviating from package labeling. Isotopic values of δ¹⁵N and δ¹⁸O showed significant differences between species and country of origin. Based on low δ¹⁵N composition and generally little variation in stable isotope values, our results imply that frogs from Vietnam were indeed farmed. In contrast, the frogs from the Indonesian supplier likely grew up under natural conditions, indicated by higher δ¹⁵N values and stronger variability in the stable isotope composition. Our results indicate that stable isotope analyses seem to be a useful tool to distinguish between naturally growing and intensively farmed frogs. We believe that this method can be used to improve the control in the international trade of frog legs, as well as for other biological products, thus supporting farming activities and decreasing pressure on wild populations. However, we examined different species from different countries and had no access to samples of individuals with confirmed origin and living conditions. Therefore, we suggest improving this method further with individuals of known origin and history, preferably including samples of the respective nutritive bases.     

Arabidopsis tic110 is essential for the assembly and function of the protein import machinery of plastids

The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.     

Functional divergence and horizontal transfer of type IV secretion systems

The type IV secretion system (TFSSs) is a multifunctional family of translocation pathways that mediate the transfer of DNA among bacteria and deliver DNA and proteins to eukaryotic cells during bacterial infections. Horizontal transmission has dominated the evolution of the TFSS, as demonstrated here by a lack of congruence between the tree topology inferred from components of the TFSS and the presumed bacterial species divergence pattern. A parsimony analysis suggests that conjugation represents the ancestral state and that the divergence from conjugation to secretion of effector molecules has occurred independently at multiple sites in the tree. The result shows that the nodes at which functional shifts have occurred coincide with those of horizontal gene transfers among distantly related bacteria. We suggest that it is the transfer between species that paved the way for the divergence of the TFSSs and discuss the general role of horizontal gene transfers for the evolution of novel gene functions.     

Different versions of the Dayhoff rate matrix

Many phylogenetic inference methods are based on Markov models of sequence evolution. These are usually expressed in terms of a matrix (Q) of instantaneous rates of change but some models of amino acid replacement, most notably the PAM model of Dayhoff and colleagues, were originally published only in terms of time-dependent probability matrices (P(t)). Previously published methods for deriving Q have used eigen-decomposition of an approximation to P(t). We show that the commonly used value of t is too large to ensure convergence of the estimates of elements of Q. We describe two simpler alternative methods for deriving Q from information such as that published by Dayhoff and colleagues. Neither of these methods requires approximation or eigen-decomposition. We identify the methods used to derive various different versions of the Dayhoff model in current software, perform a comparison of existing and new implementations, and, to facilitate agreement among scientists using supposedly identical models, recommend that one of the new methods be used as a standard.     

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80 degrees C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80 degrees C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80 degrees C with a half-life of 4 h at 90 degrees C and 1.5 h at 100 degrees C.     

Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.     

Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence

The gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causative agent of bacterial spot disease in pepper and tomato plants, which leads to economically important yield losses. This pathosystem has become a well-established model for studying bacterial infection strategies. Here, we present the whole-genome sequence of the pepper-pathogenic Xanthomonas campestris pv. vesicatoria strain 85-10, which comprises a 5.17-Mb circular chromosome and four plasmids. The genome has a high G+C content (64.75%) and signatures of extensive genome plasticity. Whole-genome comparisons revealed a gene order similar to both Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and a structure completely different from Xanthomonas oryzae pv. oryzae. A total of 548 coding sequences (12.2%) are unique to X. campestris pv. vesicatoria. In addition to a type III secretion system, which is essential for pathogenicity, the genome of strain 85-10 encodes all other types of protein secretion systems described so far in gram-negative bacteria. Remarkably, one of the putative type IV secretion systems encoded on the largest plasmid is similar to the Icm/Dot systems of the human pathogens Legionella pneumophila and Coxiella burnetii. Comparisons with other completely sequenced plant pathogens predicted six novel type III effector proteins and several other virulence factors, including adhesins, cell wall-degrading enzymes, and extracellular polysaccharides.