Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.     

Black Yeast Biota in the Mangrove, in Search of the Origin of the Lethargic Crab Disease (LCD)

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis, two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea- or Exophiala-like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae, was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis, which is probably not dependent on the host species, U. cordatus. However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves.     

Diplospory and obligate apomixis in Miconia albicans (Miconieae, Melastomataceae) and an embryological comparison with its sexual congener M. chamissois

Apomixis, or asexual reproduction through seeds, has been reported for species of the tribe Miconieae, Melastomataceae, but details of the process have yet to be described. We analyzed and compared sporogenesis and gametogenesis in the apomictic Miconia albicans and the sexual M. chamissois. The results point to some differences between species, which were related to the apomictic process. In M. albicans microsporogenesis, problems during meiosis and degeneration of its products led to total pollen sterility, while M. chamissois presented normal bicellular pollen grains in the mature anther. The absence or abnormality of meiosis in M. albicans megasporogenesis led to the formation of an unreduced embryo sac and also to egg cell parthenogenesis, which gave rise to the apomictic embryo. Embryo and endosperm development were autonomous, resulting in seeds and fruits independent of pollination and fertilization. Thus, in this species, apomixis can be classified as diplosporic and obligate. In contrast, meiosis was as expected in the sexual M. chamissois, and led to the development of a reduced embryo sac. Despite the divergent pathways, many embryological characteristics were similar between the studied species and other Melastomataceae and they seem to be conservative character states for the family.     

Genetic Analysis of the Biosynthesis of 2-Methoxy-3-Isobutylpyrazine,a Major Grape-Derived Aroma Compound Impacting Wine Quality

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.The pleasure experienced while enjoying a glass of wine is the result of sophisticated sensory, neurophysiological, and psychological processes triggered by wine aroma. Wine flavor is the result of a complex mixture of volatile compounds in the headspace of the glass that induces feelings of pleasure at the brain level (Shepherd, 2006). During the last 40 years, over 800 volatile molecules have been formally identified in wines, in concentrations ranging from hundreds of milligrams per liter down to a few picograms per liter (Ebeler and Thorngate, 2009; Styger et al., 2011). Among all of them, a relatively limited number of compounds, called varietal (or primary) aromas, play a crucial role in wine flavor and typicality. These aromas, which are related to the grape variety, belong to a limited number of chemical families, including monoterpenes, C13 norisoprenoids, volatile sulfur compounds, and methoxypyrazines (MPs; Ebeler and Thorngate, 2009). Quite frequently, they exist mostly in the grape (Vitis vinifera) berry as nonvolatile, odorless, “bound” forms that can be released by chemical and enzymatic reactions occurring during the winemaking and wine aging processes, thus enhancing wine’s varietal expression (Styger et al., 2011). Two classical examples are the glycoside precursors of the monoterpenols (Strauss et al., 1986) and the cysteinylated or glutathionylated precursors of the volatile thiols (Tominaga et al., 1998; Peña-Gallego et al., 2012). Noticeable exceptions are the MPs, which are found in grape berries exclusively as free, volatile molecules.MPs are strongly odorant volatile heterocycles, with vegetable-like fragrances, that are widely occurring in the plant kingdom (Maga, 1982). In grape, they can be detected in fruits, leaves, shoots, and roots (Dunlevy et al., 2010). They are found in different grape varieties and are particularly abundant in the so-called Bordeaux cultivars (i.e. cv Cabernet Franc, Cabernet Sauvignon [CS], Sauvignon Blanc, Merlot, and Carménère [Car]; Bayonove et al., 1975; Lacey et al., 1991; Roujou de Boubée et al., 2002; Belancic and Agosin, 2007), whereas they are rarely detected in other cultivars, such as cv Pinot Noir (PN), Chardonnay, or Petit Verdot (PV). This finding indicates a strong genotype dependency of MP biosynthesis (Koch et al., 2010). MPs are accumulated in berries until bunch closure or véraison, and then their level declines after véraison (Hashizume and Samuta, 1999; Ryona et al., 2008). MP concentration in wine is highly correlated with the grape berry content at harvest (Roujou de Boubée et al., 2002). Three MPs are found in grape berries: 2-methoxy-3-isobutylpyrazine (IBMP), which is the most abundant, and two others, 2-methoxy-3-isopropylpyrazine (IPMP) and 2-methoxy-3-sec-butylpyrazine (SBMP; Ebeler and Thorngate, 2009). Both IBMP and IPMP display very low sensory detection thresholds in the wine matrix, ranging from 1 to 16 ng L^–1.MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols (Dubourdieu et al., 2006; Lund et al., 2009). Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these extremely powerful odorants will reduce consumer acceptance (Parr et al., 2007). In red wine, MPs are considered as off-flavor, and red wines can be depreciated by concentrations above 10 ng L^–1 (Allen et al., 1991; Roujou de Boubée et al., 2000; Belancic and Agosin, 2007). Given the importance of MPs, either as typical varietal aromas or as detrimental off-flavors, deciphering the genetic and molecular determinism of their accumulation is of high interest for viticulture.In spite of this, until recently little was known about the MP biosynthesis pathway or the MP biosynthetic genes, either in grapevine or other plant species. Theoretical biosynthesis pathways have been proposed since the mid-1970s. They all start by the addition of an α-dicarbonyl on a branched amino acid (Leu for IBMP, Val for IPMP) to form a 2-hydroxy-3-alkylpyrazine, which is subsequently transformed into the corresponding MP, by a methoxylation reaction (Murray and Whitfield 1975; Gallois et al., 1988). While the initial addition step remains to be demonstrated in plants, an S-adenosyl-l-Met (SAM)-dependent O-methyltransferase (OMT), capable of converting 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP, has been detected in CS shoots, partially purified and sequenced (Hashizume et al., 2001a, 2001b; Fig. 1). Recently, Dunlevy et al. (2010) characterized two OMTs, VvOMT1 and VvOMT2, capable of methylating IBHP in vitro, albeit with high apparent K_m values. To investigate the genetic bases of MP biosynthesis in grape berries, we performed a quantitative trait loci (QTL) analysis, which has led to the identification of two previously uncharacterized OMTs termed VvOMT3 and VvOMT4. Functional characterization of these two OMTs showed that VvOMT3 was highly specific and efficient for IBHP methylation. Based on its differential expression in high-MP and low-MP grapevine varieties, we propose that VvOMT3 and, to a lesser extent, VvOMT4 are key genes for MP biosynthesis in grapevine berries.Open in a separate windowFigure 1.Putative biosynthesis pathway for IBMP adapted from Hashizume et al. (2001a). SAHcy, S-Adenosyl-l-homo-Cys.     

How does the metabolism of tumour cells differ from that of normal cells

Tumour cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbour modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so-called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumour cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumour phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumour cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3BP (3-bromopyruvate), on glycolytic enzyme targets will be presented.     

New Culicoides Latreille of the subgenus Mataemyia Vargas from Pará, Brazil (Diptera: Ceratopogonidae)

Two new species of Culicoides Latreille of the Neotropical subgenus Mataemyia Vargas are described and illustrated based on female specimens from Juruti, Pará, Brazil and compared with their similar congeners. This paper also presents a diagnosis of the subgenus Mataemyia and a systematic key for the identification of the 19 species of the subgenus.     

Phenology of mating flight in Ecitoninae (Hymenoptera: Formicidae) in a Brazilian Atlantic Forest location

The mating flight of Ecitoninae ants was studied using four light traps inspected daily during the whole year of 2004. A total of 1285 males belonging to 15 species was collected, the species number varying monthly from two to 11. For most species, the beginning of the reproductive period occurred in November or in February. Synchronism of the flight activity among species was observed in the genera Neivamyrmex and Eciton, while segregation occurred in two Labidus species. In the model tested to explain male abundance over the year, including rainfall, temperature and interaction between both factors, only temperature was significant.     

A multi-environmental study of recent breeding progress on nitrogen use efficiency in wheat (Triticum aestivum L.)

Key message

By comparing 195 varieties in eight trials, this study assesses nitrogen use efficiency improvement in high and low nitrogen conditions in European winter wheat over the last 25 years.

Abstract

In a context where European agriculture practices have to deal with environmental concerns and nitrogen (N) fertiliser cost, nitrogen use efficiency (NUE) has to be improved. This study assessed genetic progress in winter wheat (Triticum aestivum L.) NUE. Two hundred and twenty-five European elite varieties were tested in four environments under two levels of N. Global genetic progress was assessed on additive genetic values and on genotype × N interaction, covering 25 years of European breeding. To avoid sampling bias, quality, precocity and plant height were added as covariates in the analyses when needed. Genotype × environment interactions were highly significant for all the traits studied to such an extent that no additive genetic effect was detected on N uptake. Genotype × N interactions were significant for yield, grain protein content (GPC), N concentration in straw, N utilisation, and NUE. Grain yield improvement (+0.45 % year^?1) was independent of the N treatment. GPC was stable, thus grain nitrogen yield was improved (+0.39 % year^?1). Genetic progress on N harvest index (+0.12 % year^?1) and on N concentration in straw (?0.52 % year^?1) possibly revealed improvement in N remobilisation. There has been an improvement of NUE additive genetic value (+0.33 % year^?1) linked to better N utilisation (+0.20 % year^?1). Improved yield stability was detected as a significant improvement of NUE in low compared to high N conditions. The application of these results to breeding programs is discussed.     

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.     

Application of Hazard Analysis and Critical Control Points (HACCP) to the Cultivation Line of Mushroom and Other Cultivated Edible Fungi

The Hazard analysis and critical control points (HACCP) is a preventive system which seeks to ensure food safety and security. It allows product protection and correction of errors, improves the costs derived from quality defects and reduces the final overcontrol. In this paper, the system is applied to the line of cultivation of mushrooms and other edible cultivated fungi. From all stages of the process, only the reception of covering materials (stage 1) and compost (stage 3), the pre-fruiting and induction (step 6) and the harvest (stage 7) have been considered as critical control point (CCP). The main hazards found were the presence of unauthorized phytosanitary products or above the permitted dose (stages 6 and 7), and the presence of pathogenic bacteria (stages 1 and 3) and/or heavy metals (stage 3). The implementation of this knowledge will allow the self-control of their productions based on the system HACCP to any plant dedicated to mushroom or other edible fungi cultivation.