Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Temporal variation in microbial and plant biomass during summer in a Mediterranean high-mountain dry grassland

Aims

We assessed the temporal changes on microbial biomass in relation to changes in soil moisture, dissolved organic carbon and plant biomass during the summer season in a Mediterranean high-mountain grassland.

Methods

Temporal variations were tested by two-way ANOVA. The relationships among microbial biomass, plant biomass, soil water content, soil organic carbon, dissolved organic carbon and total soil nitrogen during the summer season were assessed by means of structural equation modeling.

Results

Microbial biomass did not show variation, while dissolved organic carbon and root biomass decreased throughout the summer. Aboveground plant biomass peaked in the middle of the summer, when soil water content was at its minimum. Soil water content directly and negatively affected soil microbial biomass, and positively affected dissolved organic carbon. Moreover soil microbial biomass and dissolved organic carbon were negatively related. Plant biomass effects on soil microbial biomass were driven by root biomass, which indirectly affected soil microbial biomass through effects on soil organic carbon and soil nitrogen.

Conclusions

The temporal dynamic of microbial biomass during the summer season appeared to differ from previous observations in temperate alpine communities, and indicated the drought resistance of the microbial community during the summer in Mediterranean high-mountain grasslands. During the dry period, microbial biomass may play an alternative role in soil carbon conservation.     

Geranyllinalool Synthases in Solanaceae and Other Angiosperms Constitute an Ancient Branch of Diterpene Synthases Involved in the Synthesis of Defensive Compounds

Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the terpene synthase (TPS) family and two Fabaceae GLSs that belong to the TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (Solanum lycopersicum) TPS-e/f gene, TPS46, encoding GLS (SlGLS) and its homolog (NaGLS) from Nicotiana attenuata. The K_m value of SlGLS for geranylgeranyl diphosphate was 18.7 µm, with a turnover rate value of 6.85 s^–1. In leaves and flowers of N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides, NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato, SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the TPS-e/f clade that diverged from kaurene synthases, also in the TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the GLS activity of genes in this branch in diverse eudicot families suggest that GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a TPS sequence belonging to this GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants.Geranyllinalool is an acyclic diterpene alcohol with a wide distribution in the plant kingdom; it has been identified as component of essential oils of distantly related plant species such as Jasmin grandiflorum, Michelia champaca, and Homamelis virginiana (Sandeep, 2009). Geranyllinalool is the precursor of 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), a volatile C₁₆-homoterpene emitted from the foliage of many angiosperm species including Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), maize (Zea mays), fava bean (Vicia faba), lima bean (Phaseolus lunatus), alfalfa (Medicago sativa), and Eucalyptus spp. (Van Poecke et al., 2001; Ament et al., 2004; Williams et al., 2005; Hopke et al., 1994; Leitner et al., 2010; Webster et al., 2010). In addition, various hydroxygeranyllinalool glycosides have been isolated from many Solanaceous species such as Capsicum annuum, Lycium chinense, and at least 26 Nicotiana species (Yahara et al., 1993; Iorizzi et al., 2001; Snook et al., 1997).The biosynthetic pathway leading to geranyllinalool, as for all other terpenoids, begins with the condensation of isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate. Sequential condensation of one isopentenyl diphosphate molecule with three dimethylallyl diphosphate molecules produces geranylgeranyl diphosphate (GGPP), the C-20 intermediate of the diterpenoid pathway. Next, a terpene synthase (TPS) catalyzes a two-step reaction in which carbocation formation of the C20 precursor is followed by an allylic rearrangement that results in the production of the tertiary alcohol geranyllinalool (Herde et al., 2008).Although geranyllinalool and its derivatives, TMTT and geranyllinalool glycosides, have been reported in a wide variety of plant species, a geranyllinalool synthase (GLS) involved in TMTT biosynthesis was only recently identified in Arabidopsis (Herde et al., 2008). AtTPS04 belongs to the TPS-e/f subfamily along with the previously identified Clarkia spp. linalool synthases (Chen et al., 2011). More recently, two TPSs from Vitis vinifera and one from the daisy Grindelia hirsutula, also members of the TPS-e/f subfamily, were found to exhibit GLS activity in vitro (Martin et al., 2010; Zerbe et al., 2013). However, no in planta information has been presented for these, nor any evidence showing their involvement in TMTT biosynthesis.The common characteristic of the TPS-e/f GLSs so far identified is that they lack a predicted plastidial transit peptide, and direct evidence for nonplastidial localization was obtained in Arabidopsis by observing the AtTPS04-GUS fusion protein in the cytosol and endoplasmic reticulum (Herde et al., 2008). On the other hand, two TPS-g subfamily proteins from the closely related Fabaceae species Medicago truncatula and Phaseolus lunata (MtTPS03 and PlTPS2, respectively) were shown to be plastidic and to catalyze the formation of geranyllinalool in vitro when GGPP was provided as a substrate and also when expressed in a heterologous plant species (Arimura et al., 2008; Brillada et al., 2013). However, the same enzymes also produced linalool and nerolidol when supplied with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively (Arimura et al., 2008; Brillada et al., 2013). Given the present paucity of in vivo and in vitro studies of geranyllinalool biosynthesis in plants, it is not clear whether geranyllinalool in plants is typically produced via TPS-g or TPS-e/f type TPSs, or both.The role of geranyllinalool itself in plant tissues is not well established. Often geranyllinalool coexists in floral or vegetative tissues with its homoterpene derivative TMTT. The contribution of TMTT to the floral scent of insect-pollinated species suggests a putative role in attraction of pollinators (Tholl et al., 2011). On the other hand, in many angiosperm species, including tomato, TMTT is a component of volatile blends released from vegetative tissues upon herbivore attack, sometimes in parallel with its constitutive emission from floral tissues (Hopke et al., 1994; Ament et al., 2004; de Boer et al., 2004; Kant et al., 2004; Williams et al., 2005, Herde et al., 2008). The latter case suggests that TMTT might play a defensive role in both vegetative and floral tissues. TMTT production from insect-infested plants is considered as an indirect defense mechanism because TMTT attracts insect predators of the insect herbivores (Brillada et al., 2013). Interestingly, production of TMTT, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene, from herbivore-attacked lima bean plants has been found to correlate with enhanced expression of defense genes in neighboring nonaffected control plants (Arimura et al., 2000). In these cases, homoterpenes are believed to act as stress-responsive signals that enable intraspecies plant-to-plant communication.A plant defense role has also been suggested for 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) present in leaves and flowers of Nicotiana species, with higher concentrations measured in buds (Heiling et al., 2010; Jassbi et al., 2010). Several studies have found negative correlation between total leaf HGL-DTG content and the mass of the larvae that feed on them (Jassbi et al., 2008; Dinh et al., 2013). Eleven HGL-DTGs that differ in sugar moieties and number of malonylesters have been isolated from Nicotiana attenuata. The sugar groups of these compounds are Glc and rhamnose and are conjugated to the hydroxygeranyllinalool skeleton via bonds at C3 and C17 hydroxylated carbons. Additional sugars may be added to these sugars on their hydroxyl groups at C2, C4, and C6, and manolyl esters are typically formed at the C6 hydroxyl group of the glucoses. The concentrations of these HGL-DTGs are higher in young and reproductive tissues. While their total levels appear to be constant, the concentration of individual compounds change upon herbivore attack, with a proportionally greater increase in malonylated compounds. Unlike many other defense-related specialized metabolites, the N. attenuata HGL-DTGs are not found on the leaf surface or the trichomes, but, instead, they accumulate inside the leaves (Heiling et al., 2010).Here, we show that in the Solanaceae species cultivated tomato and N. attenuata, geranyllinalool is synthesized by TPSs that belong to the TPS-e/f subfamily and that the corresponding genes are related to Arabidopsis TPS04. The tomato and N. attenuata enzymes were biochemically characterized, and the kinetic parameters were determined. We also describe a detailed quantitative expression of these genes in different parts of the plant. In addition, we establish that the expression of the geranyllinalool synthase genes correlates well with the induced emission of TMTT in tomato leaves after alamethicin and methyl jasmonate (MeJA) treatments and with the total concentrations of HGL-DTGs in N. attenuata leaves and floral organs.     

Brazilian Microbiome Project: Revealing the Unexplored Microbial Diversity—Challenges and Prospects

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world’s macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world’s catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.     

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC–LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC–LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC–LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC–LH1 complexes, as long-range order appears to be induced only through its role of configuring RC–LH1 complexes into dimers.     

Aerobic endospore-forming bacteria isolated from Antarctic soils as producers of bioactive compounds of industrial interest

Spore-forming bacteria are known to produce various enzymes and bioproducts valuable to different industries and to bear the harsh conditions found in the Antarctic environment. However, aerobic or facultative spore-forming bacterial communities found in maritime Antarctic soils yet remain poorly studied. In this study, 80 spore-forming and cold-adapted bacterial strains were isolated from nine different soil samples of King George Island, in maritime Antarctica, and further clustered into amplified ribosomal DNA restriction analysis groups within each soil. Representative strains were then identified as belonging to Bacillus, Rummeliibacillus, Paenibacillus and Sporosarcina by 16S rRNA gene sequencing. The ability to produce extracellular enzymes, antimicrobial substances and biosurfactants was determined in all isolates. The enzymatic activities most frequently found among the isolates were as follows: esterase (45 %), caseinase (30 %), amylase (16.2 %) and gelatinase (15 %). Biosurfactant production was detected in 25 % of the isolates. The growth inhibition of methicillin-resistant Staphylococcus aureus was observed in 13.7 % of the strains tested, but only two strains inhibited the growth of Candida albicans. The isolated spore-forming bacterial species were also compared with the characteristics of the different Antarctic soils sampled based on their physicochemical properties, showing that pH, C and P were the main factors correlated with the distribution of this group of bacteria in the Antarctic soils studied. These Antarctic endospore-forming bacterial strains may have a potential for industrial processes occurring at low temperatures.     

Evidence for strong seasonality in the carbon storage and carbon use efficiency of an Amazonian forest

The relative contribution of gross primary production and ecosystem respiration to seasonal changes in the net carbon flux of tropical forests remains poorly quantified by both modelling and field studies. We use data assimilation to combine nine ecological time series from an eastern Amazonian forest, with mass balance constraints from an ecosystem carbon cycle model. The resulting analysis quantifies, with uncertainty estimates, the seasonal changes in the net carbon flux of a tropical rainforest which experiences a pronounced dry season. We show that the carbon accumulation in this forest was four times greater in the dry season than in the wet season and that this was accompanied by a 5% increase in the carbon use efficiency. This seasonal response was caused by a dry season increase in gross primary productivity, in response to radiation and a similar magnitude decrease in heterotrophic respiration, in response to drying soils. The analysis also predicts increased carbon allocation to leaves and wood in the wet season, and greater allocation to fine roots in the dry season. This study demonstrates implementation of seasonal variations in parameters better enables models to simulate observed patterns in data. In particular, we highlight the necessity to simulate the seasonal patterns of heterotrophic respiration to accurately simulate the net carbon flux seasonal tropical forest.     

Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes

Background

Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing.

Results

Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection.

Conclusions

Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-23) contains supplementary material, which is available to authorized users.     

Discovery of transgene insertion sites by high throughput sequencing of mate pair libraries

Background

Transgenesis by random integration of a transgene into the genome of a zygote has become a reliable and powerful method for the creation of new mouse strains that express exogenous genes, including human disease genes, tissue specific reporter genes or genes that allow for tissue specific recombination. Nearly 6,500 transgenic alleles have been created by random integration in embryos over the last 30 years, but for the vast majority of these strains, the transgene insertion sites remain uncharacterized.

Results

To obtain a complete understanding of how insertion sites might contribute to phenotypic outcomes, to more cost effectively manage transgenic strains, and to fully understand mechanisms of instability in transgene expression, we’ve developed methodology and a scoring scheme for transgene insertion site discovery using high throughput sequencing data.

Conclusions

Similar to other molecular approaches to transgene insertion site discovery, high-throughput sequencing of standard paired-end libraries is hindered by low signal to noise ratios. This problem is exacerbated when the transgene consists of sequences that are also present in the host genome. We’ve found that high throughput sequencing data from mate-pair libraries are more informative when compared to data from standard paired end libraries. We also show examples of the genomic regions that harbor transgenes, which have in common a preponderance of repetitive sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-367) contains supplementary material, which is available to authorized users.