The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome,circular plasmid pSLE1 and linear plasmid pSLE2

Background

Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics.

Results

Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.

Conclusions

The S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1652-8) contains supplementary material, which is available to authorized users.     

Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain

Clinical and experimental studies show a modulatory role of estrogens in the brain and suggest their beneficial action in mental and neurodegenerative diseases. The estrogen receptors ER and ERβ are present in the brain and their targeting could bring selectivity and reduced risk of cancer. Implication of ERs in the effect of estradiol on dopamine, opiate and glutamate neurotransmission is reviewed. The ER agonist, PPT, is shown as estradiol to modulate hippocampal NMDA receptors and AMPA receptors in cortex and striatum of ovariectomized rats whereas the ERβ agonist DPN is inactive. Striatal DPN activity suggests implication of ERβ in estradiol modulation of D2 receptors and transporters in ovariectomized rats and is supported by the lack of effect of estradiol in ERβ knockout (ERKOβ) mice. Both ER and ERβ agonists modulate striatal preproenkephalin (PPE) gene expression in ovariectomized rats. In male mice PPT protects against MPTP toxicity to striatal dopamine; this implicates Akt/GSK3β signaling and the apoptotic regulators Bcl2 and Bad. This suggests a role for ER in striatal dopamine neuroprotection. ERKO mice are more susceptible to MPTP toxicity and not protected by estradiol; differences in ERKOβ mice are subtler. These results suggest therapeutic potential for the brain of ER specific agonists.     

Development of a semi-automatic system for pollen recognition

A semi-automatic system for pollen recognitionis studied for the european project ASTHMA. The goal of such a system is to provideaccurate pollen concentration measurements. This information can be used as well by thepalynologists, the clinicians or a forecastsystem to predict pollen dispersion. At first,our emphasis has been put on Cupressaceae, Olea, Poaceae and Urticaceae pollen types. The system is composed of two modules: pollengrain extraction and pollen grain recognition. In the first module, the pollen grains areobserved in light microscopy and are extractedautomatically from a pollen slide coloured withfuchsin and digitized in 3D. In the secondmodule, the pollen grain is analyzed forrecognition. To accomplish the recognition, itis necessary to work on 3D images and to usedetailed palynological knowledge. Thisknowledge describes the pollen types accordingto their main visible characteristerics and tothose which are important for recognition. Somepollen structures are identified like the porewith annulus in Poaceae, the reticulum in Oleaand similar pollen types or the cytoplasm inCupressaceae. The preliminary results show therecognition of some pollen types, likeUrticaceae or Poaceae or some groups of pollentypes, like reticulate group.     

Landscape context modulates alien plant invasion in Mediterranean forest edges

Natural habitats in human-altered landscapes are especially vulnerable to biological invasions, especially in their edges. We aim to understand the influence of landscape and local characteristics on biological invasions by exploring the level of plant invasion and alien species traits in forest edges in highly urbanized landscapes. We identified all plant species in 73 paired plots in the edge and 50 m towards the interior of the forest. We explored the association between alien species richness and similarity in species composition between edge and interior plots with landscape and local variables, using generalized linear models and variance partitioning techniques. Then, we performed Fourth-corner analyses to explore the association between alien plant traits and local and landscape variables. In contrast to native species richness, alien species richness was more affected by the surrounding landscape than by the local characteristics of the edge. Road proximity was positively associated with alien species richness and proportion and was its most important correlate, whereas disturbance was negatively associated with native species richness and was its most influential factor. Alien plant traits were also primarily associated with landscape characteristics. For instance, species of Mediterranean origin and introduced for agriculture were associated with higher agriculture use in the landscape. Our findings suggest that risk analyses of habitat vulnerability to invasion must consider the landscape context in order to successfully predict highly invaded areas and identify potentially successful invaders.     

Diverging patterns of mitochondrial and nuclear DNA diversity in subarctic black spruce: imprint of a founder effect associated with postglacial colonization

High-latitude ecotonal populations at the species margins may exhibit altered patterns of genetic diversity, resulting from more or less recent founder events and from bottleneck effects in response to climate oscillations. Patterns of genetic diversity were investigated in nine populations of the conifer black spruce (Picea mariana [Mill.] BSP.) in northwestern Québec, Canada, using seed-dispersed mitochondrial (mt) DNA and nuclear (nc) DNA. mtDNA diversity (mitotypes) was assessed at three loci, and ncDNA diversity was estimated for nine expressed sequence tag polymorphism (ESTP) loci. Sampling included populations from the boreal forest and the southern and northern subzones of the subarctic forest-tundra, a fire-born ecotone. For ncDNA, populations from all three vegetation zones were highly diverse with little population differentiation (thetaN = 0.014); even the northernmost populations showed no loss of rare alleles. Patterns of mitotype diversity were strikingly different: within-population diversity and population differentiation were high for boreal forest populations [expected heterozygosity per locus (HE) = 0.58 and thetaM = 0.529], but all subarctic populations were fixed for a single mitotype (HE = 0). This lack of variation suggests a founder event caused by long-distance seed establishment during postglacial colonization, consistent with palaeoecological data. The estimated movement of seeds alone (effective number of migrants per generation, NmM < 2) was much restricted compared to that estimated from nuclear variants, which including pollen movement (NmN > 17). This could account for the conservation of a founder imprint in the mtDNA of subarctic black spruce. After reduction, presumably in the early Holocene, the diversity in ncDNA would have been replenished rapidly by pollen-mediated gene flow, and maintained subsequently through vegetative layering during the current cooler period covering the last 3000 years.     

Effects of grazing on patch structure in a semi‐arid two‐phase vegetation mosaic

Abstract. Question: What are the grazing effects in the spatial organization and the internal structure of high and low cover patches from a two‐phase vegetation mosaic? Location: Patagonian steppe, Argentina. Methods: We mapped vegetation under three different grazing conditions: ungrazed, lightly grazed and heavily grazed. We analysed the spatial patterns of the dominant life forms. Also, in each patch type, we determined density, species composition, richness, diversity, size structure and dead biomass of grasses under different grazing conditions. Results: The vegetation was spatially organized in a two‐phase mosaic. High cover patches resulted from the association of grasses and shrubs and low cover patches were represented by scattered tussock grasses on bare ground. This spatial organization was not affected by grazing, but heavy grazing changed the grass species involved in high cover patches and reduced the density and cover of grasses in both patch types. Species richness and diversity in high cover patches decreased under grazing conditions, whereas in low cover patches it remained unchanged. Also, the decrease of palatable grasses was steeper in high cover patches than in low cover patches under grazing conditions. Conclusions: We suggest that although grazing promotes or inhibits particular species, it does not modify the mosaic structure of Patagonian steppe. The fact that the mosaic remained unchanged after 100 years of grazing suggests that grazing does not compromize population processes involved in maintaining patch structure, including seed dispersal, establishment or biotic interactions among life forms.     

Sodium and Proton Effects on Inward Proton Transport through Na/K Pumps

The Na/K pump hydrolyzes ATP to export three intracellular Na (Na_i) as it imports two extracellular K (K_o) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Na_o and K_o, the pump passively imports protons, generating an inward current (I_H). To elucidate the mechanisms of I_H, we used voltage-clamp techniques to investigate the [H]_o, [Na]_o, and voltage dependence of I_H in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pH_o revealed that H_o both activates I_H (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Na_o effects depend on pH_o; at pH_o where no H_o inhibition is observed, Na_o inhibits I_H at all concentrations, but when applied at pH_o that inhibits pump-mediated current, low [Na]_o activates I_H and high [Na]_o inhibits it. Our results demonstrate that I_H is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of I_H previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.     

Effect of the Gas6 c.834+7G>A Polymorphism and the Interaction of Known Risk Factors on AMD Pathogenesis in Hungarian Patients

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Numerous genetic factors contribute to the development of the multifactorial disease. We performed a case-control study to assess the risk conferred by known and candidate genetic polymorphisms on the development of AMD. We searched for genetic interactions and for differences in dry and wet AMD etiology. We enrolled 213 patients with exudative, 67 patients with dry AMD and 106 age and ethnically matched controls. Altogether 12 polymorphisms in Apolipoprotein E, complement factor H, complement factor I, complement component 3, blood coagulation factor XIII, HTRA1, LOC387715, Gas6 and MerTK genes were tested. No association was found between either the exudative or the dry form and the polymorphisms in the Apolipoprotein E, complement factor I, FXIII and MerTK genes. Gas6 c.834+7G>A polymorphism was found to be significantly protective irrespective of other genotypes, reducing the odds of wet type AMD by a half (OR = 0.50, 95%CI: 0.26–0.97, p = 0.04). Multiple regression models revealed an interesting genetic interaction in the dry AMD subgroup. In the absence of C3 risk allele, mutant genotypes of both CFH and HTRA1 behaved as strongly significant risk factors (OR = 7.96, 95%CI: 2.39 = 26.50, p = 0.0007, and OR = 36.02, 95%CI: 3.30–393.02, p = 0.0033, respectively), but reduced to neutrality otherwise. The risk allele of C3 was observed to carry a significant risk in the simultaneous absence of homozygous CFH and HTRA1 polymorphisms only, in which case it was associated with a near-five-fold relative increase in the odds of dry type AMD (OR = 4.93, 95%CI: 1.98–12.25, p = 0.0006). Our results suggest a protective role of Gas6 c.834+7G>A polymorphism in exudative AMD development. In addition, novel genetic interactions were revealed between CFH, HTRA1 and C3 polymorphisms that might contribute to the pathogenesis of dry AMD.