Closing the genome of unculturable cable bacteria using a combined metagenomic assembly of long and short sequencing reads.

Many environmentally relevant micro-organisms cannot be cultured, and even with the latest metagenomic approaches, achieving complete genomes for specific target organisms of interest remains a challenge. Cable bacteria provide a prominent example of a microbial ecosystem engineer that is currently unculturable. They occur in low abundance in natural sediments, but due to their capability for long-distance electron transport, they exert a disproportionately large impact on the biogeochemistry of their environment. Current available genomes of marine cable bacteria are highly fragmented and incomplete, hampering the elucidation of their unique electrogenic physiology. Here, we present a metagenomic pipeline that combines Nanopore long-read and Illumina short-read shotgun sequencing. Starting from a clonal enrichment of a cable bacterium, we recovered a circular metagenome-assembled genome (5.09 Mbp in size), which represents a novel cable bacterium species with the proposed name Candidatus Electrothrix scaldis. The closed genome contains 1109 novel identified genes, including key metabolic enzymes not previously described in incomplete genomes of cable bacteria. We examined in detail the factors leading to genome closure. Foremost, native, non-amplified long reads are crucial to resolve the many repetitive regions within the genome of cable bacteria, and by analysing the whole metagenomic assembly, we found that low strain diversity is key for achieving genome closure. The insights and approaches presented here could help achieve genome closure for other keystone micro-organisms present in complex environmental samples at low abundance.

The impact of chromium toxicity on the yield and quality of rice grains produced under ambient and elevated levels of CO2.

Rice is a highly valuable crop consumed all over the world. Soil pollution, more specifically chromium (Cr), decreases rice yield and quality. Future climate CO2 (eCO2) is known to affect the growth and yield of crops as well as the quality parameters associated with human health. However, the detailed physiological and biochemical responses induced by Cr in rice grains produced under eCO2 have not been deeply studied. Cr (200 and 400 mg Cr6+/Kg soil) inhibited rice yield and photosynthesis in Sakha 106, but to less extend in Giza 181 rice cultivar. Elevated CO2 reduced Cr accumulation and, consequently, recovered the negative impact of the higher Cr dose, mainly in Sakha 106. This could be explained by improved photosynthesis which was consistent with increased carbohydrate level and metabolism (starch synthases and amylase). Moreover, these increases provided a route for the biosynthesis of organic, amino and fatty acids. At grain quality level, eCO2 differentially mitigated Cr stress-induced reductions in minerals (e.g., P, Mg and Ca), proteins (prolamin, globulin, albumin, glutelin), unsaturated fatty acids (e.g., C20:2 and C24:1) and antioxidants (phenolics and total antioxidant capacity) in both cultivars. This study provided insights into the physiological and biochemical bases of eCO2-induced grain yield and quality of Cr-stressed rice.

Elevated CO2 differentially mitigates chromium (VI) toxicity in two rice cultivars by modulating mineral homeostasis and improving redox status.

Chromium (Cr) contamination reduces crop productivity worldwide. On the other hand, the expected increase in the future CO2 levels (eCO2) would improve plant growth under diverse growth conditions. However, the synergetic effect of eCO2 has not been investigated at both physiological and biochemical levels in Cr-contaminated soil. This study aims to analyze the mitigating effect of eCO2 on Cr VI phytotoxicity in two rice cultivars (Giza 181 and Sakha 106). Plants are exposed to different Cr concentrations (0, 200 and 400 mg Cr/kg Soil) at ambient (aCO2) and eCO2 (410 and 620 ppm, respectively). Unlike the stress parameters (MDA, H2O2 and protein oxidation), growth and photosynthetic reactions significantly dropped with increasing Cr concentration. However, in eCO2 conditions, plants were able to mitigate the Cr stress by inducing antioxidants as well as higher concentrations of phytochelatins to detoxify Cr. Notably, the expression levels of the genes involved in mineral nutrition i.e., OsNRAMP1, OsRT1, OsHMA3, OsLCT1 and iron chelate reductase were upregulated in Cr-stressed Giza 181 plants grown under eCO2. Mainly in Sakha 106, eCO2 induced ascorbate-glutathione (ASC/GSH)-mediated antioxidative defense system. The present study brings the first ever comprehensive assessment of how future eCO2 differentially mitigated Cr toxicity in rice.

Arabidopsis root growth and development under metal exposure presented in an adverse outcome pathway framework.

Due to human activities, soils become more and more polluted with metals, which imposes risks for human health and wildlife welfare. As most of the metals end up in the food chain through accumulation in plants, we need to establish science-based environmental criteria and risk management policies. To meet these necessities, a thorough understanding is required of how these metals accumulate in and affect plants. Many studies have been conducted towards this aim, but strikingly, only a few entries can be found in ecotoxicological databases, especially on Arabidopsis thaliana, which serves as a model species for plant (cell) physiology and genetic studies. As experimental conditions seem to vary considerably throughout literature, extrapolation or comparison of data is rather difficult or should be approached with caution. Furthermore, metal-polluted soils often contain more than one metal, yet limited studies investigated the impact of metal mixtures on plants. This review aims to compile all data concerning root system architecture under Cu, Cd and Zn stress, in single or multi-metal exposure in A. thaliana, and link it to metal-induced responses at different biological levels. Global incorporation into an adverse outcome pathway framework is presented.

Novel Mechanisms of ALK Activation Revealed by Analysis of the Y1278S Neuroblastoma Mutation.

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila.

In Drosophila melanogaster, the receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (Alk) and its ligand jelly belly (Jeb) are required to specify muscle founder cells in the visceral mesoderm. We identified a critical role for the scaffolding protein Cnk (connector enhancer of kinase suppressor of Ras) in this signaling pathway. Embryos that ectopically expressed the minimal Alk interaction region in the carboxyl terminus of Cnk or lacked maternal and zygotic cnk did not generate visceral founder cells or a functional gut musculature, phenotypes that resemble those of jeb and Alk mutants. Deletion of the entire Alk-interacting region in the cnk locus affected the Alk signaling pathway in the visceral mesoderm and not other RTK signaling pathways in other tissues. In addition, the Cnk-interacting protein Aveugle (Ave) was critical for Alk signaling in the developing visceral mesoderm. Alk signaling stimulates the MAPK/ERK pathway, but the scaffolding protein Ksr, which facilitates activation of this pathway, was not required to promote visceral founder cell specification. Thus, Cnk and Ave represent critical molecules downstream of Alk, and their loss genocopies the lack of visceral founder cell specification of Alk and jeb mutants, indicating their essential roles in Alk signaling.

Tumour-associated mutations of PA-TM-RING ubiquitin ligases RNF167/RNF13 identify the PA domain as a determinant for endosomal localization.

Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, and processes that are regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. The PA (protease-associated) domain E3 ligases, such as GodzillaCG10277 in Drosophila melanogaster and RNF167 (RING finger protein 167) in humans, have been implicated in the regulation of cellular endosome trafficking. In the present study, we have characterized point mutations in the RING (really interesting new gene) domain of human RNF13 and RNF167, which have been identified in human tumour samples, that abrogate ubiquitin ligase activity as well as function. In the present study, we have also identified a functional role for the PA domain, which is required for endosomal localization of these proteins. Although the PA domain point mutations of RNF13 and RNF167 identified in human tumours are ligase active, the resultant mutant proteins are mislocalized within the cell. Thus the PA domain E3 ligases examined in the present study appear to require both E3 ligase activity as well as an intact PA domain to efficiently target and ubiquitylate their cellular substrates.