Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

BACKGROUND: Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. RESULTS: We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. CONCLUSION: Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively. Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions

Contribution

Röbbe Wünschiers, Contribution to the discussion Synthetic Biology: Concepts, Values, and Politics, ICI Berlin, 19 September 2014, video recording, mp4, 04:51 <https://doi.org/10.25620/e140919_3

A primer on pollen assignment by nanopore-based DNA sequencing

The possibility to identify plants based on the taxonomic information coming from their pollen grains offers many applications within various biological disciplines. In the past and depending on the application or research in question, pollen origin was analyzed by microscopy, usually preceded by chemical treatment methods. This procedure for identification of pollen grains is both time-consuming and requires expert knowledge of morphological features. Additionally, these microscopically recognizable features usually have a low resolution at species-level. Since a few decades, DNA has been used for the identification of pollen taxa, as sequencing technologies evolved both in their handling and affordability. We discuss advantages and challenges of pollen DNA analyses compared to traditional methods. With readers with little experience in this field in mind, we present a hands-on primer for genetic pollen analysis by nanopore sequencing. As our lab mainly works with pollen collected within agroecological research projects, we focus on pollen collected by pollinating insects. We briefly consider sample collection, storage and processing in the laboratory as well as bioinformatic aspects. Currently, pollen metabarcoding is mostly conducted with next-generation sequencing methods that generate short sequence reads (&lt;1 kb). Increasingly, however, pollen DNA analysis is carried out using the long-read generating (several kb), low-budget and mobile MinION nanopore sequencing platform by Oxford Nanopore Technologies. Therefore, we are focusing on aspects for palynology with the MinION DNA sequencing device

Nutrilyzer: A Tool for Deciphering Atomic Stoichiometry of Differentially Expressed Paralogous Proteins

Organisms try to maintain homeostasis by balanced uptake of nutrients from their environment. From an atomic perspective this means that, for example, carbon:nitrogen:sulfur ratios are kept within given limits. Upon limitation of, for example, sulfur, its acquisition is triggered. For yeast it was shown that transporters and enzymes involved in sulfur up- take are encoded as paralogous genes that express different isoforms. Sulfur deprivation leads to up-regulation of isoforms that are poor in sulfur-containing amino acids, that is, methinone and cysteine. Accordingly, sulfur-rich isoforms are down-regulated

Experimental and bioinformatic approaches for analyzing and visualizing cyanobacterial nitrogen and hydrogen metabolism

Many cyanobacteria are capable of utilizing light energy for nitrogen fixation. As a by-product of this nitrogenase mediated catalysis, hydrogen gas is produced. Several approaches to increase hydrogen production from cyanobacteria exist. Usually, these approaches are non-targeted. Here we exemplify how DNA-microarray based gene-expression analysis and bioinformatic visualization techniques can be used to analyze nitrogen and hydrogen metabolism from the filamentous, heterocyst forming cyanobacterium Nostoc PCC 7120. We analyzed the expression of 1249 genes from major metabolic categories under nitrogen fixing and non-nitrogen fixing growth. Of the selected genes, 494 show a more than 2-fold expression difference in the two conditions analyzed. Under nitrogen-fixing conditions 465 genes, mainly involved in energy metabolism, photosynthesis, respiration and nitrogen-fixation, were found to be stronger expressed, whereas only 29 genes showed a stronger expression under non-nitrogen fixing conditions. To help understanding probe hybridization, all expression data were correlated with potential target secondary structures and probe GC-content. For the first time the expression of high light-induced stress proteins (HLIP-family) is shown to be linked to the nitrogen availability

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included

Development of a genetic biomonitoring test for the investigation of pollinator-plant-interactions

There is a world-wide decline in biodiversity recorded. Especially insects and accompanying pollinators are threatened. When the foraging behaviour of pollinators is understood in detail, future crop and floral pollination services can be sustained and it is possible to establish projects for the conservation of pollinators and plant biodiversity. With the use of nanopore sequencing methods it is possible to detect pollen species that were collected by pollinators by their genetic information. In this study, a protocol for portable nanopore sequencing of DNA from pollen that was collected by honey bees, bumble bees and wild bees is being designed. DNAmetabarcoding is used to identify species within the mixed DNA sample. The ITS2-region will be used as a barcode. We will investigate pollen preferences of three pollinator species by placing their hives or nests at the same. Based on the results, landscape management schemes are developed that target pollen preferences and nutritional requirements of managed and wild social bee species as well as solitary wild bees.Ein weltweiter Rückgang der Artenvielfalt ist zu verzeichnen. Besonders Insekten und damit einhergehend Bestäuber, sind bedroht. Wenn das Nahrungssuchverhalten von Bestäubern im Detail verstanden wird, können zukünftig die Bestäubungsleistungen von Feldfrüchten und Blumen aufrechterhalten werden. Außerdem können Projekte zum Erhalt von Bestäubern und der Pflanzenbiodiversität etabliert werden. Mit dem Einsatz der Nanoporen-Sequenzierung ist es möglich, Pollenarten, die von Bestäubern gesammelt wurden, anhand ihrer genetischen Information zu erkennen. In dieser Studie wird ein Protokoll zur portablen Nanoporen-Sequenzierung von DNA aus Pollen, der von Honigbienen, Hummeln und Wildbienen gesammelt wurde, entwickelt. Wir verwenden DNA-Metabarcoding, um Arten innerhalb der gemischten DNA-Probe zu identifizieren. Dazu wird die ITS2-Region als Barcode verwendet. Mit einer Biodiversitätsanalyse wird untersucht, wie die drei Bestäuberarten die umliegenden Pollenressourcen nutzen, wenn sich ihre Stöcke bzw. Nester am selben Standort befinden. Basierend auf den Ergebnissen werden Landschaftsmanagementpläne entwickelt, die auf die Pollenpräferenzen und den Nährstoffbedarf von bewirtschafteten und wilden sozialen Bienenarten sowie solitären Wildbienen abzielen

Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols

AbstractThe activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin. The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I50=21 nM) and E. coli thioredoxin (I50=83 nM). For the homologous cytoplasmic thioredoxins I+II an I50 of 667 nM was determined. Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect. In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin