Spatiotemporal succession of phosphorous accumulating biofilms during the first year of establishment.

Many wastewater treatment plants are dependent on the utilization of microorganisms in biofilms. Our knowledge about the establishment of these biofilms is limited, particular with respect to biofilms involved in enhanced biological phosphorus removal (EBPR). These biofilms rely on polyphosphate-accumulating organisms (PAOs), requiring alternating oxic and anaerobic conditions for phosphorous uptake. This challenge has been solved using the Hias process, which combines moving-bed biofilm-reactor (MBBR) technology with physical transfer of biofilm-carriers from oxic to anaerobic zones. We combined biofilm fractionation with temporal analyses to unveil the establishment in the Hias process. A stable phosphorous removal efficiency of >95% was reached within 16 weeks of operation. Phosphorus removal, however, was not correlated with the establishment of known PAOs. The biofilms seemed associated with an outer microbiota layer with rapid turnover and an inner layer with a slow expansion. The inner layer showed an overrepresentation of known PAOs. In conclusion, our spatiotemporal analyses of phosphorous accumulating biofilm establishment lead to a new model for biofilm growth, while the mechanisms for phosphorous removal remain largely unresolved.

Inkoo and Sindbis viruses in blood sucking insects, and a serological study for Inkoo virus in semi-domesticated Eurasian tundra reindeer in Norway.

BACKGROUND: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. MATERIALS AND METHODS: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013-2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). RESULTS: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). CONCLUSION: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norway indicate that further studies are required for monitoring this virus. SINV was not detected in the BSI in this study, however, human cases of SINV infection are yearly reported from other regions such as Rjukan in south-central Norway. It is therefore essential to monitor both viruses in the human population. Our findings are important to raise awareness regarding the geographical distribution of these mosquito-borne viruses in Northern Europe.

Spatial fractionation of phosphorus accumulating biofilm: stratification of polyphosphate accumulation and dissimilatory nitrogen metabolism.

The spatial distribution of microorganisms represents a critical issue in understanding biofilm function. The aim of the current work was to develop a method for biofilm fractionation, facilitating the analysis of individual spatial biofilm layers using metagenomic approaches. Phosphorus accumulating biofilm applied in an enhanced biological phosphorus removal wastewater treatment plant, were fractionated, and analyzed. The fractionated biofilm revealed a surprising spatial distribution of bacteria and genes, where potential polyphosphate accumulating organisms account for ∼ 47% of the inner layer microbiome. A spatial distribution of genes involved in dissimilatory nitrogen reduction was observed, indicating that different layers of the biofilm were metabolically active during the anoxic and aerobic zones of the phosphorus removal process. The physical biofilm separation into individual fractions unveiled functional layers of the biofilm, which will be important for future understanding of the phosphorus removal wastewater process.

The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens.

The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain active-site amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site.

Genetic analysis of DEFECTIVE KERNEL1 loop function in three-dimensional body patterning in Physcomitrella patens.

DEFECTIVE KERNEL1 (DEK1) of higher plants plays an essential role in position-dependent signaling and consists of a large transmembrane domain (MEM) linked to a protease catalytic domain and a regulatory domain. Here, we show that the postulated sensory Loop of the MEM domain plays an important role in the developmental regulation of DEK1 activity in the moss Physcomitrella patens. Compared with P. patens lacking DEK1 (∆dek1), the dek1∆loop mutant correctly positions the division plane in the bud apical cell. In contrast with an early developmental arrest of ∆dek1 buds, dek1∆loop develops aberrant gametophores lacking expanded phyllids resulting from misregulation of mitotic activity. In contrast with the highly conserved sequence of the protease catalytic domain, the Loop is highly variable in land plants. Functionally, the sequence from Marchantia polymorpha fully complements the dek1∆loop phenotype, whereas sequences from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) give phenotypes with retarded growth and affected phyllid development. Bioinformatic analysis identifies MEM as a member of the Major Facilitator Superfamily, membrane transporters reacting to stimuli from the external environment. Transcriptome analysis comparing wild-type and ∆dek1 tissues identifies an effect on two groups of transcripts connected to dek1 mutant phenotypes: transcripts related to cell wall remodeling and regulation of the AINTEGUMENTA, PLETHORA, and BABY BOOM2 (APB2) and APB3 transcription factors known to regulate bud initiation. Finally, sequence data support the hypothesis that the advanced charophyte algae that evolved into ancestral land plants lost cytosolic calpains, retaining DEK1 as the sole calpain in the evolving land plant lineage.

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²âº-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (∆dek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.

Short turnaround time of seven to nine hours from sample collection until informed decision for sepsis treatment using nanopore sequencing.

Bloodstream infections (BSIs) and sepsis are major health problems, annually claiming millions of lives. Traditional blood culture techniques, employed to identify sepsis-causing pathogens and assess antibiotic susceptibility, usually take 2-4 days. Early and accurate antibiotic prescription is vital in sepsis to mitigate mortality and antibiotic resistance. This study aimed to reduce the wait time for sepsis diagnosis by employing shorter blood culture incubation times for BD BACTEC™ bottles using standard laboratory incubators, followed by real-time nanopore sequencing and data analysis. The method was tested on nine blood samples spiked with clinical isolates from the six most prevalent sepsis-causing pathogens. The results showed that pathogen identification was possible at as low as 102-104 CFU/mL, achieved after just 2 h of incubation and within 40 min of nanopore sequencing. Moreover, all the antimicrobial resistance genes were identified at 103-107 CFU/mL, achieved after incubation for 5 h and only 10 min to 3 h of sequencing. Therefore, the total turnaround time from sample collection to the information required for an informed decision on the right antibiotic treatment was between 7 and 9 h. These results hold significant promise for better clinical management of sepsis compared with current culture-based methods.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

Membrane-anchored calpains - hidden regulators of growth and development beyond plants?

Calpains are modulatory proteases that modify diverse cellular substrates and play essential roles in eukaryots. The best studied are animal cytosolic calpains. Here, we focus on enigmatic membrane-anchored calpains, their structural and functional features as well as phylogenetic distribution. Based on domain composition, we identified four types of membrane-anchored calpains. Type 1 and 2 show broad phylogenetic distribution among unicellular protists and streptophytes suggesting their ancient evolutionary origin. Type 3 and 4 diversified early and are present in brown algae and oomycetes. The plant DEK1 protein is the only representative of membrane-anchored calpains that has been functionally studied. Here, we present up to date knowledge about its structural features, putative regulation, posttranslational modifications, and biological role. Finally, we discuss potential model organisms and available tools for functional studies of membrane-anchored calpains with yet unknown biological role. Mechanistic understanding of membrane-anchored calpains may provide important insights into fundamental principles of cell polarization, cell fate control, and morphogenesis beyond plants.

Massive expansion of the calpain gene family in unicellular eukaryotes.

BACKGROUND: Calpains are Ca2+-dependent cysteine proteases that participate in a range of crucial cellular processes. Dysfunction of these enzymes may cause, for instance, life-threatening diseases in humans, the loss of sex determination in nematodes and embryo lethality in plants. Although the calpain family is well characterized in animal and plant model organisms, there is a great lack of knowledge about these genes in unicellular eukaryote species (i.e. protists). Here, we study the distribution and evolution of calpain genes in a wide range of eukaryote genomes from major branches in the tree of life. RESULTS: Our investigations reveal 24 types of protein domains that are combined with the calpain-specific catalytic domain CysPc. In total we identify 41 different calpain domain architectures, 28 of these domain combinations have not been previously described. Based on our phylogenetic inferences, we propose that at least four calpain variants were established in the early evolution of eukaryotes, most likely before the radiation of all the major supergroups of eukaryotes. Many domains associated with eukaryotic calpain genes can be found among eubacteria or archaebacteria but never in combination with the CysPc domain. CONCLUSIONS: The analyses presented here show that ancient modules present in prokaryotes, and a few de novo eukaryote domains, have been assembled into many novel domain combinations along the evolutionary history of eukaryotes. Some of the new calpain genes show a narrow distribution in a few branches in the tree of life, likely representing lineage-specific innovations. Hence, the functionally important classical calpain genes found among humans and vertebrates make up only a tiny fraction of the calpain family. In fact, a massive expansion of the calpain family occurred by domain shuffling among unicellular eukaryotes and contributed to a wealth of functionally different genes.

Assessing Evolutionary Significant Units (ESU) of the Endangered Freshwater Pearl Mussel (Margaritifera margaritifera) in Southeast Norway on the Basis of Genetic Analysis.

A total of 312 specimens of freshwater pearl mussel (Margaritifera margaritifera) were sampled from 11 populations, located in four different river systems in Southeast Norway, and analyzed for 11 simple sequence repeat (SSR) (microsatellite) markers. All study populations have landlocked brown trout (Salmo trutta) as the only possible host. Several populations had experienced recruitment failure, probably due to low pH (about 6.0) and calcium concentration. STRUCTURE clustering analysis revealed two genetic clusters, of which one cluster occurred mainly in the western river systems, and totally dominated in one population (Fallselva (A-FAL)) that had higher genetic diversity than the others. Cluster 2 completely dominated in the populations of the eastern river systems, and all of them had low genetic diversity. Bottleneck events were indicated in all populations and the inbreeding coefficient FIS was significant in all populations, except for the southernmost population (Sørkedalselva (B-SØR)), which was the only population with genotypes in Hardy-Weinberg equilibrium. FIS were especially high in the populations of the eastern river systems, and maximum shell length was negatively correlated to FIS. If artificially breeding and stocking should become necessary for future preservation, it should be based on single populations; alternatively, the eastern populations should be based on cross-breeding of populations within the cluster to increase their genetic diversity.

Complement factor C5 in Atlantic salmon (Salmo salar): Characterization of cDNA, protein and glycosylation.

Complement component 5 (C5) is an essential factor of the defensive complement system in all vertebrates. We report the characterization of C5 cDNA and protein from Atlantic salmon (Salmo salar), a teleost fish species of high importance in aquaculture. The C5 cDNA cloned from liver is 5079 nucleotides long, whose translation product has a molecular weight of 190 kDa, with the classical ß-α orientation and motifs/sites for ß-α cleavage (678RPKR681) and cleavage by C5 convertases (R758). Mass spectrometric analysis show a single N-linked, biantennary, complex glycan at N1125. Moreover, the N-linked glycan displays an unusual modification in the form of acetylated sialic acid residues. Three anti-C5 antisera produced in mice using purified C5 worked in immunohistochemical analyses of formalin fixed liver tissue. The purification method, whereby inactive and activated (C5b) forms were isolated, opens for interesting studies on the complement function in fish, including possible connection to stress, disease and glycosylation.

An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing.

Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5' end of the intron and preserve the nucleotide sequence of the targeted intron.

DEK1; missing piece in puzzle of plant development.

Patterning of land plant bodies is determined by positioning of cell walls. A crucial event in land plant evolution was the ability to utilize spatial information to direct cell wall deposition. Recent studies of DEK1 in Physcomitrella patens support a role for DEK1 in position dependent cell wall orientation.

Viral suppressor proteins show varying abilities and effectiveness to suppress transgene-induced post-transcriptional gene silencing of endogenous Chalcone synthase in transgenic Arabidopsis.

Many, if not most, plant viruses encode proteins that interfere with RNA silencing pathways in plants. These proteins, known as viral suppressor proteins interfere at different key steps of the silencing pathways, and are able to suppress, to varying degree, transgene-induced silencing in plants. In this study, we report the ability and effectiveness of four different viral suppressor proteins that interfere with post-transcriptional gene silencing (PTGS) of the endogenous chalcone synthase gene (CHS) in Arabidopsis when the silencing trigger and the viral suppressor protein were expressed from the same transgene locus. The silencing trigger consisted of an inverted-repeat transgene construct that induces PTGS of the endogenous Arabidopsis CHS gene with high efficiency. Real-time PCR analyses were used to monitor the transcript levels of both the viral mRNAs and the target CHS mRNAs in transgenic Arabidopsis. Our results show that only one of the viral suppressor proteins tested, the p38 protein of Turnip Crinkle Virus, was able to efficiently, albeit to varying degrees, interfere with PTGS of CHS in individual transgenic Arabidopsis plants. Moreover, we show that the degree of CHS silencing suppression was dependent on the transcript level of p38. In contrast to earlier reports, we also found that constitutive expression of p38 in transgenic Arabidopsis is correlated with morphological defects in leaves and flowers.