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  • 1. Brodiazhenko, Tetiana
    et al.
    Johansson, Marcus J. O.
    Takada, Hiraku
    Nissan, Tracy
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Sussex, United Kingdom.
    Hauryliuk, Vasili
    Murina, Victoriia
    Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 3041Article in journal (Refereed)
    Abstract [en]

    Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosomenascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stmt in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

  • 2.
    Broman, Elias
    et al.
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. Linnaeus University, Sweden.
    Asmala, Eero
    Carstensen, Jacob
    Pinhassi, Jarone
    Dopson, Mark
    Distinct Coastal Microbiome Populations Associated With Autochthonous- and Allochthonous-Like Dissolved Organic Matter2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 2579Article in journal (Refereed)
    Abstract [en]

    Coastal zones are important transitional areas between the land and sea, where both terrestrial and phytoplankton supplied dissolved organic matter (DOM) are respired or transformed. As climate change is expected to increase river discharge and water temperatures, DOM from both allochthonous and autochthonous sources is projected to increase. As these transformations are largely regulated by bacteria, we analyzed microbial community structure data in relation to a 6-month long time-series dataset of DOM characteristics from Roskilde Fjord and adjacent streams, Denmark. The results showed that the microbial community composition in the outer estuary (closer to the sea) was largely associated with salinity and nutrients, while the inner estuary formed two clusters linked to either nutrients plus allochthonous DOM or autochthonous DOM characteristics. In contrast, the microbial community composition in the streams was found to be mainly associated with allochthonous DOM characteristics. A general pattern across the land-to-sea interface was that Betaproteobacteria were strongly associated with humic-like DOM [operational taxonomic units (OTUs) belonging to family Comamonadaceae], while distinct populations were instead associated with nutrients or abiotic variables such as temperature (Cyanobacteria genus Synechococcus) and salinity (Actinobacteria family Microbacteriaceae). Furthermore, there was a stark shift in the relative abundance of OTUs between stream and marine stations. This indicates that as DOM travels through the land-to-sea interface, different bacterial guilds continuously degrade it.

  • 3. Conroy, Brandon J.
    et al.
    Steinberg, Deborah K.
    Song, Bongkuen
    Kalmbach, Andrew
    Carpenter, Edward J.
    Foster, Rachel A.
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. University of California, United States.
    Mesozooplankton Graze on Cyanobacteria in the Amazon River Plume and Western Tropical North Atlantic2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 1436Article in journal (Refereed)
    Abstract [en]

    Diazotrophic cyanobacteria, those capable of fixing di-nitrogen (N2), are considered one of the major sources of new nitrogen (N) in the oligotrophic tropical ocean, but direct incorporation of diazotrophic N into food webs has not been fully examined. In the Amazon River-influenced western tropical North Atlantic (WTNA), diatom diazotroph associations (DDAs) and the filamentous colonial diazotrophs Trichodesmium have seasonally high abundances. We sampled epipelagic mesozooplankton in the Amazon River plume and WTNA in May-June 2010 to investigate direct grazing by mesozooplankton on two DDA populations: Richelia associated with Rhizosolenia diatoms (het-1) and Hemiaulus diatoms (het-2), and on Trichodesmium using highly specific qPCR assays targeting nitrogenase genes (nifH). Both DDAs and Trichodesmium occurred in zooplankton gut contents, with higher detection of het-2 predominantly in calanoid copepods (2.33-16.76 nifH copies organism 1). Abundance of Trichodesmium was low (2.21-4.03 nifH copies organism 1), but they were consistently detected at high salinity stations (> 35) in calanoid copepods. This suggests direct grazing on DDAs, Trichodesmium filaments and colonies, or consumption as part of sinking aggregates, is common. In parallel with the qPCR approach, a next generation sequencing analysis of 16S rRNA genes identified that cyanobacterial assemblage associated with zooplankton guts was dominated by the non-diazotrophic unicellular phylotypes Synechococcus (56%) and Prochlorococcus (26%). However, in two separate calanoid copepod samples, two unicellular diazotrophs Candidatus Atelocyanobacterium thalassa (UCYN-A) and Crocosphaera watsonii (UCYN-B) were present, respectively, as a small component of cyanobacterial assemblages (< 2%). This study represents the first evidence of consumption of DDAs, Trichodesmium, and unicellular cyanobacteria by calanoid copepods in an area of the WTNA known for high carbon export. These diazotroph populations are quantitatively important in the global N budget, widespread and hence, the next step is to accurately quantify grazing. Nonetheless, these results highlight a direct pathway of diazotrophic N into the food web and have important implications for biogeochemical cycles, particularly oligotrophic regions where N-2 fixation is the main source of new nitrogen.

  • 4.
    Cooper, Callum J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mirzaei, Mohammadali Khan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Adapting Drug Approval Pathways for Bacteriophage-Based Therapeutics2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1209Article, review/survey (Refereed)
    Abstract [en]

    The global rise of multi-drug resistant bacteria has resulted in the notion that an antibiotic apocalypse is fast approaching. This has led to a number of well publicized calls for global funding initiatives to develop new antibacterial agents. The long clinical history of phage therapy in Eastern Europe, combined with more recent in vitro and in vivo success, demonstrates the potential for whole phage or phage based antibacterial agents. To date, no whole phage or phage derived products are approved for human therapeutic use in the EU or USA. There are at least three reasons for this: (i) phages possess different biological, physical, and pharmacological properties compared to conventional antibiotics. Phages need to replicate in order to achieve a viable antibacterial effect, resulting in complex pharmacodynamics/pharmacokinetics. (ii) The specificity of individual phages requires multiple phages to treat single species infections, often as part of complex cocktails. (iii) The current approval process for antibacterial agents has evolved with the development of chemically based drugs at its core, and is not suitable for phages. Due to similarities with conventional antibiotics, phage derived products such as endolysins are suitable for approval under current processes as biological therapeutic proteins. These criteria render the approval of phages for clinical use theoretically possible but not economically viable. In this review, pitfalls of the current approval process will be discussed for whole phage and phage derived products, in addition to the utilization of alternative approval pathways including adaptive licensing and Right to try legislation.

  • 5.
    Couturier, Mohea
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lindås, Ann-Christin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 137Article in journal (Refereed)
    Abstract [en]

    DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N-6-methyladenine (m6A), N-4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

  • 6.
    Dahlin, Paul
    et al.
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. KTH Royal Institute of Technology, Sweden.
    Srivastava, Vaibhav
    Bulone, Vincent
    McKee, Lauren S.
    The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1802Article in journal (Refereed)
    Abstract [en]

    The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

  • 7. Henein, Alexandra E.
    et al.
    Hanlon, Geoffrey W.
    Cooper, Callum J.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Denyer, Stephen P.
    Maillard, Jean-Yves
    A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1198Article in journal (Refereed)
    Abstract [en]

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baurnannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 x 10(9) pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required.

  • 8. Hjelm, Linnea C.
    et al.
    Ninebrant, Johan
    Nygren, Per-Åke
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Seijsing, Johan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a Non-covalent Barnase-Barstar Interaction Bridge2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 558Article in journal (Refereed)
    Abstract [en]

    Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.

  • 9. Ivarsson, Magnus
    et al.
    Schnürer, Anna
    Bengtson, Stefan
    Neubeck, Anna
    Stockholm University, Faculty of Science, Department of Geological Sciences.
    Anaerobic Fungi: A Potential Source of Biological H-2 in the Oceanic Crust2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 674Article in journal (Refereed)
    Abstract [en]

    The recent recognition of fungi in the oceanic igneous crust challenges the understanding of this environment as being exclusively prokaryotic and forces reconsiderations of the ecology of the deep biosphere. Anoxic provinces in the igneous crust are abundant and increase with age and depth of the crust. The presence of anaerobic fungi in deep-sea sediments and on the seafloor introduces a type of organism with attributes of geobiological significance not previously accounted for. Anaerobic fungi are best known from the rumen of herbivores where they produce molecular hydrogen, which in turn stimulates the growth of methanogens. The symbiotic cooperation between anaerobic fungi and methanogens in the rumen enhance the metabolic rate and growth of both. Methanogens and other hydrogen-consuming anaerobic archaea are known from subseafloor basalt; however, the abiotic production of hydrogen is questioned to be sufficient to support such communities. Alternatively, biologically produced hydrogen could serve as a continuous source. Here, we propose anaerobic fungi as a source of bioavailable hydrogen in the oceanic crust, and a close interplay between anaerobic fungi and hydrogen-driven prokaryotes.

  • 10.
    Karyolaimos, Alexandros
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ampah-Korsah, Henry
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hillenaar, Tamara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Borras, Anna Mestre
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dolata, Katarzyna Magdalena
    Sievers, Susanne
    Riedel, Katharina
    Daniels, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1511Article in journal (Refereed)
    Abstract [en]

    Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, disulfide bond containing recombinant proteins are often targeted to the periplasm by an N -terminal signal peptide that is removed once it passes through the Sectranslocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.

  • 11.
    Khan Mirzaei, Mohammadali
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Haileselassie, Yeneneh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Navis, Marit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cooper, Callum
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Anders S.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 437Article in journal (Refereed)
    Abstract [en]

    Due to a global increase in the range and number of infections caused by multi resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum betalactamase-producing Escherichia colinumbers by 1-4 log(10) compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

  • 12.
    Klawonn, Isabell
    et al.
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
    Lavik, Gaute
    Böning, Philipp
    Marchant, Hannah K.
    Dekaezemacker, Julien
    Mohr, Wiebke
    Ploug, Helle
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
    Simple approach for the preparation of N-15-15(2)-enriched water for nitrogen fixation assessments: evaluation, application and recommendations2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 769Article in journal (Refereed)
    Abstract [en]

    Recent findings revealed that the commonly used N-15(2) tracer assay for the determination of dinitrogen (N-2) fixation can underestimate the activity of aquatic N-2-fixing organisms. Therefore, a modification to the method using pre-prepared N-15-15(2)-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of N-15-15(2)-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add N-15-15(2) gas to the water in amounts exceeding the standard N-2 solubility, followed by vigorous agitation (vortex mixing >= 5 min). Optionally, water can be degassed at low-pressure (>= 950 mbar) for 10 mm prior to the N-15-15(2) gas addition to indirectly enhance the N-15-15(2) concentration. This preparation of N-15-15(2)-enriched water can be done within 1 h using standard laboratory equipment. The final N-15-atom% excess was 5% after replacing 2-5% of the incubation volume with N-15-15(2)-enriched water. Notably, the addition of N-15-15(2)-enriched water can alter levels of trace elements in the incubation water due to the contact of N-15-15(2)-enriched water with glass, plastic and rubber ware. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L-1 in the final incubation volume, which may bias rate measurements in regions where N-2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with N-15-15(2). The N-15-15(2) was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the N-15-15(2) equilibration. This approach achieved a N-15-atom% excess of 6.6 +/- 1.7% when adding 2 mL N-15-15(2) per liter of incubation water. The herein presented methodological tests offer guidelines for the N-15(2) tracer assay and thus, are crucial to circumvent methodological draw-backs for future N-2 fixation assessments.

  • 13. Lemmens, Liesbeth
    et al.
    Tilleman, Laurentijn
    De Koning, Ezra
    Valegard, Karin
    Lindås, Ann-Christin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Van Nieuwerburgh, Filip
    Maes, Dominique
    Peeters, Eveline
    YtrA(Sa), a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 2084Article in journal (Refereed)
    Abstract [en]

    In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrA(Sa), with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrA(Sa) overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrA(Sa) was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.

  • 14. Martínez-Gómez, Estrella
    et al.
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Gil-Ramírez, Yolanda
    Zúñiga-Ripa, Amaia
    Zaccheus, Mona
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Moriyón, Ignacio
    Iriarte, Maite
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Conde-Álvarez, Raquel
    Genomic Insertion of a Heterologous Acetyltransferase Generates a New Lipopolysaccharide Antigenic Structure in Brucella abortus and Brucella melitensis2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 1092Article in journal (Refereed)
    Abstract [en]

    Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a per R mutant. In mice, the wbdR constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against Brucella.

  • 15. Poole, Anthony M.
    et al.
    Lundin, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Linnaeus University, Sweden.
    Rytkoenen, Kalle T.
    The evolution of early cellular systems viewed through the lens of biological interactions2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 1144Article in journal (Refereed)
    Abstract [en]

    The minimal cell concept represents a pragmatic approach to the question of how few genes are required to run a cell. This is a helpful way to build a parts-list, and has been more successful than attempts to deduce a minimal gene set for life by inferring the gene repertoire of the last universal common ancestor, as few genes trace back to this hypothetical ancestral state. However, the study of minimal cellular systems is the study of biological outliers where, by practical necessity, coevolutionary interactions are minimized or ignored. In this paper, we consider the biological context from which minimal genomes have been removed. For instance, some of the most reduced genomes are from endosymbionts and are the result of coevolutionary interactions with a host; few such organisms are free-living. As few, if any, biological systems exist in complete isolation, we expect that, as with modern life, early biological systems were part of an ecosystem, replete with organismal interactions. We favor refocusing discussions of the evolution of cellular systems on processes rather than gene counts. We therefore draw a distinction between a pragmatic minimal cell (an interesting engineering problem), a distributed genome (a system resulting from an evolutionary transition involving more than one cell) and the looser coevolutionary interactions that are ubiquitous in ecosystems. Finally, we consider the distributed genome and coevolutionary interactions between genomic entities in the context of early evolution.

  • 16.
    Saroj, Sunil D.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Maudsdotter, Lisa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Tavares, Raquel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1176Article in journal (Refereed)
    Abstract [en]

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics.

  • 17.
    Sigurlásdóttir, Sara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wassing, Gabriela M.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Zuo, Fanglei
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Arts, Melanie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 422Article in journal (Refereed)
    Abstract [en]

    Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (idhA) mutant had an increased level of biofilm formation. Deletion of IdhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-delicient mutant, suggesting a critical role for eDNA. Compared to wild-type, the IdhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the IdhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of IdhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

  • 18. Solanki, Manoj Kumar
    et al.
    Abdelfattah, Ahmed
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences. Università Mediterranea di Reggio Calabria, Italy.
    Britzi, Malka
    Zakin, Varda
    Wisniewski, Michael
    Droby, Samir
    Sionov, Edward
    Shifts in the Composition of the Microbiota of Stored Wheat Grains in Response to Fumigation2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1098Article in journal (Refereed)
    Abstract [en]

    While the wheat-associated microbiome is of major agricultural importance, little is known about the alterations in wheat grain microbial community composition during storage. Characterization of the bacterial and fungal communities in stored wheat grains revealed the impact of phosphine fumigation, one of the most effective methods to eliminate insects in stored commodities, on the composition of the wheat grain microbiome. High-throughput amplicon sequencing of the bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region was used to analyze the wheat grain microbiome at different times over as 6 months period of storage. Higher bacterial diversity was found across the samples during the first (immediately after harvest) and second (3 months later) time points, with a predominance of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Planctomycetes. A two-fold decrease in the number of bacterial operational taxonomic units (OTUs) was observed in wheat grains at the last time point (6 months later), following phosphine treatment. In contrast to the effect of phosphine on bacteria, it did not affect fungal diversity in stored grains. The majority of fungal sequences were assigned to Ascomycota, followed by Basidiomycota, Glomeromycota, and unidentified fungi, which were evenly distributed throughout the storage period. Alpha and beta diversity analyses were confirmed by examination of the cultured microbial taxa obtained from the stored wheat grains. Mycotoxin analysis of wheat grains collected after phosphine fumigation revealed the presence of Fusarium toxins, primarily deoxynivalenol (DON). Several mycotoxigenic Fusarium spp. were also detected in the same samples. Results of the present study indicate that microbiome of stored, whole wheat grains was strongly affected by phosphine fumigation, which changed the structure of the microbial community leading to shifts in species composition toward mycotoxigenic strains. A better understanding of the complex interactions within the microbial communities of stored grains will assist in the development of novel biocontrol strategies to overcome mycotoxin contamination.

  • 19.
    Stenegren, Marcus
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Berg, Carlo
    Padilla, Cory C.
    David, Stefan-Sebastian
    Montoya, Joseph P.
    Yager, Patricia L.
    Foster, Rachel A.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK). Max Planck Institute for Marine Microbiology, Germany; University of California, USA.
    Piecewise structural equation model (SEM) disentangles the environmental conditions favoring diatom diazotroph associations (DDAs) in the western tropical North Atlantic (WTNA)2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 810Article in journal (Refereed)
    Abstract [en]

    Diatom diazotroph associations (DDAs) are important components in the world's oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont's nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (> 10(6) cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30-35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (> 0.1 mu mol L-1) and Si (> 1.0 mu mol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 x 10(5) nifH copies L-1) than het-2 and limited to regions of oceanic SSS (> 36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (> 10(3) nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8-34.3), immeasurable DIN, moderate DIP (0.1-0.60 mu mol L-1) and higher Si (4.19-22.1 mu mol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations.

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