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  • 1.
    Ampomah, Osei
    et al.
    University of Tromso.
    Ofori-Ayeh, Emmanuel
    University of Tromso.
    Solheim, Bjorn
    University of Tromso.
    Svenning, Mette
    University of Tromso.
    Host range, symbiotic effectiveness and nodulation competitiveness of some indigenous cowpea bradyrhizobia isolates from the transitional savannah zone of Ghana2008In: African Journal of Biotechnology, ISSN 1684-5315, Vol. 7, no 8, 988-996 p.Article in journal (Refereed)
  • 2.
    Ampomah, Osei Y.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avetisyan, A.
    Hansen, E.
    Svenson, J.
    Huser, T.
    Jensen, J. B.
    Bhuvaneswari, T. V.
    The thuEFGKAB operon of Rhizobia and Agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives2013In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, no 17, 3797-3807 p.Article in journal (Refereed)
    Abstract [en]

    The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide- forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism.

  • 3.
    Ampomah, Osei Y.
    et al.
    Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    James, E. K.
    Iannetta, P. P. M.
    Kenicer, G.
    Sprent, J. I.
    Huss-Danell, K.
    Nodulation and ecological significance of indigenous legumes in Scotland and Sweden2012In: Symbiosis, ISSN 0334-5114, Vol. 57, no 3, 133-148 p.Article in journal (Refereed)
    Abstract [en]

    The ability of wild indigenous legumes to form root nodules capable of biological nitrogen (N 2) fixation has rarely been demonstrated for species in natural ecosystems in large parts of Europe. In order to understand and manage these ecosystems, it is important to demonstrate nodulation across a diverse range of environments, sites and climates. This study surveyed nodulation at a number of sites in Scotland and Sweden. Presence of nodules was noted and nodule structure and indicators of nitrogen fixation capacity were assessed using light and transmission electron microscopy. Soils from several sites were also sampled for carbon and nitrogen analysis. The collections comprised 24 species in Scotland, and 30 taxa in Sweden; 17 of these in common for both countries. Highest species numbers occurred in meadows, farmland margins, hedgerows, roadsides and wasteland. Coastal sites and sites in the mountainous region above the Arctic Circle hosted several rare species. All sampled species had features of N 2-fixing nodules such as pink colour (leghaemoglobin) when dissected and bacteroids. Nodule structure for a number of species is here reported for the first time and presence of the N 2-fixing enzyme nitrogenase is demonstrated in three previously not studied Swedish legume species. North European legumes may make significant contributions to the N-budgets of their ecosystems. Such species (and their symbionts) represent unique germplasm that may be adopted to empower advances in agriculture and conservation aimed at mitigation and adaptation to the effects of climate change.

  • 4. Ampomah, Osei Yaw
    et al.
    Huss-Danell, Kerstin
    Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden2016In: Systematic and Applied Microbiology, ISSN 0723-2020, E-ISSN 1618-0984, Vol. 39, no 3, 203-210 p.Article in journal (Refereed)
    Abstract [en]

    Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromso, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny. (C) 2016 Elsevier GmbH. All rights reserved.

  • 5.
    Ampomah, Osei Yaw
    et al.
    Swedish University of Agricultural Sciences.
    Huss-Danell, Kerstin
    Swedish University of Agricultural Sciences.
    Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden2011In: Systematic and applied microbiology, ISSN 1618-0984, Vol. 34, no 4, 267-75 p.Article in journal (Refereed)
    Abstract [en]

    Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.

  • 6.
    Ampomah, Osei Yaw
    et al.
    Swedish University of Agricultural Sciences.
    Huss-Danell, Kerstin
    Swedish University of Agricultural Sciences.
    Nodulation of Thermopsis lupinoides by a Mesorhizobium huakuii strain with a unique nodA gene in Kamtchatka, Russia2011In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 77, no 15, 5513-5516 p.Article in journal (Refereed)
    Abstract [en]

    Very little is known about rhizobia that form nodules on Thermopsis spp. We report the isolation of a Mesorhizobium huakuii strain with a unique nodA gene that form nodules on Thermopsis lupinoides in Kamtchatka, Russia. The isolate did not form nodules on Thermopsis chinensis or Thermopsis caroliniana, which suggests it may be host specific.

  • 7. Ampomah, Osei Yaw
    et al.
    Mousavi, Seyed Abdollah
    Lindstrom, Kristina
    Huss-Danell, Kerstin
    Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia2017In: Systematic and Applied Microbiology, ISSN 0723-2020, E-ISSN 1618-0984, Vol. 40, no 1, 51-58 p.Article in journal (Refereed)
    Abstract [en]

    Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. N-15 analysis suggested that the legumes in association with these isolates were actively fixing nitrogen. (C) 2016 Elsevier GmbH. All rights reserved.

  • 8.
    Andersson, Sofia
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Characterization of Bacterial Biofilms for Wastewater Treatment2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Research performed at the Division of Environmental Microbiology has over the last years resulted in the isolation of possible bacterial key-organisms with efficient nutrient removal properties (Comamonas denitrificans, Brachymonas denitrificans, Aeromonas hydrophila). Effective use of these organisms for enhanced nutrient removal in wastewater treatment applications requires the strains to be retained, to proliferate and to maintain biological activity within theprocess. This can be achieved by immobilization of the organisms using an appropriate system.Two putative immobilization systems, agar entrapment and biofilm formation, wereassessed. Surface attached biofilm growth provided better results with respect to cell retention,proliferation and microbial activity than immobilization in agar beads. Thus, biofilm physiology was further characterized using simplified systems of single, dual or multi strain bacterial consortia containing the key-organisms as well as other wastewater treatment isolates. Mechanisms for initial adherence, biofilm formation over time, dynamics and characteristics of extracellular polymeric substances (EPS) and exopolysaccharides, nutrient removal activity as well as the effect of bacterial interactions were investigated. The results showed that all theassessed bacterial strains could form single strain biofilm providing that a suitable nutrientsupply was given. Production of EPS was found to be critical for biofilm development and both EPS and polysaccharide residue composition varied with bacterial strain, culture conditions and biofilm age. Denitrification and phosphorus removal activity of the keyorganisms was maintained in biofilm growth. Co-culturing of two or more strains resulted in both synergistic and antagonistic effects on biofilm formation as well as the microbial activitywithin the biofilm. Bacterial interactions also induced the synthesis of new polysaccharideswhich were not produced in pure strain biofilms.The complexity of single and mixed strain biofilm development and the implications of interactions on biofilm performance were underlined in this study. The data presented can be useful for modeling of biofilm systems, serve as a tool for selection of bacterial strain combinations to use for bioaugmentation/bioremediation or provide a base for further experiment design.

  • 9.
    Andersson, Sofia
    et al.
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Dalhammar, Gunnel
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Kuttuva Rajarao, Gunaratna
    KTH, School of Biotechnology (BIO), Environmental Microbiology.
    Influence of microbial interactions and EPS/polysaccharide composition on nutrient removal activity in biofilms formed by strains found in wastewater treatment systems2011In: Microbiology Research, ISSN 0944-5013, E-ISSN 1618-0623, Vol. 166, no 6, 449-457 p.Article in journal (Refereed)
    Abstract [en]

    The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.

  • 10.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hallström, Björn
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Hudson, Elton Paul
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp Strain PCC 68032013In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 23, 7419-7427 p.Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are emerging as promising hosts for production of advanced biofuels such as n-butanol and alkanes. However, cyanobacteria suffer from the same product inhibition problems as those that plague other microbial biofuel hosts. High concentrations of butanol severely reduce growth, and even small amounts can negatively affect metabolic processes. An understanding of how cyanobacteria are affected by their biofuel product can enable identification of engineering strategies for improving their tolerance. Here we used transcriptome sequencing (RNA-Seq) to assess the transcriptome response of Synechocystis sp. strain PCC 6803 to two concentrations of exogenous n-butanol. Approximately 80 transcripts were differentially expressed at 40 mg/liter butanol, and 280 transcripts were different at 1 g/liter butanol. Our results suggest a compromised cell membrane, impaired photosynthetic electron transport, and reduced biosynthesis. Accumulation of intracellular reactive oxygen species (ROS) scaled with butanol concentration. Using the physiology and transcriptomics data, we selected several genes for overexpression in an attempt to improve butanol tolerance. We found that overexpression of several proteins, notably, the small heat shock protein HspA, improved tolerance to butanol. Transcriptomics-guided engineering created more solvent-tolerant cyanobacteria strains that could be the foundation for a more productive biofuel host.

  • 11.
    Anfelt, Josefine
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Kaczmarzyk, Danuta
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Shabestary, Kiyan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Renberg, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Nielsen, Jens
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. Tech Univ Denmark.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, 167Article in journal (Refereed)
    Abstract [en]

    Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

  • 12.
    Arnling Bååth, Jenny
    et al.
    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    Giummarella, Nicola
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Klaubauf, Sylvia
    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    Lawoko, Martin
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Olsson, Lisbeth
    Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
    A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds2016In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 590, no 16, 2611-2618 p.Article in journal (Refereed)
    Abstract [en]

    The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.

  • 13.
    Asem, Heba
    et al.
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Functional Materials, FNM. Karolinska Institutet (KI), Sweden.
    Zhao, Ying
    Ye, Fei
    Barrefelt, Asa
    Abedi-Valugerdi, Manuchehr
    El-Sayed, Ramy
    El-Serafi, Ibrahim
    Abu-Salah, Khalid M.
    Hamm, Jorg
    Muhammed, Mamoun
    KTH, School of Information and Communication Technology (ICT), Materials- and Nano Physics, Functional Materials, FNM.
    Hassan, Moustapha
    Biodistribution of biodegradable polymeric nano-carriers loaded with busulphan and designed for multimodal imaging2016In: Journal of Nanobiotechnology, ISSN 1477-3155, Vol. 14, no 1, 82Article in journal (Refereed)
    Abstract [en]

    Background: Multifunctional nanocarriers for controlled drug delivery, imaging of disease development and follow-up of treatment efficacy are promising novel tools for disease diagnosis and treatment. In the current investigation, we present a multifunctional theranostic nanocarrier system for anticancer drug delivery and molecular imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) as an MRI contrast agent and busulphan as a model for lipophilic antineoplastic drugs were encapsulated into poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL) micelles via the emulsion-evaporation method, and PEG-PCL was labelled with VivoTag 680XL fluorochrome for in vivo fluorescence imaging. Results: Busulphan entrapment efficiency was 83% while the drug release showed a sustained pattern over 10 h. SPION loaded-PEG-PCL micelles showed contrast enhancement in T-2*-weighted MRI with high r(2)* relaxivity. In vitro cellular uptake of PEG-PCL micelles labeled with fluorescein in J774A cells was found to be time-dependent. The maximum uptake was observed after 24 h of incubation. The biodistribution of PEG-PCL micelles functionalized with VivoTag 680XL was investigated in Balb/c mice over 48 h using in vivo fluorescence imaging. The results of real-time live imaging were then confirmed by ex vivo organ imaging and histological examination. Generally, PEG-PCL micelles were highly distributed into the lungs during the first 4 h post intravenous administration, then redistributed and accumulated in liver and spleen until 48 h post administration. No pathological impairment was found in the major organs studied. Conclusions: Thus, with loaded contrast agent and conjugated fluorochrome, PEG-PCL micelles as biodegradable and biocompatible nanocarriers are efficient multimodal imaging agents, offering high drug loading capacity, and sustained drug release. These might offer high treatment efficacy and real-time tracking of the drug delivery system in vivo, which is crucial for designing of an efficient drug delivery system.

  • 14.
    Asplund Samuelsson, Johannes
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Sundh, J.
    Dupont, C. L.
    Allen, A. E.
    McCrow, J. P.
    Celepli, N. A.
    Bergman, B.
    Ininbergs, K.
    Ekman, M.
    Diversity and expression of bacterial metacaspases in an aquatic ecosystem2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, no JUL, 1043Article in journal (Refereed)
    Abstract [en]

    Metacaspases are distant homologs of metazoan caspase proteases, implicated in stress response, and programmed cell death (PCD) in bacteria and phytoplankton. While the few previous studies on metacaspases have relied on cultured organisms and sequenced genomes, no studies have focused on metacaspases in a natural setting. We here present data from the first microbial community-wide metacaspase survey; performed by querying metagenomic and metatranscriptomic datasets from the brackish Baltic Sea, a water body characterized by pronounced environmental gradients and periods of massive cyanobacterial blooms. Metacaspase genes were restricted to ~4% of the bacteria, taxonomically affiliated mainly to Bacteroidetes, Alpha- and Betaproteobacteria and Cyanobacteria. The gene abundance was significantly higher in larger or particle-associated bacteria (<0.8 μm), and filamentous Cyanobacteria dominated metacaspase gene expression throughout the bloom season. Distinct seasonal expression patterns were detected for the three metacaspase genes in Nodularia spumigena, one of the main bloom-formers. Clustering of normalized gene expression in combination with analyses of genomic and assembly data suggest functional diversification of these genes, and possible roles of the metacaspase genes related to stress responses, i.e., sulfur metabolism in connection to oxidative stress, and nutrient stress induced cellular differentiation. Co-expression of genes encoding metacaspases and nodularin toxin synthesis enzymes was also observed in Nodularia spumigena. The study shows that metacaspases represent an adaptation of potentially high importance for several key organisms in the Baltic Sea, most prominently Cyanobacteria, and open up for further exploration of their physiological roles in microbes and assessment of their ecological impact in aquatic habitats.

  • 15. Avican, Kemal
    et al.
    Fahlgren, Anna
    Huss, Mikael
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Heroven, Ann Kathrin
    Beckstette, Michael
    Dersch, Petra
    Fallman, Maria
    Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 1Article in journal (Refereed)
    Abstract [en]

    We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.

  • 16.
    Axelsson, Karolin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Chemical signals in interactions between Hylobius abietis and associated bacteria2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The pine weevil (Hylobius abietis L.) is one of the two topmost economically important insect pests in Swedish conifer forests. The damage increase in areas were the silvicultural practice is to use clear cuttings were the insects gather and breed. During egglaying the female protects her offspring by creating a cave in roots and stumps were she puts her egg and covers it with frass, a mixture of weevil feces and chewed bark. Adult pine weevils have been observed to feed on the other side of the egg laying site and antifeedant substance has been discovered in the feces of the pine weevil. We think it is possible that microorganisms present in the frass contribute with antifeedant/repellent substances. Little is known about the pine weevils associated bacteria community and their symbiotic functions. In this thesis the bacterial community is characterized in gut and frass both from pine weevils in different populations across Europe as well as after a 28 day long diet regime on Scots pine, silver birch or bilberry. Volatile substances produced by isolated bacteria as well as from a consortium of microorganisms were collected with solid phase micro extraction (SPME) and analyzed with GC-MS. The main volatiles were tested against pine weevils using a two-choice test. Wolbachia, Rahnella aquatilis, Serratia and Pseudomonas syringae was commonly associated with the pine weevil. 2-Methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol were found in the headspace from Rahnella aquatilis when grown in substrate containing pine bark. 2-Methoxyphenol and 3-methyl-1-butanol, phenol and methyl salicylate were found in pine feces. Birch and bilberry feces emitted mainly linalool oxides and bilberry emitted also small amounts of 2-phenylethanol.

    A second part of the thesis discusses the role of fungi in forest insect interactions and the production of oxygenated monoterpenes as possible antifeedants. Spruce bark beetles (Ips typhographus L.) aggregate with the help of pheromones and with collected forces they kill weakened adult trees as a result of associated fungi growth and larval development. A fungi associated with the bark beetle, Grosmannia europhoides, was shown to produce de novo 2-methyl-3-buten-2-ol, the major component of the spruce bark beetle aggregation pheromone. Chemical defense responses against Endoconidiophora polonica and Heterobasidion parviporum were investigated using four clones of Norway spruce with different susceptibility to Heterobasidion sp. Clone specific differences were found in induced mono-, sesqui and diterpenes. A number of oxygenated monoterpenes which are known antifeedants for the pine weevil were produced in the infested areas.

  • 17.
    Axelsson, Karolin
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry. KTH, School of Biotechnology (BIO).
    Konstanzer, Vera
    KTH.
    Rajarao, Gunaratna Kuttuva
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Terenius, Olle
    Seriot, Lisa
    KTH, School of Biotechnology (BIO).
    Nordenhem, Henrik
    Nordlander, Goran
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry. Tartu University, Estonia.
    Antifeedants Produced by Bacteria Associated with the Gut of the Pine Weevil Hylobius abietis2017In: Microbial Ecology, ISSN 0095-3628, E-ISSN 1432-184X, Vol. 74, no 1, 177-184 p.Article in journal (Refereed)
    Abstract [en]

    The pine weevil, Hylobius abietis, is a severe forest pest insect as it feeds on newly planted conifer seedlings. To identify and develop an antifeedant could be one step towards the protection of seedlings from feeding damage by the pine weevil. With the aim to trace the origin of the antifeedants previously found in feces of the pine weevil, we investigated the culturable bacteria associated with the gut and identified the volatiles they produced. Bacterial isolates were identified by 16S ribosomal RNA gene analysis. The volatile emissions of selected bacteria, cultivated on NB media or on the grated phloem of Scots pine twigs dispersed in water, were collected and analyzed by solid-phase microextraction gas chromatography-mass spectrometry. The bacterial isolates released a variety of compounds, among others 2-methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, dimethyl disulfide, and dimethyl trisulfide. A strong antifeedant effect was observed by 2-phenylethanol, which could thus be a good candidate for use to protect planted conifer seedlings against feeding damage caused by H. abietis.

  • 18. Bagnoud, Alexandre
    et al.
    de Bruijn, Ino
    Andersson, Anders F.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Diomidis, Nikitas
    Leupin, Olivier X.
    Schwyn, Bernhard
    Bernier-Latmani, Rizlan
    A minimalistic microbial food web in an excavated deep subsurface clay rock2016In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 92, no 1, UNSP fiv138Article in journal (Refereed)
    Abstract [en]

    Clay rocks are being considered for radioactive waste disposal, but relatively little is known about the impact of microbes on the long-term safety of geological repositories. Thus, a more complete understanding of microbial community structure and function in these environments would provide further detail for the evaluation of the safety of geological disposal of radioactive waste in clay rocks. It would also provide a unique glimpse into a poorly studied deep subsurface microbial ecosystem. Previous studies concluded that microorganisms were present in pristine Opalinus Clay, but inactive. In this work, we describe the microbial community and assess the metabolic activities taking place within borehole water. Metagenomic sequencing and genome-binning of a porewater sample containing suspended clay particles revealed a remarkably simple heterotrophic microbial community, fueled by sedimentary organic carbon, mainly composed of two organisms: a Pseudomonas sp. fermenting bacterium growing on organic macromolecules and releasing organic acids and H-2, and a sulfate-reducing Peptococcaceae able to oxidize organic molecules to CO2. In Opalinus Clay, this microbial system likely thrives where pore space allows it. In a repository, this may occur where the clay rock has been locally damaged by excavation or in engineered backfills.

  • 19. Beck Jensen, John
    et al.
    Ampomah, Osei Yaw
    Darrah, Richard
    Kent Peters, N.
    Bhuvaneswari, T. V.
    Role of trehalose transport and utilization in Sinorhizobium meliloti-alfalfa interactions2005In: Molecular Plant-Microbe Interactions, ISSN 0894-0282, E-ISSN 1943-7706, Vol. 18, no 7Article in journal (Refereed)
    Abstract [en]

    Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rm1021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.

  • 20.
    Berasategui, Aileen
    et al.
    Dep. of Biochemistry, Max Planck institute for Chemical Ecology.
    Axelsson, Karolin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Nordlander, Göran
    Dep. of Ecology, SLU.
    Schmidt, Axel
    Max Planck Institute for Chemical Ecology.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Gershenzon, Jonathan
    Dep of Biochemistry, Max Planck institute for Chemical Ecology.
    Terenius, Olle
    Dep of Ecology, SLU.
    Kaltenpoth, Martin
    Insect Symbiosis Research Group, Max Planck institute for Chemical Ecology.
    The Gut microbiota of the pine weevil is similar across Europe and resembles that of other conifer-feeding beetles2016In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 25, no 16, 4014-4031 p.Article in journal (Refereed)
    Abstract [en]

    The pine weevil (Hylobius abietis, Coleoptera: Curculionidae) is an important pest of conifer seedlings in Europe. Despite its economic importance, little is known about the composition of its gut microbial community and the role it plays in mediating the weevil's ability to utilize conifers as a food source. Here, we characterized the gut bacterial communities of different populations of H. abietis across Europe and compared them to those of other beetles that occupy similar ecological niches. We demonstrate that the microbial community of H. abietis is similar at higher taxonomic levels (family and genus) across locations in Europe, with Wolbachia as the dominant microbe, followed by Enterobacteria and Firmicutes. Despite this similarity, we observed consistent differences between countries and locations, but not sexes. Our meta-analysis demonstrates that the gut bacterial community of the pine weevil is very similar to that of bark beetles that also exploit conifers as a food source. The Enterobacteriaceae symbionts of both host taxa are especially closely related phylogenetically. Conversely, the microbiota of H. abietis is distinct from that of closely related weevils feeding on non-conifer food sources, suggesting that the microbial community of the pine weevil is determined by the environment and may be relevant to host ecology. Furthermore, several H. abietis-associated members of the Enterobacteriaceae family are known to contain genes involved in terpenoid degradation. As such, we hypothesize that the gut microbial community is important for the utilization of conifer seedlings as a food source, either through the detoxification of plant secondary metabolites or supplementation of essential nutrients.

  • 21.
    Bi, Ran
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Lignocellulose Degradation by Soil Micro-organisms2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Lignocellulosic biomass is a sustainable resource with abundant reserves. Compared to petroleum ‐ based products, the biomass ‐ derived polymers and chemicals give better environmental profiles. A lot of research interest is focused on understanding the lignocellulose structures.

    Lignin, among the three major wood components, represents most difficulty for microbial degradation because of its complex structure and because cross ‐ linking to hemicellulose makes wood such a compact structure. Nevertheless, wood is naturally degraded by wood ‐ degrading micro ‐ organisms and modified and partly degraded residual of lignin goes into soil. Therefore soil serves as a good environment in which to search for special lignin ‐ degraders. In this thesis, different types of lignin have been used as sole carbon sources to screen for lignin ‐ degrading soil micro ‐ organisms. Eleven aerobic and three anaerobic microbe strains have been isolated and identified as able to grow on lignin. The lignin degradation patterns of selected strains have been studied and these partly include an endwise cleavage of  β‐ O ‐ 4 bonds in lignin and is more complex than simple hydrolytic degradation.

    As lignin exists in wood covalently bonded to hemicellulose, one isolated microbe strain, Phoma herbarum, has also been studied with regards to its ability to degrade covalent lignin polysaccharide networks (LCC). The results show that its culture filtrate can attack lignin ‐ polysaccharide networks in a manner different from that of the commercial enzyme product, Gammanase, possibly by selective cleavage of phenyl glucoside bonds. The effects on LCC of Phoma herbarum also enhance polymer extractability. Hot ‐ water extraction of a culture filtrate of Phoma herbarum ‐ treated fiberized spruce wood material gave an amount of extracted galactoglucomannan more than that given by the Gammanase ‐ treated material and non ‐ enzyme ‐ treated material.

    Over millions of years of natural evolution, micro ‐ organisms on the one hand develop so that they can degrade all wood components to get energy for growth, while plants on the other hand also continuously develop to defend from microbial attack. Compared with lignin and cellulose, hemicelluloses as major components of plant cell walls, are much more easily degraded, but hemicelluloses differ from cellulose in that they are acetylated to different extents. The biological functions of acetylation are not completely understood, but it is suggested is that one function is to decrease the microbial degradability of cell walls. By cultivation of soil micro ‐ organisms using mannans acetylated to deffernent degrees as sole carbon source on agar plates, we were able to see significant trends where the resistance towards microbial degradation of glucomannan and galactomannan increased with increasing degree of acetylation. Possible mechanisms and the technological significance of this are discussed. Tailoring the degree of acetylation of polysaccharide materials might slow down the biodegradation, making it possible to design a material with a degradation rate suited to its application.

  • 22.
    Bi, Ran
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Azhar, Shoaib
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Mckee, Lauren
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Culture Filtrates from a Soil Organism Enhances Extractability of Polymers from Fiberised Spruce WoodManuscript (preprint) (Other academic)
  • 23.
    Bi, Ran
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Berglund, Jennie
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    McKee, Lauren
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    Henriksson, Gunnar
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    The Degree Of Acetylation Affects The Microbial Degradability Of HemicellulosesManuscript (preprint) (Other academic)
  • 24. Bracher, J. M.
    et al.
    de Hulster, E.
    Koster, C. C.
    van den Broek, M.
    Daran, J. -MG.
    van Maris, Antonius J.A.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Delft University of Technology, Netherlands.
    Pronk, J. T.
    Laboratory evolution of a biotin-requiring Saccharomyces cerevisiae strain for full biotin prototrophy and identification of causal mutations2017In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 83, no 16, e00892-17Article in journal (Refereed)
    Abstract [en]

    Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ= 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.

  • 25.
    Dahlin, Paul
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience. Stockholm University, Sweden.
    Srivastava, Vaibhav
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Mckee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center.
    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, 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.

  • 26. Eriksson, Jens
    et al.
    Eriksson, Olaspers Sara
    Maudsdotter, Lisa
    Palm, Oskar
    KTH, School of Engineering Sciences (SCI), Theoretical Physics, Statistical Physics.
    Engman, Jakob
    Sarkissian, Tim
    Aro, Helena
    Wallin, Mats
    KTH, School of Engineering Sciences (SCI), Theoretical Physics, Statistical Physics.
    Jonsson, Ann-Beth
    Characterization of motility and piliation in pathogenic Neisseria2015In: BMC Microbiology, ISSN 1471-2180, Vol. 15, 92Article in journal (Refereed)
    Abstract [en]

    Background: The type IV pili (Tfp) of pathogenic Neisseria (i. e., N. gonorrhoeae and N. meningitidis) are essential for twitching motility. Tfp retraction, which is dependent on the ATPase PilT, generates the forces that move bacteria over surfaces. Neisseria motility has mainly been studied in N. gonorrhoeae whereas the motility of N. meningitidis has not yet been characterized. Results: In this work, we analyzed bacterial motility and monitored Tfp retraction using live- cell imaging of freely moving bacteria. We observed that N. meningitidis moved over surfaces at an approximate speed of 1.6 mu m/s, whereas N. gonorrhoeae moved with a lower speed (1.0 mu/s). An alignment of the meningococcal and gonococcal pilT promoters revealed a conserved single base pair variation in the -10 promoter element that influence PilT expression. By tracking mutants with altered pilT expression or pilE sequence, we concluded that the difference in motility speed was independent of both. Live-cell imaging using total internal reflection fluorescence microscopy demonstrated that N. gonorrhoeae more often moved with fewer visible retracting filaments when compared to N. meningitidis. Correspondingly, meningococci also displayed a higher level of piliation in transmission electron microscopy. Nevertheless, motile gonococci that had the same number of filaments as N. meningitidis still moved with a lower speed. Conclusions: These data reveal differences in both speed and piliation between the pathogenic Neisseria species during twitching motility, suggesting a difference in Tfp-dynamics.

  • 27. Farnelid, Hanna
    et al.
    Bentzon-Tilia, Mikkel
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bertilsson, Stefan
    Jost, Guenter
    Labrenz, Matthias
    Juergens, Klaus
    Riemann, Lasse
    Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea2013In: The ISME Journal, ISSN 1751-7362, Vol. 7, no 7, 1413-1423 p.Article in journal (Refereed)
    Abstract [en]

    The Baltic Sea receives large nitrogen inputs by diazotrophic (N-2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N-2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N-2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 mu m size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 x 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 x 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N-2 fixation (0.44 +/- 0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N-2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N-2 fixation could also be of importance in other suboxic regions of the world's oceans.

  • 28.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Functional characterization of cellulose and chitin synthase genes in Oomycetes2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.

     

    A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.

     

    Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.

  • 29.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Nano Biotechnology.
    Novel diagnostic microarray assay formats towards comprehensive on-site analysis2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

    In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

  • 30. Gaurivaud, P.
    et al.
    Persson, Anja
    KTH, Superseded Departments, Biotechnology.
    Le Grand, D.
    Westberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Solsona, M.
    Johansson, K. E.
    Poumarat, F.
    Variability of a glucose phosphotransferase system permease in Mycoplasma mycoides subsp mycoides Small Colony2004In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 150, 4009-4022 p.Article in journal (Refereed)
    Abstract [en]

    Intraclonal antigenic variation in pathogenic mycoplasma species is considered an important feature of host-pathogen interaction. Such intraclonal protein variation was observed for the interaction of Mycoplasma mycoides subsp. mycoides Small Colony, the agent of contagious bovine pleuropneumonia, with mAb 3F3. Colony immunostaining allows the definition of 3F3 ON- and 3F3 OFF-type variants, which revert at low frequency. Targets of mAb 3F3 were shown to be surface located, and resided on multiple polypeptides in the 58-68 kDa size range. Phage display and a genomic database were combined to determine the gene encoding the proteins recognized by mAb 3F3. A gene encoding the putative permease of the glucose phosphotransferase system was identified. Genome sequence analysis of strain PG1 revealed two highly similar copies of this gene, resulting from duplication of the chromosomal region carrying the gene. Southern blot analysis demonstrated the presence of this duplication in almost every African strain tested, but not in European strains. DNA analysis revealed that ON/OFF switching is governed by a base substitution occurring upstream of the coding region for the 3F3 epitope. This event generates a stop codon that results in the premature termination of the PtsG protein.

  • 31. Gioti, Anastasia
    et al.
    Nystedt, Björn
    Li, Wenjun
    Xu, Jun
    Andersson, Anna
    Averette, Anna F.
    Muench, Karin
    Wang, Xuying
    Kappauf, Catharine
    Kingsbury, Joanne M.
    Kraak, Bart
    Walker, Louise A.
    Johansson, Henrik J.
    Holm, Tina
    Lehtiö, Janne
    Stajich, Jason E.
    Mieczkowski, Piotr
    Kahmann, Regine
    Kennell, John C.
    Cardenas, Maria E.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Saunders, Charles W.
    Boekhout, Teun
    Dawson, Thomas L.
    Munro, Carol A.
    de Groot, Piet W. J.
    Butler, Geraldine
    Heitman, Joseph
    Scheynius, Annika
    Genomic Insights into the Atopic Eczema-Associated Skin Commensal Yeast Malassezia sympodialis2013In: mBio, ISSN 2150-7511, Vol. 4, no 1, e00572-12- p.Article in journal (Refereed)
    Abstract [en]

    Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e. g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. IMPORTANCE Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

  • 32.
    Guerriero, Gea
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Avino, Mariano
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Fugelstad, Johanna
    KTH, School of Biotechnology (BIO), Glycoscience.
    Clergeot, Pierre-Henri
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs2010In: PLOS PATHOG, ISSN 1553-7366, Vol. 6, no 8, e1001070- p.Article in journal (Refereed)
    Abstract [en]

    Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

  • 33.
    Guerzoni, Samuele
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Deplaine, Harmony
    El Haskouri, Jamal
    Amoros, Pedro
    Monleon Pradas, Manuel
    Edlund, Ulrica
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Gallego Ferrer, Gloria
    Combination of silica nanoparticles with hydroxyapatite reinforces poly (L-lactide acid) scaffolds without loss of bioactivity2014In: Journal of bioactive and compatible polymers (Print), ISSN 0883-9115, E-ISSN 1530-8030, Vol. 29, no 1, 15-31 p.Article in journal (Refereed)
    Abstract [en]

    Composite scaffolds of poly(l-lactide acid) and hydroxyapatite are of great interest in bone tissue engineering, but their mechanical properties are typically inferior to scaffolds of pure poly(l-lactide acid) due to agglomeration of the particles and weak interfacial component interaction. Fabrication strategies like double sonication of hydroxyapatite or increasing the amount of this inorganic filler do not effectively enhance the mechanical performance. In this study, poly(l-lactide acid) composites combining two types of fillers, mesoporous silica (SiO2) nanoparticles and hydroxyapatite, were developed to reinforce the poly(l-lactide acid) scaffold without any loss of bioactivity. A 5% addition of SiO2 nanoparticles to hydroxyapatite nanopowder and subjecting the scaffold formulation to double sonication increased the Young's modulus from 5 MPa (pure poly(l-lactide acid) scaffold) to almost 7 MPa (poly(l-lactide acid)/hydroxyapatite/SiO2 scaffold). In addition, the composite was able to deposit a layer of biomimetic hydroxyapatite both on the surface and interior of the scaffold after 21 days of immersion in a simulated body fluid. The manufacturing method was straightforward and economically viable and does not require any chemical modification of the particles' surfaces.

  • 34.
    Guevara-Martinez, Monica
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Gällnö, Karin Sjöberg
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Perez-Zabaleta, Mariel
    Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Regulating the production of (R)-3-hydroxybutyrate in Escherichia coil by N or P limitation2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, 844Article in journal (Refereed)
    Abstract [en]

    The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.

  • 35.
    Hammar, Petter
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Angermayr, S. Andreas
    Sjöström, Staffan L.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    van der Meer, Josefin
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hellingwerf, Klaas J.
    Hudson, Elton P.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Jönsson, Håkan N.
    Novo Nordisk Foundation Center for Biosustainability.
    Single-cell screening of photosynthetic growth and lactate production by cyanobacteria2015In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 8, 193Article in journal (Refereed)
    Abstract [en]

    Background: Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. Results: We present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for L-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive L-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells. Conclusions: The workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology.

  • 36.
    Hassan, Noor
    et al.
    KTH, School of Biotechnology (BIO).
    Nguyen, Thu-Ha
    Intanon, Montira
    Kori, Lokesh D.
    Patel, Bharat K. C.
    Haltrich, Dietmar
    Divne, Christina
    KTH, School of Biotechnology (BIO).
    Tan, Tien Chye
    KTH, School of Biotechnology (BIO).
    Biochemical and structural characterization of a thermostable beta-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis2015In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 99, no 4, 1731-1744 p.Article in journal (Refereed)
    Abstract [en]

    Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

  • 37. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Juergens, Klaus
    Phylogenetic Signals of Salinity and Season in Bacterial Community Composition Across the Salinity Gradient of the Baltic Sea2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, 1883Article in journal (Refereed)
    Abstract [en]

    Understanding the key processes that control bacterial community composition has enabled predictions of bacterial distribution and function within ecosystems. In this study, we used the Baltic Sea as a model system to quantify the phylogenetic signal of salinity and season with respect to bacterioplankton community composition. The abundances of 16S rRNA gene amplicon sequencing reads were analyzed from samples obtained from similar geographic locations in July and February along a brackish to marine salinity gradient in the Baltic Sea. While there was no distinct pattern of bacterial richness at different salinities, the number of bacterial phylotypes in winter was significantly higher than in summer. Bacterial community composition in brackish vs. marine conditions, and in July vs. February was significantly different. Non-metric multidimensional scaling showed that bacterial community composition was primarily separated according to salinity and secondly according to seasonal differences at all taxonomic ranks tested. Similarly, quantitative phylogenetic clustering implicated a phylogenetic signal for both salinity and seasonality. Our results suggest that global patterns of bacterial community composition with respect to salinity and season are the result of phylogenetically clustered ecological preferences with stronger imprints from salinity.

  • 38. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Jürgens, Klaus
    Zheng, Zongli
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria2013In: mBio, ISSN 2150-7511, Vol. 4, no 3, e00569-12- p.Article in journal (Refereed)
    Abstract [en]

    The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype's distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.

  • 39.
    Hu, Yue
    KTH, School of Biotechnology (BIO).
    Microbial DNA Sequencing in Environmental Studies2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The field of microbial ecology has just entered a new era of rapid technological development and generation of big data. The high-throughput sequencing techniques presently available provide an opportunity to extensively inventorize the blueprints of life. Now, millions of microbes of natural microbial communities can be studied simultaneously without prior cultivation. New species and new functions (genes) can be discovered just by mining sequencing data. However, there is still a tremendous number of microorganisms not yet examined, nor are the ecosystem functions these carry out. The modern genomic technologies can contribute to solve environmental problems and help us understand ecosystems, but to most efficiently do so, methods need to be continuously optimised.

     

    During my Ph. D. studies, I developed a method to survey eukaryotic microbial diversity with a higher accuracy, and applied various sequencing-based approaches in an attempt to answer questions of importance in environmental research and ecology. In PAPER-I, we developed a set of 18S rRNA gene PCR primers with high taxonomic coverage, meeting the requirements of currently popular sequencing technologies and matching the richness of 18S rRNA reference sequences accumulated so far. In PAPER-II, we conducted the first sequencing-based spatial survey on the combined eukaryotic and bacterial planktonic community in the Baltic Sea to uncover the relationship of microbial diversity and environmental conditions. Here, the 18S primers designed in PAPER-I and a pair of broad-coverage 16S primers were employed to target the rRNA genes of protists and bacterioplankton for amplicon sequencing. In PAPER-III, we integrated metagenomic, metabarcoding, and metatranscriptomic data in an effort to scrutinise the protein synthesis potential (i.e., activity) of microbes in the sediment at a depth of 460 m in the Baltic Sea and, thus, disclosing microbial diversity and their possible ecological functions within such an extreme environment. Lastly, in PAPER-IV, we compared the performance of E. coli culturing, high-throughput sequencing, and portable real-time sequencing in tracking wastewater contamination in an urban stormwater system. From the aspects of cost, mobility and accuracy, we evaluated the usage of sequencing-based approaches in civil engineering, and for the first time, validated the real-time sequencing device in use within water quality monitoring.

     

    In summary, these studies demonstrate how DNA sequencing of microbial communities can be applied in environmental monitoring and ecological research.

  • 40.
    Hu, Yue O. O.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Karlson, Bengt
    Charvet, Sophie
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Diversity of Pico- to Mesoplankton along the 2000 km Salinity Gradient of the Baltic Sea2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, 679Article in journal (Refereed)
    Abstract [en]

    Microbial plankton form the productive base of both marine and freshwater ecosystems and are key drivers of global biogeochemical cycles of carbon and nutrients. Plankton diversity is immense with representations from all major phyla within the three domains of life. So far, plankton monitoring has mainly been based on microscopic identification, which has limited sensitivity and reproducibility, not least because of the numerical majority of plankton being unidentifiable under the light microscope. High-throughput sequencing of taxonomic marker genes offers a means to identify taxa inaccessible by traditional methods; thus, recent studies have unveiled an extensive previously unknown diversity of plankton. Here, we conducted ultra-deep Illumina sequencing (average 105 sequences/sample) of rRNA gene amplicons of surface water eukaryotic and bacterial plankton communities sampled in summer along a 2000 km transect following the salinity gradient of the Baltic Sea. Community composition was strongly correlated with salinity for both bacterial and eukaryotic plankton assemblages, highlighting the importance of salinity for structuring the biodiversity within this ecosystem. In contrast, no clear trends in alpha-diversity for bacterial or eukaryotic communities could be detected along the transect. The distribution of major planktonic taxa followed expected patterns as observed in monitoring programs, but groups novel to the Baltic Sea were also identified, such as relatives to the coccolithophore Erniliana huxleyi detected in the northern Baltic Sea. This study provides the first ultra-deep sequencing-based survey on eukaryotic and bacterial plankton biogeography in the Baltic Sea.

  • 41.
    Hugerth, Luisa
    KTH, School of Biotechnology (BIO), Gene Technology. Science for Life Laboratory.
    High-throughput DNA Sequencingin Microbial Ecology: Methods and Applications2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Microorganisms play central roles in planet Earth’s geochemical cycles, in food production, and in health and disease of humans and livestock. In spite of this, most microbial life forms remain unknown and unnamed, their ecological importance and potential technological applications beyond the realm of speculation. This is due both to the magnitude of microbial diversity and to technological limitations. Of the many advances that have enabled microbiology to reach new depth and breadth in the past decade, one of the most important is affordable high-throughput DNA sequencing. This technology plays a central role in each paper in this thesis.

    Papers I and II are focused on developing methods to survey microbial diversity based on marker gene amplification and sequencing. In Paper I we proposed a computational strategy to design primers with the highest coverage among a given set of sequences and applied it to drastically improve one of the most commonly used primer pairs for ecological surveys of prokaryotes. In Paper II this strategy was applied to an eukaryotic marker gene. Despite their importance in the food chain, eukaryotic microbes are much more seldom surveyed than bacteria. Paper II aimed at making this domain of life more amenable to high-throughput surveys.

    In Paper III, the primers designed in papers I and II were applied to water samples collected up to twice weekly from 2011 to 2013 at an offshore station in the Baltic proper, the Linnaeus Microbial Observatory. In addition to tracking microbial communities over these three years, we created predictive models for hundreds of microbial populations, based on their co-occurrence with other populations and environmental factors.

    In paper IV we explored the entire metagenomic diversity in the Linnaeus Microbial Observatory. We used computational tools developed in our group to construct draft genomes of abundant bacteria and archaea and described their phylogeny, seasonal dynamics and potential physiology. We were also able to establish that, rather than being a mixture of genomes from fresh and saline water, the Baltic Sea plankton community is composed of brackish specialists which diverged from other aquatic microorganisms thousands of years before the formation of the Baltic itself.

  • 42.
    Hugerth, Luisa
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. Science for Life Laboratory.
    Lindh, Markus
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Sjöqvist, Conny
    KTH, School of Biotechnology (BIO), Gene Technology.
    Carina, Bunse
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Legrand, Catherine
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Pinhassi, Jarone
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology.
    Seasonal dynamics and interactions among Baltic Sea prokaryoticand eukaryotic plankton assemblagesManuscript (preprint) (Other academic)
    Abstract [en]

    One of the main goals of microbial ecology is to identify the mechanismsthat regulate patterns in community structure at temporal scalescompatible with populations’ turnover times across complete seasonalcycles. Here, we examined high-frequency temporal dynamics of marineplankton from a sampling effort covering 2011-2013, roughly twice weekly,comprising 144 samples. Bacterial and eukaryotic communities wereprofiled by 16S and 18S high-throughput sequencing, respectively.Interestingly, we found that no combination of the measured environmentalparameters could predict a significant proportion of the variation inpopulation dynamics of bacterioplankton, and even less so for eukaryoticplankton. Large differences in physicochemical conditions and communitycomposition typical of temperate climates mean that different regimes canquickly succeed each other over the year, with the relative importance ofdifferent drivers changing equally rapidly. Nevertheless, our approachrevealed interesting recurrent co-occurrence patterns across distinctenvironmental changes. Hence, we could make abundance predictions formore than half of the most frequent OTUs based on interactions with otherOTUs. These results suggests that a complex set of biotic interactions arecontributing to temporal patterns among planktonic assemblages despiterapid changes in environmental conditions.

  • 43.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Analysing Microbial Community Composition through Amplicon Sequencing: From Sampling to Hypothesis Testing2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, 1561Article, review/survey (Refereed)
    Abstract [en]

    Microbial ecology as a scientific field is fundamentally driven by technological advance. The past decade's revolution in DNA sequencing cost and throughput has made it possible for most research groups to map microbial community composition in environments of interest. However, the computational and statistical methodology required to analyse this kind of data is often not part of the biologist training. In this review, we give a historical perspective on the use of sequencing data inmicrobial ecology and restate the current need for this method; but also highlight the major caveats with standard practices for handling these data, from sample collection and library preparation to statistical analysis. Further, we outline the main new analytical tools that have been developed in the past few years to bypass these caveats, as well as highlight the major requirements of common statistical practices and the extent to which they are applicable to microbial data. Besides delving into the meaning of select alpha- and beta-diversity measures, we give special consideration to techniques for finding the main drivers of community dissimilarity and for interaction network construction. While every project design has specific needs, this review should serve as a starting point for considering what options are available.

  • 44.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Larsson, John
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindh, Markus V.
    Legrand, Catherine
    Pinhassi, Jarone
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Metagenome-assembled genomes uncover a global brackish microbiome2015In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16, 279Article in journal (Refereed)
    Abstract [en]

    Background: Microbes are main drivers of biogeochemical cycles in oceans and lakes. Although the genome is a foundation for understanding the metabolism, ecology and evolution of an organism, few bacterioplankton genomes have been sequenced, partly due to difficulties in cultivating them. Results: We use automatic binning to reconstruct a large number of bacterioplankton genomes from a metagenomic time-series from the Baltic Sea, one of world's largest brackish water bodies. These genomes represent novel species within typical freshwater and marine clades, including clades not previously sequenced. The genomes' seasonal dynamics follow phylogenetic patterns, but with fine-grained lineage-specific variations, reflected in gene-content. Signs of streamlining are evident in most genomes, and estimated genome sizes correlate with abundance variation across filter size fractions. Comparing the genomes with globally distributed metagenomes reveals significant fragment recruitment at high sequence identity from brackish waters in North America, but little from lakes or oceans. This suggests the existence of a global brackish metacommunity whose populations diverged from freshwater and marine relatives over 100,000 years ago, long before the Baltic Sea was formed (8000 years ago). This markedly contrasts to most Baltic Sea multicellular organisms, which are locally adapted populations of freshwater or marine counterparts. Conclusions: We describe the gene content, temporal dynamics and biogeography of a large set of new bacterioplankton genomes assembled from metagenomes. We propose that brackish environments exert such strong selection that lineages adapted to them flourish globally with limited influence from surrounding aquatic communities.

  • 45.
    Innocenti, Nicolas
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Data Analysis and Next Generation Sequencing : Applications in Microbiology.2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Next Generation Sequencing (NGS) is a new technology that has revolutionized the way we study living organisms. Where previously only a few genes could be studied at a time through targeted direct probing, NGS offers the possibility to perform measurements for a whole genome at once. The drawback is that the amount of data generated in the process is large and extracting useful information from it requires new methods to process and analyze it.

    The main contribution of this thesis is the development of a novel experimental method coined tagRNA-seq, combining 5’tagRACE, a previously developed technique, with RNA-sequencing technology. Briefly, tagRNA-seq makes it possible to identify the 5’ ends of RNAs in bacteria and directly probe for their type, primary or processed, by ligating short RNA sequences, the tags, to the beginnings of RNA molecules. We used the method to directly probe for transcription start and processing sites in two bacterial species, Escherichiacoli and Enterococcus faecalis. It was also used to study polyadenylation in E. coli, where the ability to identify processed RNA molecules proved to be useful to separate direct and indirect regulatory effects of this mechanism. We also demonstrate how data from tagRNA-seq experiments can be used to increase confidence on the discovery of anti-sense transcripts in bacteria. Analyses of RNA-seq data obtained in the context of these experiments revealed subtle artifacts in the coverage signal towards gene ends, that we were able to explain and quantify based Kolmogorov’s broken stick model. We also discovered evidences for circularization of a few RNA transcripts, both in our own data sets and publicly available data.

    Designing the tags used in tagRNA-seq led us to the problem of words absent from a text. We focus on a particular subset of these, the minimal absent words (MAWs), and develop a theory providing a complete description of their size distribution in random text. We also show that MAWs in genomes from viruses and living organisms almost always exhibit a behavior different from random texts in the tail of the distribution, and that MAWs from this tail are closely related to sequences present in the genome that preferentially appear in regions with important regulatory functions.

    Finally, and independently from tagRNA-seq, we propose a new approach to the problem of bacterial community reconstruction in metagenomic, based on techniques from compressed sensing. We provide a novel algorithm competing with state-of-the-art techniques in the field.

  • 46.
    Innocenti, Nicolas
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Golumbeanu, Monica
    Department of Biosystems Science and Engineering, ETH Zürich, CH-4058, Basel, Switzerland.
    Fouquier d'Hérouël, Aymeric
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362, Esch-sur-Alzette, Luxembourg.
    Lacoux, Caroline
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Bonnin, Rémy A.
    Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, UMR8621, F-91405, Orsay, France.
    Kennedy, Sean P.
    INRA, MetaGenoPolis US1367, Domaine de Vilvert, F-78350, Jouy-en-Josas, France.
    Wessner, Francoise
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Serror, Pascale
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Bouloc, Philippe
    Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, UMR8621, F-91405, Orsay, France.
    Repoila, Francis
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis2015In: RNA, ISSN 1355-8382Article in journal (Refereed)
    Abstract [en]

    Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small-and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.

  • 47.
    Innocenti, Nicolas
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Repoila, Francis
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France}\affiliation{AgroParisTech, UMR Micalis, Domaine de Vilvert, F-78350, Jouy-en-Josas, France.
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Detection and quantitative estimation of spurious double stranded DNA formation during reverse transcription in bateria using tagRNA-seq2015In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584Article in journal (Refereed)
    Abstract [en]

    Standard RNA-seq has a well know tendency to generate "ghost" antisense reads due to formation of spurious second strand cDNA in the sequencing process. We recently reported on a novel variant of RNA-seq coined "tagRNA-seq" introduced for the purpose of distinguishing primary from processed transcripts in bacteria. Incidentally, the additional information provided by the tag is also very suitable for detection of true anti-sense RNA transcripts and quantification of spurious antisense signals in a sample. We briefly explain how to perform such a detection and illustrate on previously published datasets.

  • 48. Jarvis, Å.
    et al.
    Sundberg, Cecilia
    Swedish University of Agricultural Sciences, Sweden.
    Milenkovski, S.
    Pell, M.
    Smårs, S.
    Lindgren, P. -E
    Hallin, S.
    Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste2009In: Journal of Applied Microbiology, ISSN 1364-5072, E-ISSN 1365-2672, Vol. 106, no 5, 1502-1511 p.Article in journal (Refereed)
    Abstract [en]

    Aims: To monitor emissions of NH3 and N2O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB). Methods and Results: A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH 3 emissions peaked when pH started to increase. Small amounts of N2O and CH4 were also produced. In total, 16% and less than 1% of the initial N was lost as NH3-N and N2O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase. Conclusions: Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH3 emissions. Small but significant amounts of N2O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear. Significance and Impact of Study: This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH3 emissions.

  • 49.
    Kroll, Jens
    et al.
    Westfälische Wilhelms-Universität Münster, Germany.
    Klinter, Stefan
    Westfälische Wilhelms-Universität Münster, Germany.
    Schneider, Cornelia
    Westfälische Wilhelms-Universität Münster, Germany.
    Voß, Isabella
    Westfälische Wilhelms-Universität Münster, Germany.
    Steinbüchel, Alexander
    Westfälische Wilhelms-Universität Münster, Germany.
    Plasmid addiction systems: Perspectives and applications in biotechnology2010In: Microbial Biotechnology, ISSN 1751-7907, Vol. 3, no 6, 634-657 p.Article, review/survey (Refereed)
    Abstract [en]

    Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications.

  • 50.
    Kroll, Jens
    et al.
    Westfälische Wilhelms-Universität Münster, Germany.
    Klinter, Stefan
    Westfälische Wilhelms-Universität Münster, Germany.
    Steinbüchel, Alexander
    Westfälische Wilhelms-Universität Münster, Germany.
    A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium2011In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 89, no 3, 593-604 p.Article in journal (Refereed)
    Abstract [en]

    Introduction: Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. Material and methods: In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Results: Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Discussion: Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.

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