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  • 151.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Baker-Austin, Craig
    Bond, Philip L
    First use of two-dimensional polyacrylamide gel electrophoresis to determine phylogenetic relationships.2004In: J Microbiol Methods, ISSN 0167-7012, Vol. 58, no 3, p. 297-302Article in journal (Refereed)
    Abstract [en]

    Methods for microbial classification are not always capable of distinguishing between isolates at the species level. We have previously characterised four Ferroplasma isolates that were >98.9% similar at the 16S rDNA level, the isolates showed marked phenotypic differences, and one isolate was borderline on the 70% species boundary from DNA-DNA similarity data. In this study we have used statistical comparisons of two-dimensional polyacylamide gel electrophoresis gels for classification of closely related isolates. From the protein profile similarities an un-rooted tree was constructed that was congruent with a tree derived from DNA-DNA similarities.

  • 152.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Baker-Austin, Craig
    Hind, Andrew
    Bowman, John P
    Bond, Philip L
    Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.2004In: Appl Environ Microbiol, ISSN 0099-2240, Vol. 70, no 4, p. 2079-88Article in journal (Refereed)
    Abstract [en]

    Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y(T) by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate "Ferroplasma acidarmanus" Fer1(T) required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42 degrees C and the pH optima were 1.0 to 1.7, and "F. acidarmanus" Fer1(T) was capable of growing at pH 0. The optimum yeast extract concentration for "F. acidarmanus" Fer1(T) was higher than that for the other three isolates. Phenotypic results suggested that isolate "F. acidarmanus" Fer1(T) is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y(T) group as a single species. "F. acidarmanus" Fer1(T) groups separately, and we propose the new species "F. acidarmanus" Fer1(T) sp. nov.

  • 153.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Halinen, Anna-Kaisa
    Rahrmen, Nelli
    Boström, Dan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics, Energy Technology and Thermal Process Chemistry.
    Sundkvist, Jan-Eric
    Riekkola-Vanhanen, Marja
    Kaksonen, Anna H.
    Puhakka, Jaakko A.
    Silicate mineral dissolution during heap bioleaching2008In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 99, no 4, p. 811-820Article in journal (Refereed)
    Abstract [en]

    Silicate minerals are present in association with metal sulfides in ores and their dissolution occurs when the sulfide minerals are bioleached in heaps for metal recovery. It has previously been suggested that silicate mineral dissolution can affect mineral bioleaching by acid consumption, release of trace elements, and increasing the viscosity of the teach solution. In this study, the effect of silicates present in three separate samples in conjunction with chalcopyrite and a complex multi-metal sulfide ore on heap bioleaching was evaluated in column bioreactors. Fe2+ oxidation was inhibited in columns containing chalcopyrite samples A and C that leached 1.79 and 1.11 mM fluoride, respectively but not in sample B that contained 0.14 mM fluoride. Microbial Fe2+ oxidation inhibition experiments containing elevated fluoride concentrations and measurements of fluoride release from the chalcopyrite ores supported that inhibition of Fe2+ oxidation during column leaching of two of the chalcopyrite ores was due to fluoride toxicity. Column bioleaching of the complex sulfide ore was carried out at various temperatures (7-50 degrees C) and pH values (1.5-3.0). Column leaching at pH 1.5 and 2.0 resulted in increased acid consumption rates and silicate dissolution such that it became difficult to filter the leach solutions and for the leach liquor to percolate through the column. However, column temperature (at pH 2.5) only had a minor effect on the acid consumption and silicate dissolution rates. This study demonstrates the potential negative impact of silicate mineral dissolution on heap bioleaching by microbial inhibition and liquid flow.

  • 154.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Halinen, Anna-Kaisa
    Rahunen, Nelli
    Ozkaya, Bestamin
    Sahinkaya, Erkan
    Kaksonen, Anna H
    Lindström, Börje
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Puhakka, Jaakko A
    Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms.2007In: Biotechnol Bioeng, ISSN 0006-3592, Vol. 97, no 5, p. 1205-15Article in journal (Refereed)
    Abstract [en]

    An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C. (c) 2006 Wiley Periodicals, Inc.

  • 155.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kupka, Daniel
    Halinen, Anna-Kaisa
    Rahunen, Nelli
    Özkaya, Bestamin
    Sahinkaya, Erkan
    Rzhepishevska, Olena I
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kaksonen, Anna H
    Karnachuk, Olia V
    Tuovinen, IH
    Puhakka, Jaakko A
    Iron oxidation and bioleaching potential at low temperatures2007In: Biohydrometallurgy: from the single cell to the environment / [ed] Axel Schippers, Zurich: Trans Tech Publications Inc., 2007, p. 578-578Conference paper (Refereed)
  • 156.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lindström, E Börje
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Analysis of community composition during moderately thermophilic bioleaching of pyrite, arsenical pyrite and chalcopyrite2004In: Microbial Ecology, Vol. 48, p. 19-28Article in journal (Other (popular science, discussion, etc.))
  • 157.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lövgren, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Boström, Dan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics, Energy Technology and Thermal Process Chemistry.
    Silicate mineral dissolution in the presence of acidophilic microorganisms: implications for heap bioleaching2009In: Hydrometallurgy, ISSN 0304-386X, E-ISSN 1879-1158, Vol. 96, no 4, p. 288-293Article in journal (Refereed)
    Abstract [en]

    Silicate minerals are found with sulfide minerals and therefore, can be present during heap bioleaching for metal extraction. The weathering of silicate minerals by chemical and biological means is variable depending on the conditions and microorganisms tested. In low pH metal rich environments their dissolution can influence the solution chemistry by increasing pH, releasing toxic trace elements, and thickening of the leach liquor. The amenity of five silicate minerals to chemical and biological dissolution was tested in the presence of either ‘Ferroplasma acidarmanus’ Fer1 or Acidithiobacillus ferrooxidans with olivine and hornblende being the most and least amenable, respectively. A number of the silicates caused the pH of the leach liquor to increase including augite, biotite, hornblende, and olivine. For the silicate mineral olivine, the factors affecting magnesium dissolution included addition of microorganisms and Fe2+. XRD analysis identified secondary minerals in several of the experiments including jarosite from augite and hornblende when the medium contained Fe2+. Despite acidophiles preferentially attaching to sulfide minerals, the increase in iron coupled with very low Fe2+ concentrations present at the end of leaching during dissolution of biotite, olivine, hornblende, and microcline suggested that these minerals supported growth. Weathering of the tested silicates would affect heap bioleaching by increasing the pH with olivine, fluoride release from biotite, and production of jarosite during augite and hornblende dissolution that may have caused passivation. These data have increased knowledge of silicate weathering under bioleaching conditions and provided insights into the effects on solution chemistry during heap bioleaching.

  • 158.
    Dopson, Mark
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Sundkvist, J-E
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Lindström, Börje
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Toxicity of metal extraction and flotation chemicals to Sulfolobus metallicus and chalcopyrite bioleaching.2006In: Hydrometallurgy, no 81, p. 205-213Article in journal (Refereed)
  • 159. Drotz, Marcus K.
    et al.
    Brodin, Tomas
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Saura, Anssi
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Giles, Barbara E.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Ecotype Differentiation in the Face of Gene Flow within the Diving Beetle Agabus bipustulatus (Linnaeus, 1767) in Northern Scandinavia2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2, p. e31381-Article in journal (Refereed)
    Abstract [en]

    The repeated occurrence of habitat-specific polyphyletic evolved ecotypes throughout the ranges of widely distributed species implies that multiple, independent and parallel selection events have taken place. Ecological transitions across altitudinal gradients over short geographical distances are often associated with variation in habitat-related fitness, these patterns suggest the action of strong selective forces. Genetic markers will therefore contribute differently to differences between ecotypes in local hybrid zones. Here we have studied the adaptive divergence between ecotypes of the water beetle Agabus bipustulatus along several parallel altitudinal gradients in northern Scandinavia. This water beetle is well known for its remarkable morphological variation associated with mountain regions throughout the western Palaearctic. Two morphological ecotypes are recognised: a montane type with reduced flight muscles and a lowland type with fully developed muscles. Using a multilocus survey of allozyme variation and a morphological analysis with landmark-based morphometrics, across thirty-three populations and seven altitudinal gradients, we studied the local adaptive process of gene flow and selection in detail. Populations were sampled at three different elevations: below, at and above the tree line. The results indicate that the levels of divergence observed between ecotypes in morphology and allele frequencies at alpha-Glycerophosphate dehydrogenase relative to those shown by neutral molecular markers reflects local diversifying selection in situ. Four main lines of evidence are shown here: (1) A repeated morphological pattern of differentiation is observed across all altitudinal transects, with high reclassification probabilities. (2) Allele and genotype frequencies at the alpha-Gpdh locus are strongly correlated with altitude, in sharp contrast to the presumable neutral markers. (3) Genetic differentiation is two to three times higher among populations across the tree line than among populations at or below. (4) Genetic differentiation between ecotypes within independent mountain areas is reflected by different sets of allozymes.

  • 160. Drotz, Marcus K.
    et al.
    Savolainen, Eino
    Saura, Anssi
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stahls, Gunilla
    The genetic population structure of lotic and lentic mayflies of the Baetis vernus group (Ephemeroptera: Baetidae)2012In: Canadian Entomologist, ISSN 0008-347X, E-ISSN 1918-3240, Vol. 144, no 5, p. 679-690Article in journal (Refereed)
    Abstract [en]

    Nymphs of lotic mayflies live in environments that are expected to give rise to different degrees of population structuring. Here we investigate two taxa adapted to different lifestyles. Baetis macani Kimmins (Ephemeroptera: Baetidae) lives in flowing water; brooks that may periodically dry out in the summer or freeze to the bottom in winter. Baetis jaervii Savolainen is mostly found in sedge belts along the shores of lakes. Most insects living in flowing water show low levels of among-population genetic differentiation within and among catchments. Levels of differentiation in the lotic species are therefore assumed to be lower than in lentic B. jaervii. Here we test this hypothesis. Mitochondrial DNA and allele frequencies of nuclear genes were used to detect population structure in specimens originating from an extensive area from northern Finland. The genetic differentiation among populations of the lotic B. macani is more than twice the corresponding value for the lentic B. jaervii (F-ST 0.33 versus 0.15, while the mean F-ST between species was 0.33 and significant). The result is congruent within the cytochrome c oxidase subunit I gene (COI) partial gene frequencies. We argue that the significant genetic population structure, which only was found in the lotic B. macani, is differentiated as a consequence to the unpredictable environment as contrasted to the stable environment in standing bodies of water.

  • 161. Dufe, Veronica T
    et al.
    Ingner, Daniel
    Heby, Olle
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Khomutov, Alex R
    Persson, Lo
    Al-Karadaghi, Salam
    A structural insight into the inhibition of human and Leishmania donovani ornithine decarboxylases by 1-amino-oxy-3-aminopropane.2007In: Biochem J, ISSN 1470-8728, Vol. 405, no 2, p. 261-8Article in journal (Refereed)
    Abstract [en]

    The critical role of polyamines in key processes such as cell growth, differentiation and macromolecular synthesis makes the enzymes involved in their synthesis potential targets in the treatment of certain types of cancer and parasitic diseases. Here we present a study on the inhibition of human and Leishmania donovani ODC (ornithine decarboxylase), the first committed enzyme in the polyamine biosynthesis pathway, by APA (1-amino-oxy-3-aminopropane). The present study shows APA to be a potent inhibitor of both human and L. donovani ODC with a K(i) value of around 1.0 nM. We also show that L. donovani ODC binds the substrate, the co-enzyme pyridoxal 5'-phosphate and the irreversible inhibitor alpha-difluoromethylornithine (a curative agent of West African sleeping sickness) with less affinity than human ODC. We have also determined the three-dimensional structure of human ODC in complex with APA, which revealed the mode of the inhibitor binding to the enzyme. In contrast with earlier reports, the structure showed no indication of oxime formation between APA and PLP (pyridoxal 5'-phosphate). Homology modelling suggests a similar mode of binding of APA to L. donovani ODC. A comparison of the ODC-APA-PLP structure with earlier ODC structures also shows that the protease-sensitive loop (residues 158-168) undergoes a large conformational change and covers the active site of the protein. The understanding of the structural mode of APA binding may constitute the basis for the development of more specific inhibitors of L. donovani ODC.

  • 162. Dukuzumuremyi, Jean-Marie
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Åkerström, Bo
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Schesser, Kurt
    The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 45, p. 35281-35290Article in journal (Refereed)
    Abstract [en]

    The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

  • 163. Durand, Jérôme M B
    et al.
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri2009In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 155, no Pt 8, p. 2498-2508Article in journal (Refereed)
    Abstract [en]

    This paper shows that compounds in defined growth media strongly influence the expression of the effectors of virulence in the human invasive pathogen Shigella flexneri. Ornithine in conjunction with uracil reduces the haemolytic ability of wild-type cultures more than 20-fold and the expression of the type III secretion system more than 8-fold, as monitored by an mxiC : : lacZ transcriptional reporter. mxiC gene expression is further decreased by the presence of methionine or branched-chain amino acids (15-fold or 25-fold at least, respectively). Lysine and a few other aminated metabolites (cadaverine, homoserine and diaminopimelate) counteract the ornithine-mediated inhibition of haemolytic activity and of the expression of a transcriptional activator virF reporter. The complete abolition of invasion of HeLa cells by wild-type bacteria by ornithine, uracil, methionine or branched-chain amino acids establishes that these metabolites are powerful effectors of virulence. These findings provide a direct connection between metabolism and virulence in S. flexneri. The inhibitory potential exhibited by the nutritional environment is stronger than temperature, the classical environmental effector of virulence. The implications and practical application of this finding in prophylaxis and treatment of shigellosis are discussed.

  • 164.
    Dwivedi, Gaurav Dutta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning and Expression of Streptococcal Recombinant Protein G.2015Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    •  
  • 165. Díaz-Salazar, Carlos
    et al.
    Calero, Patricia
    Espinosa-Portero, Rocío
    Jiménez-Fernández, Alicia
    Wirebrand, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Velasco-Domínguez, María G.
    López-Sánchez, Aroa
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Govantes, Fernando
    The stringent response promotes biofilm dispersal in Pseudomonas putida2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 18055Article in journal (Refereed)
    Abstract [en]

    Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

  • 166.
    Edgren, Tomas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III Secretion in Yersinia: Injectisome or Not?2012In: PLoS pathogens, ISSN 1553-7374, Vol. 8, no 5, p. e1002669-Article in journal (Refereed)
  • 167. Edqvist, Petra
    et al.
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Minimal secretion of the YopB and YopD translocators is sufficient for Yop-effector translocation by YersiniaManuscript (Other academic)
  • 168.
    Edqvist, Petra J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

    The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

    Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

    Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

  • 169.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Liu, Junfa
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells.2007In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 9, no 2, p. 224-233Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.

  • 170.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Betts, Helen J
    Forsberg, Ake
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Pallen, Mark J
    Francis, Matthew S
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.2006In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, no 1, p. 31-44Article in journal (Refereed)
    Abstract [en]

    Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

  • 171.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Åhlund, Monika K
    Forsberg, Åke
    Francis, Matthew S
    Functional insights into the YopD C-terminus through comprehensive site-directed mutagenesisManuscript (Other academic)
  • 172.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Examination of LcrH type III secretion chaperone function during Yersinia-eukaryotic cell contactManuscript (Other academic)
  • 173.
    Edqvist, Petra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lavander, Moa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Sundberg, Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lloyd, Scott
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System2003In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 185, no 7, p. 2259-2266Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

  • 174.
    Edwin, Aaron
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Stier, Gunter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Domain isolation, expression, purification and proteolytic activity of the metalloprotease PrtV from Vibrio cholerae2014In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 96, p. 39-47Article in journal (Refereed)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the bacteria's ability to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102kDa) multidomain pre-pro-protein that so far has only been expressed in V. cholerae. Structural studies require high amounts of soluble protein with high purity. Previous attempts for recombinant expression have been hampered by low expression and solubility of protein fragments. Here, we describe results from parallel cloning experiments in Escherichia coli where fusion tagged constructs of PrtV fragments were designed, and protein products tested for expression and solubility. Of more than 100 designed constructs, three produced protein products that expressed well. These include the N-terminal domain (residues 23-103), the PKD1 domain (residues 755-839), and a 25kDa fragment (residues 581-839). The soluble fusion proteins were captured with Ni(2+) affinity chromatography, and subsequently cleaved with tobacco etch virus protease. Purification protocols yielded ∼10-15mg of pure protein from 1L of culture. Proper folding of the shorter domains was confirmed by heteronuclear NMR spectra recorded on (15)N-labeled samples. A modified protocol for the native purification of the secreted 81kDa pro-protein of PrtV is provided. Proteolytic activity measurements suggest that the 37kDa catalytic metalloprotease domain alone is sufficient for activity.

  • 175.
    Ekestubbe, Sofie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Timing and targeting of Type III secretion translocation of virulence effectors in Yersinia2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

  • 176.
    Ekestubbe, Sofie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Edgren, Tomas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

  • 177. Enerly, Espen
    et al.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Lambertsson, Andrew
    Reverse genetics in Drosophila: from sequence to phenotype using UAS-RNAi transgenic flies.2002In: Genesis, ISSN 1526-954X, Vol. 34, no 1-2, p. 152-5Article in journal (Other academic)
  • 178.
    Engström, Patrik
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chemical genetics discloses the importance of heme and glucose metabolism in Chlamydia trachomatis pathogenesis2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Chlamydiae are important human bacterial pathogens with an intracellular life cycle that consists of two distinct bacterial forms, an infectious form (EB) that infects the eukaryotic host cell, and a non-infectious form (RB) that allows intracellular proliferation. To be successful, chlamydiae need to alternate between EB and RB to generate infectious EB’s which are competent to infect new host cells.

    Chemical genetics is an attractive approach to study bacterial pathogenesis; in principal this approach relies on an inhibitory compound that specifically inhibits a protein of interest. An obstacle in using this approach is target identification, however whole genome sequencing (WGS) of spontaneous mutants resistant to novel inhibitory compounds has significantly extended the utility of chemical genetic approaches by allowing the identification of their target proteins and/or biological pathways.

    In this thesis, a chemical genetics approach is used, I have found that heme and glucose metabolism of C. trachomatis is specifically important for the transition from the RB form to the infectious EB form. Heme and glucose metabolism are both coupled to energy metabolism, which suggests a common link between the RB-to-EB transitions. In connection with the above findings I have developed strategies that enable the isolation of isogenic C. trachomatis mutant strains. These strategies are based on WGS of spontaneous mutant populations and subsequent genotyping of clonal strains isolated from these mutant populations. Experiments with the mutant strains suggest that the uptake of glucose-6-phosphate (G-6-P) regulates the RB-to-EB transition, representing one of the first examples where genetics has been used to study C. trachomatis pathogenesis. Additional experiments with the mutant strains indicate that G-6-P promotes bacterial growth during metabolic stress.

    In concert with other findings presented in this thesis, I have fine-tuned methods that could be employed to reveal how novel inhibitory chemical compounds affect chlamydiae. In a broader context, I suggest that C. trachomatis could be used as a model organism to understand how new inhibitory drugs affect other bacterial pathogens.

    In addition, I observed that C. pneumoniae infections resulted in generalized bone loss in mice and that these mice display a cytokine profile similar to infected bone cells in vitro. Thus, this study indicates that C. pneumoniae potentially can infect bone cells in vivo, resulting in bone loss, alternatively, the inflammatory responses seen in vivo could be the causative factor of the bone loss observed.

  • 179.
    Eriksson, Björn
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Ann-Sofie
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Roos, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Levan, Göran
    Holmberg, Dan
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Establishment and characterization of a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia1999In: Experimental Hematology, ISSN 0301-472X, E-ISSN 1873-2399, Vol. 27, no 4, p. 682-688Article in journal (Refereed)
    Abstract [en]

    In this study, a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia with an early onset and high incidence was established and characterized. All tumors analyzed were found to express the alpha,beta T-cell receptor, and the majority of them had a mature, CD3+CD4+CD8- immunophenotype. In a few cases, tumors with a more immature CD3+CD4+CD8+ phenotype were isolated. Expanded phenotyping using a broad panel of lymphocyte differentiation markers confirmed the mature T-cell phenotype of the tumors. Histologic and cell cycle analysis of the tumors revealed an aggressive lymphoblastic malignancy with a very high proliferation rate and widespread engagement of bone marrow and lymphoid as well as nonlymphoid organs. Thus, the TLL mouse strain represents a unique model for the analysis of the oncogenesis and progression of mature T-cell tumors and for the development of therapeutic measures to combat such tumors.

  • 180.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer-Eriksson, A Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Small Molecule Screening for Inhibitors of the YopH Phosphatase of Yersinia pseudotuberculosis2012In: Advances in Yersinia Research, New York: Springer, 2012, Vol. 954, p. 357-363Chapter in book (Refereed)
    Abstract [en]

    Bacterial virulence systems are attractive targets for development of new antibacterial agents. Yersinia spp. utilize the type III secretion (T3S) system to secrete and translocate Yersinia outer proteins (Yop effectors) into the cytosol of the target cell and thereby overcome host defenses to successfully establish an infection. Thus, the Yop effectors constitute attractive targets for drug development. In the present study we apply small molecule screening to identify inhibitors of one of the secreted proteins YopH, a tyrosine phosphatase required for virulence. Characterization of seven inhibitors indicated that both competitive and noncompetitive inhibitors were identified with IC50 values of 6–20 μM.

  • 181.
    Eriksson, Sara
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Quality by Design (QbD) in Biochemical Applications: Possibilities and Limitations2013Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
  • 182.
    Eriksson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Varshney, Gaurav
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aspenström, Pontus
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster2010In: BMC Developmental Biology, ISSN 1471-213X, E-ISSN 1471-213X, Vol. 10, p. 86-Article in journal (Refereed)
    Abstract [en]

    Background

    In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.

    Results

    Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype.

    Conclusions

    Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm.

  • 183.
    Esberg, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Functional aspects of wobble uridine modifications in yeast tRNA2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications.

    The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes.

    The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons.

    Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

  • 184.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Huang, Bo
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Johansson, Marcus J O
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.2006In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 24, no 1, p. 139-148Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis, and formation of modified wobble uridines in tRNA. Therefore it was important to clarify whether Elongator has three distinct functions or whether it regulates one key process that leads to multiple downstream effects. Here, we show that the phenotypes of Elongator-deficient cells linking the complex to transcription and exocytosis are suppressed by increased expression of two tRNA species. Elongator is required for formation of the mcm(5) group of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) in these tRNAs. Hence, in cells with normal levels of these tRNAs, presence of mcm(5)s(2)U is crucial for posttranscriptional expression of gene products important in transcription and exocytosis. Our results indicate that the physiologically relevant function of the evolutionary-conserved Elongator complex is in formation of modified nucleosides in tRNAs.

  • 185.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moqtaderi, Zarmik
    Fan, Xiaochun
    Lu, Jian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Struhl, Kevin
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Iwr1 protein is important for preinitiation complex formation by all three nuclear RNA polymerases in Saccharomyces cerevisiae2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e20829-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II.

    PRINCIPAL FINDINGS: Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect.

    CONCLUSIONS: Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.

  • 186.
    Evander, Magnus
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Eriksson, Irene
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Pettersson, Lisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Juto, Per
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Olsson, Gert E
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bucht, Göran
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Allard, Annika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Puumala hantavirus viremia diagnosed by real-time reverse transcriptase PCR using samples from patients with hemorrhagic fever and renal syndrome.2007In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 45, no 8, p. 2491-7Article in journal (Refereed)
    Abstract [en]

    Puumala virus (PUUV) is the endemic hantavirus in northern Sweden and causes nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome. There is a need for fast and reliable diagnostics to differentiate the disease from other infections. By aligning virus RNA sequences isolated from 11 different bank voles and one human patient, we designed a real-time reverse transcriptase (RT) PCR method for detection of PUUV RNA. The real-time RT-PCR assay showed linearity from 20 to 2 x 10(6) virus copies with a correlation coefficient above 0.98 to 0.99 for all experiments. The detection threshold for PUUV cDNA was two copies per reaction. A two-step qualitative RT-PCR to detect PUUV RNA showed 100% concordance with the real-time RT-PCR assay. PUUV RNA viremia was detected in 33 of 34 PUUV immunoglobulin M (IgM)-positive patients with typical clinical NE disease from the region of endemicity. One PUUV IgM-negative sample had PUUV RNA, and 4 days later, the patient was IgM positive. Of samples with indeterminate IgM, 43% were PUUV RNA positive. The kinetics of antibody titers and PUUV viremia were studied, and five of six NE patients displayed a decrease in PUUV viremia a few days after disease outbreak coupled with an increase in PUUV IgM and IgG. In one patient with continuously high PUUV RNA levels but low IgM and no IgG response, the infection was lethal. These findings demonstrated that real-time RT-PCR is a useful method for diagnosis of PUUV viremia and for detecting PUUV RNA at early time points, before the appearance of IgM antibodies.

  • 187. Fabrik, Ivo
    et al.
    Link, Marek
    Härtlova, Anetta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dankova, Vera
    Rehulka, Pavel
    Stulik, Jiri
    Application of SILAC labeling to primary bone marrow-derived dendritic cells reveals extensive GM-CSF-dependent arginine metabolism2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 2, p. 752-762Article in journal (Refereed)
    Abstract [en]

    Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the into the field of DC research would therefore be highly beneficial. quantitative proteomic technique of metabolic labeling (SILAC) To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of C-13/N-15-labeled amino acids.. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, similar to 20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of N-15 from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of >= 90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

  • 188. Fadeev, Andrey
    et al.
    Mendoza-Garcia, Patricia
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Irion, Uwe
    Guan, Jikui
    Pfeifer, Kathrin
    Wiessner, Stephanie
    Serluca, Fabrizio
    Singh, Ajeet Pratap
    Nuesslein-Volhard, Christiane
    Palmer, Ruth H.
    ALKALs are in vivo ligands for ALK family receptor tyrosine kinases in the neural crest and derived cells2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 4, p. E630-E638Article in journal (Refereed)
    Abstract [en]

    Mutations in anaplastic lymphoma kinase (ALK) are implicated in somatic and familial neuroblastoma, a pediatric tumor of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related leukocyte tyrosine kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signaling events. Overexpression of the three DrAlkals cause ectopic iridophore development, whereas loss-of-function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss-of-function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

  • 189. Fang, Hanwei
    et al.
    Gomes, Ana Rita
    Klages, Natacha
    Pino, Paco
    Maco, Bohumil
    Walker, Eloise M.
    Zenonos, Zenon A.
    Angrisano, Fiona
    Baum, Jake
    Doerig, Christian
    Baker, David A.
    Billker, Oliver
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Brochet, Mathieu
    Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4248Article in journal (Refereed)
    Abstract [en]

    In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

  • 190. Farto, R
    et al.
    Milton, Debra L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bermúdez, M B
    Nieto, T P
    Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection2011In: Diseases of Aquatic Organisms, ISSN 0177-5103, E-ISSN 1616-1580, Vol. 95, p. 167-173Article in journal (Refereed)
    Abstract [en]

    Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.

  • 191.
    Faucillion, Marie-Line
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Increased expression of X-linked genes in mammals is associated with a higher stability of transcripts and an increased ribosome density2015In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653, Vol. 7, no 4, p. 1039-1052Article in journal (Refereed)
    Abstract [en]

    Mammalian sex chromosomes evolved from the degeneration of one homolog of a pair of ancestral autosomes, the proto-Y. This resulted in a gene dose imbalance that is believed to be restored (partially or fully) through up-regulation of gene expression from the single active X-chromosome in both sexes by a dosage compensatory mechanism. We analyzed multiple genome-wide RNA stability datasets and found significantly longer average half-lives for X-chromosome transcripts than for autosomal transcripts in various human cell lines, both male and female, and in mice. Analysis of ribosome profiling data shows that ribosome density is higher on X-chromosome transcripts than on autosomal transcripts in both humans and mice, suggesting that the higher stability is causally linked to a higher translation rate. Our results and observations are in accordance with a dosage compensatory upregulation of expressed X-linked genes. We therefore propose that differential mRNA stability and translation rates of the autosomes and sex chromosomes contribute to an evolutionarily conserved dosage compensation mechanism in mammals.

  • 192. Fernebro, Jenny
    et al.
    Blomberg, Christel
    Morfeldt, Eva
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Normark, Staffan
    Normark, Birgitta Henriques
    The influence of in vitro fitness defects on pneumococcal ability to colonize and to cause invasive disease.2008In: BMC microbiology, ISSN 1471-2180, Vol. 8, p. 65-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Streptococcus pneumoniae is a genetically diverse major human pathogen, yet a common colonizer of the nasopharynx. Here we analyzed the influence of defects affecting in vitro growth rate, on the ability of S. pneumoniae to colonize and to cause invasive disease in vivo. RESULTS: Of eleven different clinical isolates one serotype 14 carrier isolate showed a significantly longer generation time as compared to other isolates, and was severely attenuated in mice. To directly investigate the impact of growth rate on virulence, a panel of mutants in five non-essential housekeeping genes was constructed in the virulent TIGR4 background by insertion-deletion mutagenesis. Three of these mutants (ychF, hemK and yebC) were, to different degrees, growth defective, and showed a reduced invasiveness in an intranasal murine challenge model that correlated to their in vitro growth rate, but remained capable of colonizing the upper airways. The growth defect, as well as virulence defect of the hemK insertion-deletion mutant, was mediated by polarity effects on the downstream yrdC gene, encoding a probable chaperone in ribosome assembly. CONCLUSION: We conclude that large fitness defects are needed to completely prevent pneumococci from causing invasive disease after intranasal challenge. However, even severe growth defects still allow pneumococci to persistently colonize the upper airways.

  • 193. Fernández, S
    et al.
    Shingler, V
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    De Lorenzo, V
    Cross-regulation by XylR and DmpR activators of Pseudomonas putida suggests that transcriptional control of biodegradative operons evolves independently of catabolic genes.1994In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 176, no 16Article in journal (Refereed)
    Abstract [en]

    The Pu promoter of the toluene degradation plasmid pWW0 of Pseudomonas putida drives expression of an operon involved in the sequential oxidation of toluene and m- and p-xylenes to benzoate and toluates, respectively. Similarly, the Po promoter of plasmid pVI150 controls expression of an operon of Pseudomonas sp. strain CF600 which is required for the complete catabolism of phenol and cresols. These promoters, which both belong to the sigma 54-dependent class, are regulated by their cognate activators, XylR and DmpR, respectively. XylR and DmpR are homologous proteins, and both require aromatic compounds as effector molecules for activity. However, these two proteins respond to different profiles of aromatic compounds. The activity of each promoter in the presence of the heterologous regulator was monitored using lacZ and luxAB reporter systems. Genetic evidence is presented that the two activators can functionally substitute each other in the regulation of their corresponding promoters by binding the same upstream DNA segment. Furthermore, when coexpressed, the two proteins appear to act simultaneously on each of the promoters, expanding the responsiveness of these systems to the presence of effectors of both proteins. Potential mechanisms for the occurrence of evolutionary divergence between XylR and DmpR are discussed in view of the DNA sequence similarities among Pu, Po, and a third XylR-responsive promoter, Ps.

  • 194.
    Figueiredo, Margarida
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Epigenetics and targeting mechanisms in Drosophila melanogaster2015Doctoral thesis, comprehensive summary (Other academic)
  • 195.
    Figueiredo, Margarida L. A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kim, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå University, SE-90187 Umeå, Sweden.
    Allgardsson, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå UniversityUmeå, Sweden; Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions2014In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, p. e1004865-Article in journal (Refereed)
    Abstract [en]

    Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4thchromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4thchromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

  • 196.
    Figueiredo, Margarida L. A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The Male-Specific Lethal complex interacts with non-roX RNAs in Drosophila melanogasterManuscript (preprint) (Other academic)
  • 197.
    Figueiredo, Margarida L A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster2012In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 8, no 11, p. e1003061-Article in journal (Refereed)
    Abstract [en]

    Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.

  • 198. Flentie, Kelly
    et al.
    Harrison, Gregory A.
    Tükenmez, Hasan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Livny, Jonathan
    Good, James A. D.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sarkar, Souvik
    Zhu, Dennis X.
    Kinsella, Rachel L.
    Weiss, Leslie A.
    Solomon, Samantha D.
    Schene, Miranda E.
    Hansen, Mette R.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cairns, Andrew G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kulén, Martina
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wixe, Torbjörn
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Anders E. G.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hultgren, Scott J.
    Larsson, Christer
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Stallings, Christina L.
    Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis2019In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 21, p. 10510-10517Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

  • 199.
    Forshell, Linus Plym
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Li, Yongmei
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Plym Forshell, Tacha Zi
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rudelius, Martina
    Department of Pathology, Technical University of Munich, Munich, Germany.
    Nilsson, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Keller, Ulrich
    Department of Medicine, Technical University of Munich, Munich, Germany.
    Nilsson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The direct Myc target Pim3 cooperates with other Pim kinases in supporting viability of Myc-induced B-cell lymphomas2011In: Oncotarget, ISSN 1949-2553, Vol. 2, no 6, p. 448-460Article in journal (Refereed)
    Abstract [en]

    The Pim kinases are weak oncogenes. However, when co-expressed with a strong oncogene, such as c-Myc, Pim kinases potentiate the oncogenic effect resulting in an acceleration of tumorigenesis. In this study we show that the least studied Pim kinase, Pim-3, is encoded by a gene directly regulated by c-Myc via binding to one of the conserved E-boxes within the Pim3 gene. Accordingly, lymphomas arising in Myc-transgenic mice and Burkitt lymphoma cell lines exhibit elevated levels of Pim-3. Interestingly, inhibition of Pim kinases by a novel pan-Pim kinase inhibitor, Pimi, in Myc-induced lymphoma results in cell death that appears independent of caspases. The data indicate that Pim kinase inhibition could be a viable treatment strategy in certain human lymphomas that rely on Pim-3 kinase expression.

  • 200.
    Forslund, Anna-Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification of new virulence factors in Francisella tularensis2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq.

    The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain.

    In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.

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