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  • 151. Jiménez-Leiva, Andrea
    et al.
    Cabrera, Juan J.
    Bueno, Emilio
    Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
    Torres, María J.
    Salazar, Sergio
    Bedmar, Eulogio J.
    Delgado, María J.
    Mesa, Socorro
    Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1926Article in journal (Refereed)
    Abstract [en]

    Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK(2)-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK(2) and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c(550) soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc(1) complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK(2) as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK(2) and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.

  • 152. Joers, Arvi
    et al.
    Vind, Kristiina
    Hernandez, Sara B.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Maruste, Regina
    Pereira, Marta
    Brauer, Age
    Remm, Maido
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tenson, Tanel
    Muropeptides Stimulate Growth Resumption from Stationary Phase in Escherichia coli2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 18043Article in journal (Refereed)
    Abstract [en]

    When nutrients run out, bacteria enter a dormant metabolic state. This low or undetectable metabolic activity helps bacteria to preserve their scant reserves for the future needs, yet it also diminishes their ability to scan the environment for new growth-promoting substrates. However, neighboring microbial growth is a reliable indicator of a favorable environment and can thus serve as a cue for exiting dormancy. Here we report that for Escherichia coli and Pseudomonas aeruginosa this cue is provided by the basic peptidoglycan unit (i.e. muropeptide). We show that several forms of muropeptides from a variety of bacterial species can stimulate growth resumption of dormant cells and the sugar-peptide bond is crucial for this activity. These results, together with previous research that identifies muropeptides as a germination signal for bacterial spores, and their detection by mammalian immune cells, show that muropeptides are a universal cue for bacterial growth.

  • 153.
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    RNA molecules: More than mere information intermediaries2005In: ASM news (Washington), ISSN 0044-7897, Vol. 71, no 11, p. 515-520Article in journal (Refereed)
    Abstract [en]

    RNA molecules have always been overlooked when it comes to their role in living cells. They have been considered as merely assisting in the flow of information from genes to functional molecules in living cells. However, new functions are being identified including gene regulation and expression.

  • 154.
    Johansson Söderberg, Jenny
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The streptococcal IgG degrading enzyme IdeS: studies on host-pathogen interactions2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The important human pathogen Streptococcus pyogenes causes both mild infections such as pharyngitis and impetigo but also severe life threatening invasive infections.  Specific antibodies (IgG) recognize pathogens and are important mediators for pathogen clearance by the immune defence. S.ipyogenes expresses a highly effective and specific IgG endopeptidase called IdeS (immunoglobulin degrading enzyme of S.ipyogenes). IdeS rescues bacteria from opsonising IgG by cleavage of IgG generating two fragments F(ab´)2 and ½Fc. Moreover, IdeS block ROS production by neutrophils. In this thesis I have studied (i) allelic variants of IdeS and their biological potential, (ii) consequences of ½Fc production for host-pathogen interactions and (iii) IdeS processing by streptococcal and neutrophil proteases.

    When investigating the allelic variants of IdeS we could show that in respect to IgG degradation and inhibition of ROS production the allelic variants where indistinguishable, however the allelic variant of serotype M28 appears to be an unique exception as this protein was deficient in IgG cleavage but still inhibited ROS production. Further, the ½Fc fragments produced when IgG is cleaved by IdeS were shown to prime human neutrophils and under ex vivo experimental conditions this increased the bactericidal activity of the neutrophils. Finally, we made the interesting finding that IdeS is N-terminally processed by neutrophil proteases and by the streptococcal protease SpeB, but retain enzymatic activity and was less immunogenic compared to the full length protein.

  • 155. Johnning, Anna
    et al.
    Kristiansson, Erik
    Fick, Jerker
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Weijdegard, Birgitta
    Larsson, D. G. Joakim
    Resistance Mutations in gyrA and parC are Common in Escherichia Communities of both Fluoroquinolone-Polluted and Uncontaminated Aquatic Environments2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 1355Article in journal (Refereed)
    Abstract [en]

    Alterations in the target proteins of fluoroquinolones, especially in GyrA and ParC, are known to cause resistance. Here, we investigated environmental Escherichia communities to explore the possible link between the abundance of mutations, and the exposure to fluoroquinolones. Sediment samples were collected from a relatively pristine lake, up and downstream from a sewage treatment plant, and from several industrially polluted sites. The quinolone resistance-determining regions of gyrA and parC were analyzed using amplicon sequencing of metagenomic DNA. Five non-synonymous substitutions were present in all samples, and all of these mutations have been previously linked to fluoroquinolone resistance in Escherichia coli. In GyrA, substitutions S83L and D87N were on average detected at frequencies of 86 and 32%, respectively, and 31% of all amplicons encoded both substitutions. In ParC, substitutions S80I, E84G, and E84V were detected in 42, 0.9, and 6.0% of the amplicons, respectively, and 6.5% encoded double substitutions. There was no significant correlation between the level of fluoroquinolone pollution and the relative abundance of resistance mutations, with the exception of the most polluted site, which showed the highest abundance of said substitutions in both genes. Our results demonstrate that resistance mutations can be common in environmental Escherichia, even in the absence of a fluoroquinolone selective pressure.

  • 156. Joint, Ian
    et al.
    Tait, Karen
    Callow, Maureen E.
    Callow, James A.
    Milton, Debra L.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Williams, Paul
    Cámara, Miguel
    Cell-to-cell communication across the prokaryote-eukaryote boundary2002In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 298, no 5596, p. 1207-1207Article in journal (Refereed)
  • 157. Kalbina, Irma
    et al.
    Lagerqvist, Nina
    Moiane, Belisario
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Andersson, Soren
    Strid, Ake
    Falk, Kerstin I.
    Arabidopsis thaliana plants expressing Rift Valley fever virus antigens: Mice exhibit systemic immune responses as the result of oral administration of the transgenic plants2016In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 127, p. 61-67Article in journal (Refereed)
    Abstract [en]

    The zoonotic Rift Valley fever virus affects livestock and humans in Africa and on the Arabian Peninsula. The economic impact of this pathogen due to livestock losses, as well as its relevance to public health, underscores the importance of developing effective and easily distributed vaccines. Vaccines that can be delivered orally are of particular interest. Here, we report the expression in transformed plants (Arabidopsis thaliana) of Rift Valley fever virus antigens. The antigens used in this study were the N protein and a deletion mutant of the Gn glycoprotein. Transformed lines were analysed for specific mRNA and protein content by RT-PCR and Western blotting, respectively. Furthermore, the plant-expressed antigens were evaluated for their immunogenicity in mice fed the transgenic plants. After oral intake of fresh transgenic plant material, a proportion of the mice elicited specific IgG antibody responses, as compared to the control animals that were fed wild-type plants and of which none sero-converted. Thus, we show that transgenic plants can be readily used to express and produce Rift Valley Fever virus proteins, and that the plants are immunogenic when given orally to mice. These are promising findings and provide a basis for further studies on edible plant vaccines against the Rift Valley fever virus.

  • 158. Karlsson, Edvin
    et al.
    Golovliov, Igor
    Lärkeryd, Adrian
    Granberg, Malin
    Larsson, Eva
    Öhrman, Caroline
    Niemcewicz, Marcin
    Birdsell, Dawn
    Wagner, David M.
    Forsman, Mats
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clonality of erythromycin resistance in Francisella tularensis2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 10, p. 2815-2823Article in journal (Refereed)
    Abstract [en]

    Objectives: We analysed diverse strains of Francisella tularensis subsp. holarctica to assess if its division into biovars I and II is associated with specific mutations previously linked to erythromycin resistance and to determine the distribution of this resistance trait across this subspecies. Methods:Three-hundred and fourteen F. tularensis subsp. holarctica strains were tested for erythromycin susceptibility and whole-genome sequences for these strains were examined for SNPs in genes previously associated with erythromycin resistance. Each strain was assigned to a global phylogenetic framework using genome-wide canonical SNPs. The contribution of a specific SNP to erythromycin resistance was examined using allelic exchange. The geographical distribution of erythromycin-resistant F. tularensis strains was further investigated by literature search. Results:There was a perfect correlation between biovar II strains (erythromycin resistance) and the phylogenetic group B.12. Only B.12 strains had an AaEuroS -> aEuroSC SNP at position 2059 in the three copies of the rrl gene. Introducing 2059C into an rrl gene of an erythromycin-susceptible F. tularensis strain resulted in resistance. An additional 1144 erythromycin-resistant strains were identified from the scientific literature, all of them from Eurasia. Conclusions:Erythromycin resistance in F. tularensis is caused by an A2059C rrl gene mutation, which exhibits a strictly clonal inheritance pattern found only in phylogenetic group B.12. This group is an extremely successful clone, representing the most common type of F. tularensis throughout Eurasia.

  • 159.
    Khan, Ghazanfar Ali
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Monitoring anti-infectives and antibiotic resistance genes: with focus on analytical method development, effects of antibiotics and national perspectives2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Antibiotics are biologically active and are globally used in humans and animal medicine for treatment and in sub-therapeutic amounts as growth promoters in animal husbandry, aquaculture and agriculture. After excretion, inappropriate disposal and discharge from drug production facilities they enter into water bodies either as intact drugs, metabolites or transformed products. In water environments they promote development of antibiotic resistance genes (ARGs) which could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. Measurement of antibiotics has been revolutionized with the usage of solid phase extraction (SPE) for enrichment followed by Liquid chromatography mass spectrometry (LC-MS). On-line SPE coupled to LC-MS/MS has the advantages of high sample throughput, low sample preparation time and minimal solvent utilization.  Constructed wetlands (CWs) are potential alternatives to conventional treatment plants to remove organic pollutants. A study at Plönninge, Halmstad was performed to assess the impact of bacterial community pattern and development of resistance in spiked (n=4) and control (n=4). CWs were spiked with antibiotics at environmentally relevant concentrations continuously for 25 days. Shannon Index (H’) were used to determine the bacterial diversity and real-time PCR detected and quantified antibiotic resistance genes (ARGs) sulI, tetA, tetB, erm, dfrA1, qnrS and vanB and class 1 integrons intI1. No significant differences in bacterial compositions or in ARGs or integron concentrations could be discerned between exposed and control wetlands. A study conducted in Northern Pakistan showed that the antibiotic levels in most studied rivers were comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics were detected in the river in close vicinity of the 10 million city Lahore, e.g. 4600 ng L−1 sulfamethoxazole. Highest detected levels were at one of the drug formulation facilities, with measured levels up to 49000 ng L−1 of sulfamethoxazole for example. The highest levels of ARGs detected, sul1 and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. In the study in UK, sewage epidemiology surveillance is used to measure the oseltamivir carboxylate (OC), metabolite of oseltamivir (parent drug) in twenty four time proportional hourly influent samples from two WWTPs and then back-calculations were made to assess the compliance of drug.  Predicted users of oseltamivir, based on measured OC in waste water, ranged from 3-4 and 120-154 people for the two WWTP catchments, respectively, which are consistent with the projected use from national antiviral allocation statistics, 3-8 and 108-270, respectively. Scenario analysis suggests compliance was likely between 45-60% in the study regions. 

  • 160.
    Kisand, Veljo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cuadros, Rocio
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Phylogeny of culturable estuarine bacteria catabolizing riverine organic matter in the northern Baltic Sea2002In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 68, no 1, p. 379-388Article in journal (Refereed)
    Abstract [en]

    The objective of our study was to isolate and determine the phylogenetic affiliation of culturable estuarine bacteria capable of catabolizing riverine dissolved organic matter (RDOM) under laboratory conditions. Additions of RDOM consistently promoted the growth of estuarine bacteria in carbon-limited dilution cultures, with seasonal variation in growth rates and yields. At least 42 different taxa were culturable on solid agar media and, according to quantitative DNA-DNA hybridizations, constituted 32 to 89% of the total bacterial number in the enriched treatments. Five species in the Cytophaga-Flexibacter-Bacteroides group and one in the gamma-proteobacteria phylogenetic group (Marinomonas sp.) were numerically dominant during the stationary phase of the RDOM-enriched dilution cultures but not in the control cultures. Four of the isolates in Cytophaga-Flexibacter-Bacteroides group were putatively affiliated with the genus Flavobacterium. All dominating isolates were determined to be new species based on comparison to the current databases. The same group of species dominated independently of the season investigated, suggesting a low diversity of bacteria catabolizing RDOM in the estuary. It also suggested a broad tolerance of the dominating species to seasonal variation in hydrography, chemistry, and competition with other species. Taken together, our results suggest that a limited group of bacteria, mainly in the Flavobacterium genus, played an important role in introducing new energy and carbon to the marine system in the northern Baltic Sea.

  • 161.
    Kisand, Veljo
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine dissolved organic matter2003In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 69, no 6, p. 3607-3616Article in journal (Refereed)
    Abstract [en]

    Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated beta-proteobacteria to be RDOM consumers.

  • 162. Knöppel, Anna
    et al.
    Lind, Peter A
    Lustig, Ulrika
    Näsvall, Joakim
    Andersson, Dan I
    Minor fitness costs in an experimental model of horizontal gene transfer in bacteria.2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 5Article in journal (Refereed)
    Abstract [en]

    Genes introduced by horizontal gene transfer (HGT) from other species constitute a significant portion of many bacterial genomes, and the evolutionary dynamics of HGTs are important for understanding the spread of antibiotic resistance and the emergence of new pathogenic strains of bacteria. The fitness effects of the transferred genes largely determine the fixation rates and the amount of neutral diversity of newly acquired genes in bacterial populations. Comparative analysis of bacterial genomes provides insight into what genes are commonly transferred, but direct experimental tests of the fitness constraints on HGT are scarce. Here, we address this paucity of experimental studies by introducing 98 random DNA fragments varying in size from 0.45 to 5 kb from Bacteroides, Proteus, and human intestinal phage into a defined position in the Salmonella chromosome and measuring the effects on fitness. Using highly sensitive competition assays, we found that eight inserts were deleterious with selection coefficients (s) ranging from ≈ -0.007 to -0.02 and 90 did not have significant fitness effects. When inducing transcription from a PBAD promoter located at one end of the insert, 16 transfers were deleterious and 82 were not significantly different from the control. In conclusion, a major fraction of the inserts had minor effects on fitness implying that extra DNA transferred by HGT, even though it does not confer an immediate selective advantage, could be maintained at selection-transfer balance and serve as raw material for the evolution of novel beneficial functions.

  • 163. Konradt, Christoph
    et al.
    Frigimelica, Elisabetta
    Nothelfer, Katharina
    Puhar, Andrea
    Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, France, and INSERM U786, Institut Pasteur, 25–28 Rue du Dr Roux, 75724 Paris Cedex 15, France .
    Salgado-Pabon, Wilmara
    di Bartolo, Vincenzo
    Scott-Algara, Daniel
    Rodrigues, Cristina D
    Sansonetti, Philippe J
    Phalipon, Armelle
    The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism2011In: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 9, no 4, p. 263-272Article in journal (Refereed)
    Abstract [en]

    Shigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function.

  • 164. Kovach, Kristin
    et al.
    Davis-Fields, Megan
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Biology and Biochemistry, The University of Bath, Claverton Down, Bath, UK.
    Jain, Kanishk
    Doorwar, Shashvat
    Vuong, Katherine
    Dhamani, Numa
    Mohanty, Kishore
    Touhami, Ahmed
    Gordon, Vernita D.
    Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production2017In: NPJ BIOFILMS AND MICROBIOMES, ISSN 2055-5008, Vol. 3, article id UNSP 1Article in journal (Refereed)
    Abstract [en]

    Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances, largely polysaccharides. Multiple types of extracellular polymeric substances can be produced by a single bacterial strain. The distinct polymer components of biofilms are known to provide chemical protection, but little is known about how distinct extracellular polysaccharides may also protect biofilms against mechanical stresses such as shear or phagocytic engulfment. Decades-long infections of Pseudomonas. aeruginosa biofilms in the lungs of cystic fibrosis patients are natural models for studies of biofilm fitness under pressure from antibiotics and the immune system. In cystic fibrosis infections, production of the extracellular polysaccharide alginate has long been known to increase with time and to chemically protect biofilms. More recently, it is being recognized that chronic cystic fibrosis infections also evolve to increase production of another extracellular polysaccharide, Psl; much less is known about Psl's protective benefits to biofilms. We use oscillatory bulk rheology, on biofilms grown from longitudinal clinical isolates and from genetically-manipulated lab strains, to show that increased Psl stiffens biofilms and increases biofilm toughness, which is the energy cost to cause the biofilm to yield mechanically. Further, atomic force microscopy measurements reveal greater intercellular cohesion for higher Psl expression. Of the three types of extracellular polysaccharides produced by P. aeruginosa, only Psl increases the stiffness. Stiffening by Psl requires CdrA, a protein that binds to mannose groups on Psl and is a likely cross-linker for the Psl components of the biofilm matrix. We compare the elastic moduli of biofilms to the estimated stresses exerted by neutrophils during phagocytosis, and infer that increased Psl could confer a mechanical protection against phagocytic clearance.

  • 165. Kraupner, Nadine
    et al.
    Ebmeyer, Stefan
    Bengtsson-Palme, Johan
    Fick, Jerker
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kristiansson, Erik
    Flach, Carl-Fredrik
    Larsson, D. G. Joakim
    Selective concentration for ciprofloxacin resistance in Escherichia coli grown in complex aquatic bacterial biofilms2018In: Environment International, ISSN 0160-4120, E-ISSN 1873-6750, Vol. 116, p. 255-268Article in journal (Refereed)
    Abstract [en]

    There is concern that antibiotics in the environment can select for and enrich bacteria carrying acquired antibiotic resistance genes, thus increasing the potential of those genes to emerge in a clinical context. A critical question for understanding and managing such risks is what levels of antibiotics are needed to select for resistance in complex bacterial communities. Here, we address this question by examining the phenotypic and genotypic profiles of aquatic communities exposed to ciprofloxacin, also evaluating the within-species selection of resistant E. coli in complex communities. The taxonomic composition was significantly altered at ciprofloxacin exposure concentrations down to 1 mu g/L. Shotgun metagenomic analysis indicated that mobile quinolone resistance determinants (qnrD, qnrS and qnrB) were enriched as a direct consequence of ciprofloxacin exposure from 1 mu g/L or higher. Only at 5-10 mu g/L resistant E. coli increased relative to their sensitive counterparts. These resistant E. coli predominantly harbored non-transferrable, chromosomal triple mutations (gyrA S83 L, D87N and parC S80I), which confer high-level resistance. In a controlled experimental setup such as this, we interpret effects on taxonomic composition and enrichment of mobile quinolone resistance genes as relevant indicators of risk. Hence, the lowest observed effect concentration for resistance selection in complex communities by ciprofloxacin was 1 mu g/L and the corresponding no observed effect concentration 0.1 mu g/L. These findings can be used to define and implement discharge or surface water limits to reduce risks for selection of antibiotic resistance in the environment.

  • 166. Kubler, Andre
    et al.
    Larsson, Christer
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Luna, Brian
    Andrade, Bruno B.
    Amaral, Eduardo P.
    Urbanowski, Michael
    Orandle, Marlene
    Bock, Kevin
    Ammerman, Nicole C.
    Cheung, Laurene S.
    Winglee, Kathryn
    Halushka, Marc
    Park, Jin Kyun
    Sher, Alan
    Friedland, Jon S.
    Elkington, Paul T.
    Bishai, William R.
    Cathepsin K Contributes to Cavitation and Collagen Turnover in Pulmonary Tuberculosis2016In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 213, no 4, p. 618-627Article in journal (Refereed)
    Abstract [en]

    Cavitation in tuberculosis enables highly efficient person-to-person aerosol transmission. We performed transcriptomics in the rabbit cavitary tuberculosis model. Among 17 318 transcripts, we identified 22 upregulated proteases. Five type I collagenases were overrepresented: cathepsin K (CTSK), mast cell chymase-1 (CMA1), matrix metalloproteinase 1 (MMP-1), MMP-13, and MMP-14. Studies of collagen turnover markers, specifically, collagen type I C-terminal propeptide (CICP), urinary deoxypyridinoline (DPD), and urinary helical peptide, revealed that cavitation in tuberculosis leads to both type I collagen destruction and synthesis and that proteases other than MMP-1, MMP-13, and MMP-14 are involved, suggesting a key role for CTSK. We confirmed the importance of CTSK upregulation in human lung specimens, using immunohistochemical analysis, which revealed perigranulomatous staining for CTSK, and we showed that CTSK levels were increased in the serum of patients with tuberculosis, compared with those in controls (3.3 vs 0.3 ng/mL; P = .005).

  • 167.
    Kumar, Anjani
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Manisha, --
    Sangha, Gurkamaljit Kaur
    Shrivastava, Anju
    Kaur, Jagdeep
    The immunosuppressive effects of a novel recombinant LipQ (Rv2485c) protein of Mycobacterium tuberculosis on human macrophage cell lines2017In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 107, p. 361-367Article in journal (Refereed)
    Abstract [en]

    Mycobacterium tuberculosis (MTB), an intracellular pathogen, still represents a major global health challenge. A number of mycobacterial macromolecules have been shown to target biological processes within host macrophages; however, the exact mechanism for the majority of these host pathogen interactions is still poorly understood. Moreover, the lipid metabolic pathway is one of the most important physiologic pathways that plays a vital role in the survival and infection of Mycobacterium tuberculosis. In present study, we investigated the effect of rLipQ from Mycobacterium tuberculosis H37Rv on macrophage functions invitro.Our results demonstrate that rLipQ significantly lowers the expression level of pro-inflammatory cytokines (TNF-alpha& IFN-gamma) and augments the level of anti inflammatory cytokines such as IL-4 & IL-10as compared to LPS stimulated macrophages. An up-regulation of anti-inflammatory and down-regulation of pro-inflammatory cytokines levels in rLipQ pretreated macrophages implies immuno-modulatory functions in TB patients. Interestingly, rLipQ also inhibited the expression of iNOS, TLR-2 and transcription factor NF-kB in LPS stimulated macrophages whereas the expression of TLR-4 remains unchanged. The inhibition in the expression of these signaling molecules has been correlated to the inhibition of NO production in macrophages. Taken together, these studies demonstrate that rLipQ is a novel lipase that is highly immunogenic and may play an important role in the virulence and pathogenesis of M.tuberculosis infection, by altering the balance of cytokines, which might help to assess prognosis and contribute to a better understanding against host-pathogen interactions.

  • 168.
    Kumar, Keshav
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chromatographic analysis of peptidoglycan samples with the aid of a chemometric technique: introducing a novel analytical procedure to classify bacterial cell wall collection2019In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 11, no 12, p. 1671-1679Article in journal (Refereed)
    Abstract [en]

    The technical development of liquid chromatography has provided the necessary sensitivity to characterise peptidoglycan samples. However, the analysis of large numbers of complex chromatographic data sets without the aid of a proper chemometric technique is a laborious task, carrying a high risk of losing important biochemical information. The present work describes the development of a simple analytical procedure using self-organising map (SOM) analysis to analyse the large number of complex chromatographic data sets from bacterial peptidoglycan samples. SOM analysis essentially maps the samples to a hexagonal sheet based on their compositional similarity, and thus provides an approach to classify the bacterial cell wall collection in an unsupervised manner. The utility of the proposed approach was successfully validated by analysing peptidoglycan samples belonging to the Alphaproteobacterium class. The classification results achieved with SOM analysis were found to correlate well with their relative similarity in peptidoglycan compositions. In summary, the SOM analysis-based analytical procedure is shown to be useful towards automatising the analyses of chromatographic data sets of peptidoglycan samples from bacterial collections.

  • 169. Kumru, Ozan S.
    et al.
    Bunikis, Ignas
    Sorokina, Irina
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Zueckert, Wolfram R.
    Specificity and role of the borrelia burgdorferi CtpA protease in outer membrane protein processing2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 20, p. 5759-5765Article in journal (Refereed)
    Abstract [en]

    To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used site-directed mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.

  • 170. Kupka, Daniel
    et al.
    Liljeqvist, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Nurmi, Pauliina
    Puhakka, Jaakko A
    Tuovinen, Olli H
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Oxidation of elemental sulfur, tetrathionate and ferrous iron by the psychrotolerant Acidithiobacillus strain SS3.2009In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 160, no 10, p. 767-74Article in journal (Refereed)
    Abstract [en]

    Mesophilic iron and sulfur-oxidizing acidophiles are readily found in acid mine drainage sites and bioleaching operations, but relatively little is known about their activities at suboptimal temperatures and in cold environments. The purpose of this work was to characterize the oxidation of elemental sulfur (S(0)), tetrathionate (S4O6(2-)) and ferrous iron (Fe2+) by the psychrotolerant Acidithiobacillus strain SS3. The rates of elemental sulfur and tetrathionate oxidation had temperature optima of 20 degrees and 25 degrees C, respectively, determined using a temperature gradient incubator that involved narrow (1.1 degrees C) incremental increases from 5 degrees to 30 degrees C. Activation energies calculated from the Arrhenius plots were 61 and 89 kJ mol(-1) for tetrathionate and 110 kJ mol(-1) for S(0) oxidation. The oxidation of elemental sulfur produced sulfuric acid at 5 degrees C and decreased the pH to approximately 1. The low pH inhibited further oxidation of the substrate. In media with both S(0) and Fe2+, oxidation of elemental sulfur did not commence until all available ferrous iron was oxidized. These data on sequential oxidation of the two substrates are in keeping with upregulation and downregulation of several proteins previously noted in the literature. Ferric iron was reduced to Fe2+ in parallel with elemental sulfur oxidation, indicating the presence of a sulfur:ferric iron reductase system in this bacterium.

  • 171.
    Kurhade, Chaitanya
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lee, Yi-Ping
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Schreier, Sarah
    Zegenhagen, Loreen
    Kröger, Andrea
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Correlation of disease severity in human tick-borne encephalitis and pathogenicity in miceManuscript (preprint) (Other academic)
  • 172. Küchler, Robert
    et al.
    Schröder, Björn
    Jaeger, Simon U
    Stange, Eduard F
    Wehkamp, Jan
    Antimicrobial activity of high-mobility-group box 2: a new function to a well-known protein.2013In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 57, no 10, p. 4782-93Article in journal (Refereed)
    Abstract [en]

    The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against Escherichia coli. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable in vitro in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.

  • 173. Labenski, Verena
    et al.
    Suerth, Julia D
    Barczak, Elke
    Heckl, Dirk
    Levy, Camille
    Bernadin, Ornellie
    Charpentier, Emmanuelle
    Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Williams, David A
    Fehse, Boris
    Verhoeyen, Els
    Schambach, Axel
    Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.2016In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 97, p. 97-109Article in journal (Refereed)
    Abstract [en]

    Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.

  • 174. Langenheder, Silke
    et al.
    Kisand, Veljo
    Lindström, Eva S
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tranvik, Lars J
    Growth dynamics within bacterial communities in riverine and estuarine batch cultures2004In: Aquatic Microbial Ecology, ISSN 0948-3055, E-ISSN 1616-1564, Vol. 37, no 2, p. 137-148Article in journal (Refereed)
    Abstract [en]

    We investigated temporal changes in community composition of bacteria growing on riverine dissolved organic carbon. Batch cultures were adjusted to riverine or estuarine salinity levels and inoculated with bacteria from these 2 environments to test whether growth patterns of bacterial taxa are influenced by salinity and/or the source of the inoculum. Changes in bacterial community composition at different stages of the growth phase were studied by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Furthermore, the growth dynamics of 7 bacteria previously isolated from the estuary were followed by quantitative DNA-DNA hybridization. Growth dynamics within bacterial communities were significantly influenced by the source of the inoculum but not by salinity, suggesting that slight changes in salinity, to which riverine bacteria are exposed when discharged into the Northern Baltic Sea, are not a major regulating factor of community dynamics. Additionally, our results indicated only minor differences in the appearance and growth of bacteria when examined by quantitative DNA-DNA hybridization, whereas DGGE banding patterns suggested that there were fast- and slow-growing types of bacteria.

  • 175. Langenheder, Silke
    et al.
    Kisand, Veljo
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Tranvik, Lars J
    Salinity as a structuring factor for the composition and performance of bacterioplankton degrading riverine DOC2003In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 45, no 2, p. 189-202Article in journal (Refereed)
    Abstract [en]

    The impact of salinity on the composition and functional performance (biomass production, growth efficiency and growth rates) of bacterial communities was investigated using batch cultures growing on dissolved organic carbon from a river draining into the Northern Baltic Sea. The cultures were adjusted to riverine or estuarine salinity levels and inoculated with bacteria from these two environments. Bacterial growth efficiencies differed in response to salinity and the origin of the inoculum. When salinity was adjusted to correspond to the salinity at the site where the inoculum was retrieved, growth efficiency was relatively high (11.5 +/- 2.6%). However, when bacteria were confronted with a shift in salinity, growth efficiency was lower (7.5 +/- 2.0%) and more of the utilized carbon was respired. In contrast, growth rates were higher when bacteria were exposed to a change in salinity. The composition of the bacterial communities developing in the batch cultures differed, as shown by 16S rDNA DGGE, depending on the origin of the inoculum and salinity. Reverse and direct DNA-DNA hybridization revealed salinity optima in the growth of specific bacterial strains as well as broader phylogenetic groups. Strains belonging to the alpha- and beta-Proteobacteria, Actinobacteria and gamma-Proteobacteria other than the genus Pseudomonas showed higher relative abundance under freshwater conditions, whereas strains of the genus Pseudomonas and the Cytophaga-Flavobacterium-Bacteroides group were favored by estuarine conditions. Generally, our results demonstrate functional changes associated with changes in community composition. We suggest that even moderate changes in salinity affect bacterial community composition, which subsequently leads to altered growth characteristics. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

  • 176.
    Le Rhun, Anais
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Lecrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany.
    Reimegard, Johan
    Proux-Wera, Estelle
    Broglia, Laura
    Della Beffa, Cristina
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany; Humboldt University, D-10115 Berlin, Germany.
    Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 5, p. 2329-2340Article in journal (Refereed)
    Abstract [en]

    A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5' and 3' ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3' overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.

  • 177.
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Multifaceted RNA-mediated regulatory mechanisms in Streptococcus pyogenes2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated.

    Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y.

    Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems.

    In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

  • 178.
    Le Rhun, Anaïs
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Beer, Yan Yan
    Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Reimegård, Johan
    Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-75003 Uppsala, Sweden.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Max F. Perutz Laboratories (MFPL), University of Vienna, A-1030 Vienna, Austria.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, Germany; Hannover Medical School (MHH), Hannover, Germany; Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany.
    RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes2016In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 13, no 2, p. 177-195Article in journal (Refereed)
    Abstract [en]

    Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

  • 179.
    Leary, Sophie E. C.
    et al.
    Biomedical Sciences Department, Defence Evaluation and Research Agency, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K..
    Griffin, Kate F.
    Biomedical Sciences Department, Defence Evaluation and Research Agency, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K..
    Galyov, Edouard E.
    Institute for Animal Health, Compton, Nr Newbury, Berkshire RG20 7NN, U.K..
    Hewer, Jason
    Biomedical Sciences Department, Defence Evaluation and Research Agency, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K..
    Williamson, E. Diane
    Biomedical Sciences Department, Defence Evaluation and Research Agency, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K..
    Holmström, Anna
    Department of Microbiology, Defence Research Establishment, S-901 87 Umeå, Sweden.
    Forsberg, Åke Forsberg
    Department of Microbiology, Defence Research Establishment, S-901 87 Umeå, Sweden.
    Titball, Richard W.
    Biomedical Sciences Department, Defence Evaluation and Research Agency, Porton Down, Salisbury, Wiltshire, SP4 0JQ, U.K..
    Yersinia outer proteins (YOPS) E, K and N are antigenic but non-protective compared to V antigen, in a murine model of bubonic plague1999In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 26, no 3, p. 159-169Article in journal (Refereed)
    Abstract [en]

    The pathogenic Yersiniae produce a range of virulence proteins, encoded by a 70 kb plasmid, which are essential for infection, and also form part of a contact-dependent virulence mechanism. One of these proteins, V antigen, has been shown to confer a high level of protection against parenteral infection with Y. pestis in murine models, and is considered to be a protective antigen. In this study, the protective efficacy of V antigen has been compared in the same model with that of other proteins (YopE, YopK and YopN), which are part of the contact-dependent virulence mechanism. Mice immunised with two intraperitoneal doses of V antigen or each of the Yops, administered with either Alhydrogel or interleukin-12, produced high antigen-specific serum IgG titres. As shown in previous studies, V+Alhydrogel was fully protective, and 5/5 mice survived a subcutaneous challenge with 90 or 9x10(3) LD50's of Y. pestis GB. In addition, these preliminary studies also showed that V+IL-12 was partially protective: 4/5 or 3/5 mice survived a challenge with 90 or 9x10(3) LD50's, respectively. In contrast, none of the mice immunised with the Yops survived the challenges, and there was no significant delay in the mean time to death compared to mice receiving a control protein. These results show that using two different vaccine regimens, Yops E, K and N, failed to elicit protective immune responses in a murine model of plague, whereas under the same conditions, V antigen was fully or partially protective.

  • 180. Leganés, Francisco
    et al.
    Blanco-Rivero, Amaya
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid.
    Fernández-Piñas, Francisca
    Redondo, Miguel
    Fernández-Valiente, Eduardo
    Fan, Qing
    Lechno-Yossef, Sigal
    Wolk, C Peter
    Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins2005In: Archives of Microbiology, ISSN 0302-8933, E-ISSN 1432-072X, Vol. 184, no 4, p. 234-248Article in journal (Refereed)
    Abstract [en]

    A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase-transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.

  • 181. Lenman, Annasara
    et al.
    Liaci, A. Manuel
    Frängsmyr, Lars
    Liu, Yan
    Blaum, Bärbel S.
    Podgorski, Iva I.
    Harrach, Balázs
    Benkő, Mária
    Feizi, Ten
    Stehle, Thilo
    Arnberg, Niklas
    Human adenovirus 52 short fiber binds to polysialic acidManuscript (preprint) (Other academic)
  • 182. Levi, Laura
    et al.
    Toyooka, Tatsushi
    Patarroyo, Manuel
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 4, article id e0124119Article in journal (Refereed)
    Abstract [en]

    Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin β1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.

  • 183.
    Li, Yunlong
    et al.
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    Hu, Yangbo
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Chen, Shiyun
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
    RcsB positively regulates the Yersinia Ysc-Yop type III secretion system by activating expression of the master transcriptional regulator LcrF2015In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 17, no 4, p. 1219-1233Article in journal (Refereed)
    Abstract [en]

    The Rcs phosphorelay is a complex signaling pathway used by the family Enterobacteriaceae to sense, respond and adapt to environmental changes during free-living or host-associated lifestyles. In this study, we show that the Rcs phosphorelay pathway positively regulates the virulence plasmid encoded Ysc-Yop type III secretion system (T3SS) in the enteropathogen Yesinia pseudotuberculosis. Both the overexpression of the wild-type Rcs regulator RcsB or the constitutive active RscB(D56E) variant triggered more abundant Ysc-Yop synthesis and secretion, whereas the non-phosphorylatable mutant RcsB(D56Q) negated this. Congruently, enhanced Yops expression and secretion occurred in an in cis rscB(D56E) mutant but not in an isogenic rscB(D56Q) mutant. Screening for regulatory targets of RcsB identified the virG-lcrF operon that encodes for LcrF, the Ysc-Yop T3SS master regulator. Protein-DNA binding assays confirmed that RcsB directly bound to this operon promoter, which subsequently caused stimulated lcrF transcription. Moreover, active RcsB enhanced the ability of bacteria to deliver Yop effectors into immune cells during cell contact, and this promoted an increase in bacterial viability. Taken together, our study demonstrates the role of the Rcs system in regulating the Ysc-Yop T3SS in Yersinia and reports on RcsB being the first transcriptional activator known to directly control lcrF transcription.

  • 184.
    Li, Yunlong
    et al.
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Li, Lamei
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Huang, Li
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hu, Yangbo
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Chen, Shiyun
    Center for Emerging Infectious Diseases, Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan, China.
    Yersinia Ysc-Yop type III secretion feedback inhibition is relieved through YscV-dependent recognition and secretion of LcrQ2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 91, no 3, p. 494-507Article in journal (Refereed)
    Abstract [en]

    Human pathogenic Yersinia species share a virulence plasmid encoding the Ysc-Yop type III secretion system (T3SS). A plasmid-encoded anti-activator, LcrQ, negatively regulates the expression of this secretion system. Under inducible conditions, LcrQ is secreted outside of bacterial cells and this activates the T3SS, but the mechanism of targeting LcrQ for type III secretion remains largely unknown. In this study, we characterized the regulatory role of the export apparatus component YscV. Depletion or overexpression of YscV compromised Yop synthesis and this primarily prevented secretion of LcrQ. It followed that a lcrQ deletion reversed the repressive effects of excessive YscV. Further characterization demonstrated that the YscV residues 493–511 located within the C-terminal soluble cytoplasmic domain directly bound with LcrQ. Critically, YscV-LcrQ complex formation was a requirement for LcrQ secretion, since YscVΔ493–511 failed to secrete LcrQ. This forced a cytoplasmic accumulation of LcrQ, which predictably caused the feedback inhibition of Yops synthesis. Based on these observations, we proposed a model for the YscV-dependent secretion of LcrQ and its role in regulating Yop synthesis in Yersinia.

  • 185.
    Liljeqvist, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Genomics, physiology and applications of cold tolerant acidophiles2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Psychrotolerant acidophiles have gained increasing interest because of their importance in biomining operations in environments where the temperature falls well below 10°C during large parts of the year.

    Acidithiobacillus ferrivorans is the only characterized acidophile with the ability to live a psychrotrophic lifestyle and is able to oxidize ferrous iron and inorganic sulfur compounds at low temperature. The A. ferrivorans SS3 genome sequence mirrors its low temperature chemolithotrophic lifestyle and indicates ecological flexibility. Two enzyme systems for the synthesis of the protective molecule trehalose as well as multiple cold shock proteins suggest its psychrotolerance. In addition to genes coding for ferrous iron and inorganic sulfur compound oxidation enzymes, candidate genes for molecular hydrogen utilization were identified. Also, A. ferrivorans SS3 was suggested to have the ability to switch between nitrogen fixation and nitrogen uptake. Characterization of ferrous iron and inorganic sulfur compound oxidation during low temperature growth showed that both substrates were efficiently oxidized and revealed the potential of using ferric iron as an alternative electron acceptor. Gene transcript analyses also revealed constitutive expression of genes involved in ferrous iron oxidation and their rapid increase in expression when ferrous iron became available. Growth experiments further suggested ferrous iron was preferred over inorganic sulfur compounds during bioleaching. This phenomenon was especially evident during A. ferrivorans-mediated bioleaching of chalcopyrite as sulfur accumulated and eventually inhibited further leaching. A potential way to alleviate this problem is addition of low temperature, obligate inorganic sulfur compound oxidizing acidophiles. Although, these microorganisms have not been identified, analysis of a cold, acidic biofilm from Kristineberg mine suggested additional psychrotolerant inorganic sulfur compound oxidation oxidizers might be present. Psychrotolerant acidophiles from Kristineberg mine have also been demonstrated to remove inorganic sulfur compounds from mining process water at in situ temperatures. This use of indigenous microorganisms for removal of environmental pollutants is a big step towards greener mining.

  • 186.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ossandon, Francisco J.
    Gonzalez, Carolina
    Rajan, Sukithar
    Stell, Adam
    Valdes, Jorge
    Holmes, David S.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Metagenomic analysis reveals adaptations to a cold-adapted lifestyle in a low-temperature acid mine drainage stream2015In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 91, no 4, article id fiv011Article in journal (Refereed)
    Abstract [en]

    An acid mine drainage (pH 2.5-2.7) stream biofilm situated 250 m below ground in the low-temperature (6-10 degrees C) Kristineberg mine, northern Sweden, contained a microbial community equipped for growth at low temperature and acidic pH. Metagenomic sequencing of the biofilm and planktonic fractions identified the most abundant microorganism to be similar to the psychrotolerant acidophile, Acidithiobacillus ferrivorans. In addition, metagenome contigs were most similar to other Acidithiobacillus species, an Acidobacteria-like species, and a Gallionellaceae-like species. Analyses of the metagenomes indicated functional characteristics previously characterized as related to growth at low temperature including cold-shock proteins, several pathways for the production of compatible solutes and an anti-freeze protein. In addition, genes were predicted to encode functions related to pH homeostasis and metal resistance related to growth in the acidic metal-containing mine water. Metagenome analyses identified microorganisms capable of nitrogen fixation and exhibiting a primarily autotrophic lifestyle driven by the oxidation of the ferrous iron and inorganic sulfur compounds contained in the sulfidic mine waters. The study identified a low diversity of abundant microorganisms adapted to a low-temperature acidic environment as well as identifying some of the strategies the microorganisms employ to grow in this extreme environment.

  • 187.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ossandon, Francisco J.
    Gonzalez, Carolina
    Rajan, Sukithar
    Stell, Adam
    Valdes, Jorge
    Holmes, David S.
    Dopson, Mark
    Metagenomic analysis reveals adaptations to a psychrotrophic lifestyle in a low temperature acid mine drainage streamManuscript (preprint) (Other academic)
  • 188.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rzhepishevska, Olena I
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gene Identification and Substrate Regulation Provide Insights into Sulfur Accumulation during Bioleaching with the Psychrotolerant Acidophile Acidithiobacillus ferrivorans2013In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 3, p. 951-957Article in journal (Refereed)
    Abstract [en]

    The psychrotolerant acidophile Acidithiobacillus ferrivorans has been identified from cold environments and has been shown to use ferrous iron and inorganic sulfur compounds as its energy sources. A bioinformatic evaluation presented in this study suggested that Acidithiobacillus ferrivorans utilized a ferrous iron oxidation pathway similar to that of the related species Acidithiobacillus ferrooxidans. However, the inorganic sulfur oxidation pathway was less clear, since the Acidithiobacillus ferrivorans genome contained genes from both Acidithiobacillus ferrooxidans and Acidithiobacillus caldus encoding enzymes whose assigned functions are redundant. Transcriptional analysis revealed that the petA1 and petB1 genes (implicated in ferrous iron oxidation) were downregulated upon growth on the inorganic sulfur compound tetrathionate but were on average 10.5-fold upregulated in the presence of ferrous iron. In contrast, expression of cyoB1 (involved in inorganic sulfur compound oxidation) was decreased 6.6-fold upon growth on ferrous iron alone. Competition assays between ferrous iron and tetrathionate with Acidithiobacillus ferrivorans SS3 precultured on chalcopyrite mineral showed a preference for ferrous iron oxidation over tetrathionate oxidation. Also, pure and mixed cultures of psychrotolerant acidophiles were utilized for the bioleaching of metal sulfide minerals in stirred tank reactors at 5 and 25°C in order to investigate the fate of ferrous iron and inorganic sulfur compounds. Solid sulfur accumulated in bioleaching cultures growing on a chalcopyrite concentrate. Sulfur accumulation halted mineral solubilization, but sulfur was oxidized after metal release had ceased. The data indicated that ferrous iron was preferentially oxidized during growth on chalcopyrite, a finding with important implications for biomining in cold environments.

  • 189.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rzhepishevska, Olena I.
    Dopson, Mark
    Gene identification and substrate regulation provides insights into sulfur accumulation during bioleaching with the psychrotolerant Acidithiobacillus ferrivoransManuscript (preprint) (Other academic)
  • 190.
    Liljeqvist, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Valdes, Jorge
    Holmes, David S
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS32011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 16, p. 4304-4305Article in journal (Refereed)
    Abstract [en]

    Acidithiobacillus ferrivorans SS3 is a psychrotolerant acidophile capable of growth in the range of 5° to 30°C (optimum, ≈25°C). It gains energy from the oxidation of ferrous iron and inorganic sulfur compounds and obtains organic carbon from carbon dioxide. Here, we present the draft genome sequence of A. ferrivorans SS3 that will permit investigation of genes involved in growth in acidic environments at low temperatures.

  • 191. Lin, Po-Chi
    et al.
    Puhar, Andrea
    Biochemisches Institut, Universität Zürich, Zurich, Switzerland, Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris Cedex 15, France.
    Steuber, Julia
    NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica2008In: Archives of Microbiology, ISSN 0302-8933, E-ISSN 1432-072X, Vol. 190, no 4, p. 471-480Article in journal (Refereed)
    Abstract [en]

    It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na+ uptake, indicating that a redox-driven, primary Na+ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 micromol min(-1) mg(-1)) to Na+ translocation (2.0 micromol min(-1) mg(-1)). NADH-driven Na+ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na+ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na+ gradient established by complex I.

  • 192. Lin, Po-Chi
    et al.
    Puhar, Andrea
    Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
    Türk, Karin
    Piligkos, Stergios
    Bill, Eckhard
    Neese, Frank
    Steuber, Julia
    A vertebrate-type ferredoxin domain in the Na+-translocating NADH dehydrogenase from Vibrio cholerae2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 24, p. 22560-22563Article in journal (Refereed)
    Abstract [en]

    The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. Mössbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.

  • 193.
    Lind, Peter A
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Repeatability and predictability in experimental evolution2019In: Evolution, origin of life, concepts and methods / [ed] Pierre Pontarotti, Springer, 2019, p. 57-83Chapter in book (Refereed)
    Abstract [en]

    Independent populations often use the same phenotypic and genetic solutions to adapt to a selective challenge, suggesting that evolution is surprisingly repeatable. This observation has inspired a shift in focus for evolutionary biology towards predictive studies, but progress is impeded by a lack of insight into the causes for repeatability, which prevents tests of forecasting models outside the original biological systems. Experimental evolution with microbes could provide a way to identify the causes of repeated evolution, directly test forecasting ability and develop methodology, but a range of difficulties limits successful prediction. This chapter discusses the limitations on forecasting of experimental evolution, what can and cannot be predicted on different biological levels and why predictions will often fail. Focusing on experimental populations of bacteria, the importance of selection, mutational biases and genotype-to-phenotype maps in determining evolutionary outcomes is discussed, as well as the potential for including these factors in forecasting models. The chapter concludes with a discussion on the desired properties of experimental evolution models suitable for testing forecasting models.

  • 194. Lind, Peter A
    et al.
    Andersson, Dan I
    Fitness costs of synonymous mutations in the rpsT gene can be compensated by restoring mRNA base pairing.2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5Article in journal (Refereed)
    Abstract [en]

    We previously reported that the distribution of fitness effects for non-synonymous and synonymous mutations in Salmonella typhimurium ribosomal proteins S20 and L1 are similar, suggesting that fitness constraints are present at the level of mRNA. Here we explore the hypothesis that synonymous mutations confer their fitness-reducing effect by alterating the secondary structure of the mRNA. To this end, we constructed a set of synonymous substitutions in the rpsT gene, encoding ribosomal protein S20, that are located in predicted paired regions in the mRNA and measured their effect on bacterial fitness. Our results show that for 3/9 cases tested, the reduced fitness conferred by a synonymous mutation could be fully or partly restored by introducing a second synonymous substitution that restore base pairing in a mRNA stem. In addition, random mutations in predicted paired regions had larger fitness effects than those in unpaired regions. Finally, we did not observe any correlation between fitness effects of the synonymous mutations and their rarity. These results suggest that for ribosomal protein S20, the deleterious effects of synonymous mutations are not generally due to codon usage effects, but that mRNA secondary structure is a major fitness constraint.

  • 195. Lind, Peter A
    et al.
    Andersson, Dan I
    Whole-genome mutational biases in bacteria.2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 46Article in journal (Refereed)
    Abstract [en]

    A fundamental biological question is what forces shape the guanine plus cytosine (GC) content of genomes. We studied the specificity and rate of different mutational biases in real time in the bacterium Salmonella typhimurium under conditions of strongly reduced selection and in the absence of the major DNA repair systems involved in repairing common spontaneous mutations caused by oxidized and deaminated DNA bases. The mutational spectrum was determined by whole-genome sequencing of two S. typhimurium mutants that were serially passaged for 5,000 generations. Analysis of 943 identified base pair substitutions showed that 91% were GC-to-TA transversions and 7% were GC-to-AT transitions, commonly associated with 8-oxoG- and deamination-induced damages, respectively. Other types of base pair substitutions constituted the remaining 2% of the mutations. With regard to mutational biases, there was a significant increase in C-to-T transitions on the nontranscribed strand, and for highly expressed genes, C/G-to-T mutations were more common than expected; however, no significant mutational bias with regard to leading and lagging strands of replication or chromosome position were found. These results suggest that, based on the experimentally determined mutational rates and specificities, a bacterial genome lacking the relevant DNA repair systems could, as a consequence of these underlying mutational biases, very rapidly reduce its GC content.

  • 196.
    Lind, Peter A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden.
    Arvidsson, Lars
    Berg, Otto G
    Andersson, Dan I
    Variation in mutational robustness between different proteins and the predictability of fitness effects2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 2, p. 408-418Article in journal (Refereed)
    Abstract [en]

    Random mutations in genes from disparate protein classes may have different distributions of fitness effects (DFEs) depending on different structural, functional and evolutionary constraints. We measured the fitness effects of 156 single mutations in the genes encoding AraC (transcription factor), AraD (enzyme), and AraE (transporter) used for bacterial growth on L- arabinose. Despite their different molecular functions these genes all had bimodal DFEs with most mutations either being neutral or strongly deleterious, providing a general expectation for the DFE. This contrasts with the unimodal DFEs previously obtained for ribosomal protein genes where most mutations were slightly deleterious. Based on theoretical considerations, we suggest that the 33-fold higher average mutational robustness of ribosomal proteins is due to stronger selection for reduced costs of translational and transcriptional errors. While the large majority of synonymous mutations were deleterious for ribosomal proteins genes, no fitness effects could be detected for the AraCDE genes. Four mutations in AraC and AraE increased fitness, suggesting that slightly advantageous mutations make up a significant fraction of the DFE, but that they often escape detection due to the limited sensitivity of commonly used fitness assays. We show that the fitness effects of amino acid substitutions can be predicted based on evolutionary conservation, but that weakly deleterious mutations are less reliably detected. This suggests that large-effect mutations and the fraction of highly deleterious mutations can be computationally predicted, but that experiments are required to characterize the DFE close to neutrality, where many mutations ultimately fixed in a population will occur.

  • 197. Lind, Peter A
    et al.
    Berg, Otto G
    Andersson, Dan I
    Mutational robustness of ribosomal protein genes.2010In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 330, no 6005Article in journal (Refereed)
    Abstract [en]

    The distribution of fitness effects (DFE) of mutations is of fundamental importance for understanding evolutionary dynamics and complex diseases and for conserving threatened species. DFEs estimated from DNA sequences have rarely been subject to direct experimental tests. We used a bacterial system in which the fitness effects of a large number of defined single mutations in two ribosomal proteins were measured with high sensitivity. The obtained DFE appears to be unimodal, where most mutations (120 out of 126) are weakly deleterious and the remaining ones are potentially neutral. The DFEs for synonymous and nonsynonymous substitutions are similar, suggesting that in some genes, strong fitness constraints are present at the level of the messenger RNA.

  • 198.
    Lind, Peter A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Massey Univ Albany, New Zealand Inst Adv Study, Auckland, New Zealand; Massey Univ Albany, Allan Wilson Ctr Mol Ecol & Evolut, Auckland, New Zealand.
    Farr, Andrew D
    New Zealand Institute for Advanced Study and Allan Wilson Centre for Molecular Ecology and Evolution, Massey University.
    Rainey, Paul B
    New Zealand Institute for Advanced Study and Allan Wilson Centre for Molecular Ecology and Evolution, Massey University; Department of Microbial Population Biology, Max Planck Institute for Evolutionary Biology; Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris-Tech), PSL Research University.
    Evolutionary convergence in experimental Pseudomonas populations2017In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 11, no 3, p. 589-600Article in journal (Refereed)
    Abstract [en]

    Model microbial systems provide opportunity to understand the genetic bases of ecological traits, their evolution, regulation and fitness contributions. Experimental populations of Pseudomonas fluorescens rapidly diverge in spatially structured microcosms producing a range of surface-colonising forms. Despite divergent molecular routes, wrinkly spreader (WS) niche specialist types overproduce a cellulosic polymer allowing mat formation at the air–liquid interface and access to oxygen. Given the range of ways by which cells can form mats, such phenotypic parallelism is unexpected. We deleted the cellulose-encoding genes from the ancestral genotype and asked whether this mutant could converge on an alternate phenotypic solution. Two new traits were discovered. The first involved an exopolysaccharide encoded by pgaABCD that functions as cell–cell glue similar to cellulose. The second involved an activator of an amidase (nlpD) that when defective causes cell chaining. Both types form mats, but were less fit in competition with cellulose-based WS types. Surprisingly, diguanylate cyclases linked to cellulose overexpression underpinned evolution of poly-beta-1,6-N-acetyl-d-glucosamine (PGA)-based mats. This prompted genetic analyses of the relationships between the diguanylate cyclases WspR, AwsR and MwsR, and both cellulose and PGA. Our results suggest that c-di-GMP regulatory networks may have been shaped by evolution to accommodate loss and gain of exopolysaccharide modules facilitating adaptation to new environments.

  • 199.
    Lind, Peter A
    et al.
    New Zealand Institute for Advanced Study, Massey, New; Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Auckland.
    Farr, Andrew D
    Rainey, Paul B
    Experimental evolution reveals hidden diversity in evolutionary pathways2015In: eLIFE, E-ISSN 2050-084X, Vol. 4, article id e07074Article in journal (Refereed)
    Abstract [en]

    Replicate populations of natural and experimental organisms often show evidence of parallel genetic evolution, but the causes are unclear. The wrinkly spreader morph of Pseudomonas fluorescens arises repeatedly during experimental evolution. The mutational causes reside exclusively within three pathways. By eliminating these, 13 new mutational pathways were discovered with the newly arising WS types having fitnesses similar to those arising from the commonly passaged routes. Our findings show that parallel genetic evolution is strongly biased by constraints and we reveal the genetic bases. From such knowledge, and in instances where new phenotypes arise via gene activation, we suggest a set of principles: evolution proceeds firstly via pathways subject to negative regulation, then via promoter mutations and gene fusions, and finally via activation by intragenic gain-of-function mutations. These principles inform evolutionary forecasting and have relevance to interpreting the diverse array of mutations associated with clinically identical instances of disease in humans.

  • 200.
    Lind, Peter A
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). New Zealand Institute for Advanced Study, Massey University at Albany, Auckland, New Zealand.
    Libby, Eric
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics. New Zealand Institute for Advanced Study, Massey University at Albany, Auckland, New Zealand ; 3 Santa Fe Institute, New Mexico, United States.
    Herzog, Jenny
    Rainey, Paul B
    Predicting mutational routes to new adaptive phenotypes2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, p. 1-31, article id e38822Article in journal (Refereed)
    Abstract [en]

    Predicting evolutionary change poses numerous challenges. Here we take advantage of the model bacterium Pseudomonas fluorescens in which the genotype-to-phenotype map determining evolution of the adaptive ‘wrinkly spreader’ (WS) type is known. We present mathematical descriptions of three necessary regulatory pathways and use these to predict both the rate at which each mutational route is used and the expected mutational targets. To test predictions, mutation rates and targets were determined for each pathway. Unanticipated mutational hotspots caused experimental observations to depart from predictions but additional data led to refined models. A mismatch was observed between the spectra of WS-causing mutations obtained with and without selection due to low fitness of previously undetected WS-causing mutations. Our findings contribute toward the development of mechanistic models for forecasting evolution, highlight current limitations, and draw attention to challenges in predicting locus-specific mutational biases and fitness effects.

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