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  • 201.
    Garmendia, Eva
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Bueno-Galera, Concepción
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Huseby, Douglas L.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The Selective Advantage of Replichore Balance in Salmonella Typhimurium.Manuskript (preprint) (Övrigt vetenskapligt)
  • 202.
    Garmendia, Eva
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Phenotypic and Genotypic Responses to Relocating a Highly-Expressed Bacterial Operon.Manuskript (preprint) (Övrigt vetenskapligt)
  • 203.
    Garoff, Linnéa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Exploring the Ciprofloxacin Resistome2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    This thesis presents an exploration of the resistance evolution in Escherichia coli towards the antibiotic ciprofloxacin. High level ciprofloxacin resistance is typically acquired by an accumulation of mutations and plasmid borne genes reducing drug target binding, increasing drug efflux, and modifying the drug.

    Paper I describes the finding that novel mutations in tRNA synthetase gene leuS conferred resistance to ciprofloxacin. We also provided evidence for a mechanism, where the leuS mutations induced global changes in transcription that generated a net effect of increased drug efflux.

    In Paper II we observed that the evolutionary trajectory towards high level ciprofloxacin resistance in E. coli is repeatable and predictable in in vitro evolution experiments. However, the types and order of appearance of selected mutations was highly dependent on the bottleneck size used. In addition to the findings in Paper I, we found that mutations involved in transcription and translation were repeatedly selected upon subjection to high concentrations of ciprofloxacin.

    Paper III explored the resistance capacity of the plasmid-borne gene qnr, which reduces ciprofloxacin susceptibility by a target protection mechanism. We found that upon increased expression, the gene qnrS was able to bring E. coli to clinically resistant levels of ciprofloxacin without the addition of other resistance elements.  

    In Paper IV we aimed for a similar study as described above but with another plasmid-borne gene, the inner-membrane efflux pump qepA. However, we ran into the interesting finding of a potentially undescribed regulatory mechanism of qepA expression, which we are currently investigating.

    The work in this thesis presents a new addition of mutations causing ciprofloxacin resistance, and evidence that the dogma of accumulative mutations being a requirement to develop clinical resistance to ciprofloxacin in E. coli can be circumvented. This shows that there is still much to explore, even with a drug used for several decades with an already well documented resistome. We need to learn more about the evolutionary trajectories leading to antibiotic resistance, in order to slow down its development towards existing and future antibiotics to the furthest extent possible.

    Delarbeten
    1. Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli
    Öppna denna publikation i ny flik eller fönster >>Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli
    2018 (Engelska)Ingår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, nr 12, s. 3285-3292Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.

    Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.

    Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.

    Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).

    Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.

    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-361197 (URN)10.1093/jac/dky356 (DOI)000452916600009 ()30239743 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet, 2013-02904Vetenskapsrådet, 2016-04449Vetenskapsrådet, 2017-03593
    Tillgänglig från: 2018-09-21 Skapad: 2018-09-21 Senast uppdaterad: 2019-01-10Bibliografiskt granskad
    2. Evolutionary Trajectories Dependent on Bottleneck Size and a New Class of Genes Selected During the Development of Ciprofloxacin Resistance in Escherichia coli
    Öppna denna publikation i ny flik eller fönster >>Evolutionary Trajectories Dependent on Bottleneck Size and a New Class of Genes Selected During the Development of Ciprofloxacin Resistance in Escherichia coli
    Visa övriga...
    2018 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The evolution of resistance to ciprofloxacin (CIP) in Escherichia coli is strongly associated with the accumulation of multiple chromosomal mutations. Mutations are selected in genes encoding subunits of the target enzymes, and genes encoding direct or indirect regulators of drug efflux. We asked whether and how transmission bottleneck size would affect the evolutionary trajectory of chromosomal mutation accumulation. Independent lineages of E. coli were selected for growth at increasing concentrations of ciprofloxacin up to and above the clinical resistance breakpoint. Evolution experiments were made with three different transmission bottlenecks: single cell, ≈ 3x108, and ≈ 3x1010 cfu. Whole genome sequencing was used to analyse selected clones and populations at different stages during evolution. Under all conditions mutations in gyrA were the first to be selected and to approach or reach fixation. Evolution with the largest population bottleneck selected combinations of mutations similar to those found in resistant clinical isolates (gyrA S83, D87, with parC S80). As predicted by population genetics theory, evolution with a single cell bottleneck resulted in a greater diversity of mutations. Mutations were selected in genes directly regulating drug efflux, and in novel genes involved in transcription and translation, at least some of which are known to indirectly affect drug efflux. Evolution with the intermediate bottleneck, ≈ 3x108, also selected for mutations in a wide variety of genes, similar to the profile associated with the single cell bottleneck. The data suggest that the order of chromosomal mutations accumulated under selection for resistance to ciprofloxacin is highly predictable but the precise evolutionary trajectories differ significantly as a function of transmission bottleneck size.

    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-361198 (URN)
    Tillgänglig från: 2018-09-21 Skapad: 2018-09-21 Senast uppdaterad: 2018-09-21
    3. Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli
    Öppna denna publikation i ny flik eller fönster >>Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli
    2018 (Engelska)Ingår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, nr 2, s. 348-352Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Background: Ciprofloxacin, a fluoroquinolone, targets two essential bacterial enzymes, DNA gyrase and topoisomerase IV. Plasmid-borne qnr genes, encoding proteins that protect DNA gyrase and topoisomerase IV from inhibition by fluoroquinolones, contribute to resistance development. However, the presence of a plasmid-borne qnr gene alone is insufficient to confer clinical resistance. Objectives: We asked whether the level of expression of qnr was a limiting factor in its ability to confer clinical resistance and whether expression could be increased without reducing fitness or viability. Methods: qnrB and qnrS were recombineered onto the chromosome of Escherichia coli under the control of constitutive promoters of various strengths. Expression was measured by qPCR, MIC and relative fitness as a function of expression level were determined. Results: For both qnr genes there was a positive relationship between the level of qnr mRNA and the MIC of ciprofloxacin. The highest MICs achieved with qnrB or qnrS as the sole resistance determinant were 0.375 and 1 mg/L, respectively, and were reached at expression levels that did not affect growth rate or viability. The qnrS-mediated MIC is above the EUCAST clinical breakpoint for resistance to ciprofloxacin. In the absence of Lon protease activity, overexpression of qnr genes was associated with high fitness cost, possibly explaining observations of toxicity in other genetic backgrounds. Conclusions: The ability to generate a high MIC without incurring a fitness cost shows that, in an appropriate genetic context, qnrS has the potential to generate clinical resistance to ciprofloxacin in one step.

    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-361199 (URN)10.1093/jac/dkx375 (DOI)000424144300010 ()29106520 (PubMedID)1460-2091 (Electronic) 0305-7453 (Linking) (ISBN)
    Forskningsfinansiär
    Vetenskapsrådet, 2013-02904Vetenskapsrådet, 2016-04449
    Tillgänglig från: 2018-09-21 Skapad: 2018-09-21 Senast uppdaterad: 2019-06-24Bibliografiskt granskad
    4. The qepA Gene is Dependent on Upstream Sequences to Reduce Susceptibility to Ciprofloxacin
    Öppna denna publikation i ny flik eller fönster >>The qepA Gene is Dependent on Upstream Sequences to Reduce Susceptibility to Ciprofloxacin
    2018 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The plasmid-borne qepA gene encodes a quinolone efflux pump that confers resistance to ciprofloxacin in clinical isolates of Escherichia coli. According to published data qepA is transcribed from a promoter 31 bp upstream of the coding sequence. We asked whether ciprofloxacin MIC associated with qepA in E. coli would vary as a function of mRNA expression level. To our surprise we found that the annotated qepA coding sequence was not sufficient to increase MIC. We decided to investigate the role played by surrounding sequences in determining resistance to ciprofloxacin. The qepA region was engineered onto the E. coli chromosome and mutations were generated to test the significance of surrounding sequences for expression of resistance. MIC was measured by broth microdilution. A 3.2 kb fragment from a resistance plasmid, including the annotated qepA coding sequence (1.5 kb), generated an 8-fold ciprofloxacin MIC increase when recombined into the E. coli chromosome. Deletion analysis revealed that the MIC increase was dependent on sequences upstream of qepA, annotated as ∆int1/groEL and ∆dfr2. Combining sequence analysis and mutagenesis, we identified a promoter within the ∆int1/groEL sequence, required for expression of resistance. The predicted transcript included two open reading frames (orf1 261 bp, orf2 189 bp) upstream of qepA. Deletion analysis revealed the essentiality of orf2 for the MIC increase. In conclusion, we have identified a new promoter for qepA, provided evidence that expression of the qepA coding sequence is not sufficient for resistance, and that resistance is influenced by sequences upstream of the qepA coding sequence. Details of the resistance mechanism remain to be elucidated.

    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-361200 (URN)
    Tillgänglig från: 2018-09-21 Skapad: 2018-09-21 Senast uppdaterad: 2018-09-21
  • 204.
    Garoff, Linnéa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The qepA Gene is Dependent on Upstream Sequences to Reduce Susceptibility to Ciprofloxacin2018Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The plasmid-borne qepA gene encodes a quinolone efflux pump that confers resistance to ciprofloxacin in clinical isolates of Escherichia coli. According to published data qepA is transcribed from a promoter 31 bp upstream of the coding sequence. We asked whether ciprofloxacin MIC associated with qepA in E. coli would vary as a function of mRNA expression level. To our surprise we found that the annotated qepA coding sequence was not sufficient to increase MIC. We decided to investigate the role played by surrounding sequences in determining resistance to ciprofloxacin. The qepA region was engineered onto the E. coli chromosome and mutations were generated to test the significance of surrounding sequences for expression of resistance. MIC was measured by broth microdilution. A 3.2 kb fragment from a resistance plasmid, including the annotated qepA coding sequence (1.5 kb), generated an 8-fold ciprofloxacin MIC increase when recombined into the E. coli chromosome. Deletion analysis revealed that the MIC increase was dependent on sequences upstream of qepA, annotated as ∆int1/groEL and ∆dfr2. Combining sequence analysis and mutagenesis, we identified a promoter within the ∆int1/groEL sequence, required for expression of resistance. The predicted transcript included two open reading frames (orf1 261 bp, orf2 189 bp) upstream of qepA. Deletion analysis revealed the essentiality of orf2 for the MIC increase. In conclusion, we have identified a new promoter for qepA, provided evidence that expression of the qepA coding sequence is not sufficient for resistance, and that resistance is influenced by sequences upstream of the qepA coding sequence. Details of the resistance mechanism remain to be elucidated.

  • 205.
    Garoff, Linnéa
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Huseby, Douglas L
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Praski Alzrigat, Lisa
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli2018Ingår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, nr 12, s. 3285-3292Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.

    Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.

    Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.

    Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).

    Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.

  • 206.
    Garoff, Linnéa
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Yadav, Kavita
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli2018Ingår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, nr 2, s. 348-352Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Ciprofloxacin, a fluoroquinolone, targets two essential bacterial enzymes, DNA gyrase and topoisomerase IV. Plasmid-borne qnr genes, encoding proteins that protect DNA gyrase and topoisomerase IV from inhibition by fluoroquinolones, contribute to resistance development. However, the presence of a plasmid-borne qnr gene alone is insufficient to confer clinical resistance. Objectives: We asked whether the level of expression of qnr was a limiting factor in its ability to confer clinical resistance and whether expression could be increased without reducing fitness or viability. Methods: qnrB and qnrS were recombineered onto the chromosome of Escherichia coli under the control of constitutive promoters of various strengths. Expression was measured by qPCR, MIC and relative fitness as a function of expression level were determined. Results: For both qnr genes there was a positive relationship between the level of qnr mRNA and the MIC of ciprofloxacin. The highest MICs achieved with qnrB or qnrS as the sole resistance determinant were 0.375 and 1 mg/L, respectively, and were reached at expression levels that did not affect growth rate or viability. The qnrS-mediated MIC is above the EUCAST clinical breakpoint for resistance to ciprofloxacin. In the absence of Lon protease activity, overexpression of qnr genes was associated with high fitness cost, possibly explaining observations of toxicity in other genetic backgrounds. Conclusions: The ability to generate a high MIC without incurring a fitness cost shows that, in an appropriate genetic context, qnrS has the potential to generate clinical resistance to ciprofloxacin in one step.

  • 207. Gerecht, A. C.
    et al.
    Šupraha, Luka
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Geovetenskapliga sektionen, Institutionen för geovetenskaper, Paleobiologi.
    Edvardsen, B.
    Langer, G.
    Henderiks, Jorijntje
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Geovetenskapliga sektionen, Institutionen för geovetenskaper, Paleobiologi.
    Phosphorus availability modifies carbon production in Coccolithus pelagicus (Haptophyta)2015Ingår i: Journal of Experimental Marine Biology and Ecology, ISSN 0022-0981, E-ISSN 1879-1697, Vol. 472, s. 24-31Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Abstract The coccolithophore Coccolithus pelagicus (Wallich) Schiller fixes CO2 into particulate organic carbon (POC) through photosynthesis and into particulate inorganic carbon (PIC) in the form of calcite. To examine the role of phosphorus (P) availability in the production of POC and PIC, C. pelagicus subsp. braarudii (Gaarder) Geisen et al. was grown in semi-continuous cultures at three initial phosphate concentrations (P-replete, 1, and 0.5 μM [P]). Reduced P-availability (1 and 0.5 μM [P]) decreased POC production, while PIC production only decreased when phosphate concentrations became growth limiting (0.5 μM [P]). This decrease has not been observed previously in batch cultures, highlighting the inadequacy of the batch culture approach with regard to determining carbon production. The reduction in growth rate by 50% at 0.5 μM [P] was accompanied by a doubling in cell volume (and POC). PIC production was halved, resulting in a lowered PIC to POC ratio. The average number of coccoliths per cell (and PIC content) remained the same among treatments, despite the significant change in cell size. Our data suggest that POC production in C. pelagicus is more sensitive towards a moderate reduction in phosphorus availability than PIC production. Once phosphorus availability limits cell division, however, phosphorus resources are invested into POC rather than PIC production. This reduces cell density and sinking rates, indicating that coccoliths do not act as ballast for reaching deeper nutrient-rich layers under nutrient limitation.

  • 208.
    Giachou, Paraskevi
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Identifying the best antibiotic-target genes in essential biochemical pathways2018Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
  • 209. Gillet, Reynald
    et al.
    Kirsebom, Leif AUppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Mikrobiologi.
    Functional diversity of RNA2007Konferensmeddelanden, proceedings (Refereegranskat)
  • 210. Giraud, A
    et al.
    Radman, M
    Matic, I
    Taddei, F
    The rise and fall of mutator bacteria.2001Ingår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 4, nr 5, s. 582-5Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacteria with elevated mutation rates are frequently found among natural isolates. This is probably because of their ability to generate genetic variability, the substrate for natural selection. However, such high mutation rates can lead to the loss of vital functions. The evolution of bacterial populations may happen through alternating periods of high and low mutation rates. The cost and benefits of high mutation rates in the course of bacterial adaptive evolution are reviewed.

  • 211.
    Giraud, Antoine
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. INSERM, U571, Paris, France; Université Paris Descartes, Faculté de Médecine René Descartes, IFR94, Paris, France; NRA UEPSD, Jouy-en-Josas, France,.
    Arous, Safia
    De Paepe, Marianne
    Gaboriau-Routhiau, Valérie
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Université Paris Descartes, Faculté de Médecine René Descartes, IFR94, Paris, France; INRA UEPSD, Jouy-en-Josas, France; INSERM, U793, Paris, France.
    Bambou, Jean-Christophe
    Rakotobe, Sabine
    Lindner, Ariel B.
    Taddei, Francois
    Cerf-Bensussan, Nadine
    Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut2008Ingår i: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 4, nr 1, artikel-id e2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.

  • 212. Giraud, Antoine
    et al.
    Matic, I
    Tenaillon, O
    Clara, A
    Radman, M
    Fons, M
    Taddei, F
    Costs and benefits of high mutation rates: adaptive evolution of bacteria in the mouse gut.2001Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 291, nr 5513, s. 2606-8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have shown that bacterial mutation rates change during the experimental colonization of the mouse gut. A high mutation rate was initially beneficial because it allowed faster adaptation, but this benefit disappeared once adaptation was achieved. Mutator bacteria accumulated mutations that, although neutral in the mouse gut, are often deleterious in secondary environments. Consistently, the competitiveness of mutator bacteria is reduced during transmission to and re-colonization of similar hosts. The short-term advantages and long-term disadvantages of mutator bacteria could account for their frequency in nature.

  • 213.
    Graells, Tiscar
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Ishak, Helena
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Larsson, Madeleine
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Guy, Lionel
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    The all-intracellular order Legionellales is unexpectedly diverse, globally distributed and lowly abundant.2018Ingår i: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 94, nr 12, artikel-id fiy185Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Legionellales is an order of the Gammaproteobacteria, only composed of host-adapted, intracellular bacteria, including the accidental human pathogens Legionella pneumophila and Coxiella burnetii. Although the diversity in terms of lifestyle is large across the order, only a few genera have been sequenced, owing to the difficulty to grow intracellular bacteria in pure culture. In particular, we know little about their global distribution and abundance.Here, we analyze 16/18S rDNA amplicons both from tens of thousands of published studies and from two separate sampling campaigns in and around ponds and in a silver mine. We demonstrate that the diversity of the order is much larger than previously thought, with over 450 uncultured genera. We show that Legionellales are found in about half of the samples from freshwater, soil and marine environments, and quasi-ubiquitous in man-made environments. Their abundance is low, typically 0.1%, with few samples up to 1%. Most Legionellales OTUs are globally distributed, while many do not belong to a previously identified species.This study sheds a new light on the ubiquity and diversity of one major group of host-adapted bacteria. It also emphasizes the need to use metagenomics to better understand the role of host-adapted bacteria in all environments.

  • 214.
    Graham, Emily B.
    et al.
    Univ Colorado, Inst Arctic & Alpine Res, Boulder, CO 80309 USA.;Pacific NW Natl Lab, Div Biol Sci, Richland, WA 99352 USA..
    Knelman, Joseph E.
    Univ Colorado, Inst Arctic & Alpine Res, Boulder, CO 80309 USA.;Joint Genome Inst, US Dept Energy, Walnut Creek, CA USA..
    Schindlbacher, Andreas
    Bundesforsch & Ausblldungszentrum VVald, Fed Res & Tr 3Thing Ctr Forests, Dept Forest Ecol, Vienna, Austria..
    Siciliano, Steven
    Univ Saskatchewan, Dept Soil Sci, Saskatoon, SK, Canada..
    Breulmann, Marc
    Helmholtz Ctr Environm Res, Ctr Environm Biotechnol, Leipzig, Germany..
    Yannarell, Anthony
    Univ Illinois, Dept Nat Resources & Environm Sci, Urbana, IL USA..
    Bemans, J. M.
    Univ Calif Merced, Life & Environm Sci & Sierra Nevada Res Inst, Merced, CA USA..
    Abell, Guy
    Flinders Univ S Australia, Sch Med, Adelaide, SA 5001, Australia..
    Philippot, Laurent
    Inst Natl Rech Agron Agroecol, Dijon, France..
    Prosser, James
    Univ Aberdeen, Inst Biol & Environm Sci, Aberdeen, Scotland..
    Foulquier, Arnaud
    UR MALY, Irstea, Ctr Lyon Villeurbanne, Villeurbanne, France..
    Yuste, Jorge C.
    CSIC, Museo Nacl Ciencias Nat, Dept Biogeog & Global Change, Madrid, Spain..
    Glanville, Helen C.
    Bangor Univ, Environm Ctr Wales, Gwynedd, England..
    Jones, Davey L.
    Bangor Univ, Environm Ctr Wales, Gwynedd, England..
    Angel, Foey
    Univ Vienna, Dept Microbiol & Ecosyst Sci, Vienna, Austria..
    Salminen, Janne
    Hame Univ Appl Sci, Hameenlinna, Finland..
    Newton, Ryan J.
    Univ Wisconsin, Sch Freshwater Sci, Milwaukee, WI 53201 USA..
    Buergmann, Helmut
    Eawag Swiss Fed Inst Aquat Sci & Technol, Dept Surface Waters, Kastanienbaum, Switzerland..
    Ingram, Lachlan J.
    Univ Sydney, Ctr Carbon Water & Food, Sydney, NSW 2006, Australia..
    Hamer, Ute
    Univ Munster, Inst Landscape Ecol, D-48149 Munster, Germany..
    Siljanen, Henri M. P.
    Univ Eastern Finland, Dept Environm & Biol Sci, Kuopio, Finland..
    Peltoniemi, Krista
    Nat Resources Inst, Vantaa, Finland..
    Potthast, Karin
    Tech Univ Dresden, Inst Soil Sci & Site Ecol, D-01062 Dresden, Germany..
    Baneras, Lluis
    Univ Girona, Fac Ciencies, Inst Aquat Ecol, Girona, Spain..
    Hartmann, Martin
    Inst Sustainabil Sci Agroscope, Zurich, Switzerland..
    Banerjee, Samiran
    CSIRO Agr Flagship, Crace, ACT, Australia..
    Yu, Ri-Qing
    Univ Texas Tyler, Dept Biol, Tyler, TX 75799 USA..
    Nogaro, Geraldine
    EDF R&D, Alat Hydraul & Environm Lab, Chatou, France..
    Richter, Andreas
    Univ Vienna, Dept Microbiol & Ecosyst Sci, Vienna, Austria..
    Koranda, Marianne
    Univ Vienna, Dept Microbiol & Ecosyst Sci, Div Terr Ecosyst Res, Vienna, Austria..
    Castle, Sarah C.
    Univ Montana, Dept Ecosyst & Conservat Sci, Missoula, MT 59812 USA..
    Goberna, Marta
    CSIC, Ctr Invest & Docencia Econ, Valencia, Spain..
    Song, Bongkeun
    Virginia Inst Marine Sci, Dept Biol Sci, Gloucester Point, VA USA..
    Chatterjee, Amitava
    N Dakota State Univ, AES Sch Nat Resources Sci, Fargo, ND 58105 USA..
    Nunes, Olga C.
    Lopes, Ana R.
    Univ Porto, Fac Engn, Lab Proc Engn Environm Biotechnol & Energy, LEPABE, Rua Campo Alegre 823, P-4100 Oporto, Portugal..
    Cao, Yiping
    Southern Calif Coastal Water Res Project Author, Costa Mesa, CA USA..
    Kaisermann, Aurore
    INRA Bordeaux, Interact Sol Plante Atmosphere, UMR, Villenave Dornon, France..
    Hallin, Sara
    Swedish Univ Agr Sci, Dept Forest Mycol & Plant Pathol, Uppsala, Sweden..
    Strickland, Michael S.
    State Univ, Virginia Polytech Inst, Dept Biol Sci, Blacksburg, VA USA..
    Garcia-Pausas, Jordi
    Ctr Tecnol Forestal Catalunya, Solsona, Spain..
    Barba, Josep
    Ctr Recerca Ecol & Aplicac Forestals, Barcelona, Spain..
    Kang, Hojeong
    Yonsei Univ, Sch Civil & Environm Engn, Seoul 120749, South Korea..
    Isobe, Kazuo
    Univ Tokyo, Dept Appl Biol Chem, Tokyo, Japan..
    Papaspyrou, Sokratis
    Univ Cadiz, Dept Biomed Biotechnol & Publ Hlth, Puerto Real, Spain..
    Pastorelli, Roberta
    Res Ctr Agrobiol & Pedol, Florence, Italy..
    Lagomarsino, Alessandra
    Res Ctr Agrobiol & Pedol, Florence, Italy..
    Lindström, Eva S.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi.
    Basiliko, Nathan
    Laurentian Univ, Vale Living Lakes Ctr, Sudbury, ON P3E 2C6, Canada.;Laurentian Univ, Dept Biol, Sudbury, ON P3E 2C6, Canada..
    Nemergut, Diana R.
    Univ Colorado, Inst Arctic & Alpine Res, Boulder, CO 80309 USA.;Duke Univ, Dept Biol, Durham, NC USA..
    Microbes as Engines of Ecosystem Function: When Does Community Structure Enhance Predictions of Ecosystem Processes?2016Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, artikel-id 214Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Microorganisms are vital in mediating the earth's biogeochemical cycles; yet, despite our rapidly increasing ability to explore complex environmental microbial communities, the relationship between microbial community structure and ecosystem processes remains poorly understood. Here, we address a fundamental and unanswered question in microbial ecology: 'When do we need to understand microbial community structure to accurately predict function?' We present a statistical analysis investigating the value of environmental data and microbial community structure independently and in combination for explaining rates of carbon and nitrogen cycling processes within 82 global datasets. Environmental variables were the strongest predictors of process rates but left 44% of variation unexplained on average, suggesting the potential for microbial data to increase model accuracy. Although only 29% of our datasets were significantly improved by adding information on microbial community structure, we observed improvement in models of processes mediated by narrow phylogenetic guilds via functional gene data, and conversely, improvement in models of facultative microbial processes via community diversity metrics. Our results also suggest that microbial diversity can strengthen predictions of respiration rates beyond microbial biomass parameters, as 53% of models were improved by incorporating both sets of predictors compared to 35% by microbial biomass alone. Our analysis represents the first comprehensive analysis of research examining links between microbial community structure and ecosystem function. Taken together, our results indicate that a greater understanding of microbial communities informed by ecological principles may enhance our ability to predict ecosystem process rates relative to assessments based on environmental variables and microbial physiology.

  • 215. Gran Stadniczenko, Sandra
    et al.
    Šupraha, Luka
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Geovetenskapliga sektionen, Institutionen för geovetenskaper, Paleobiologi.
    Egge, Elianne
    Edvardsen, Bente
    Comparing high-throughput sequencing and scanning electron microscopy to investigate haptophyte communities in Oslofjorden (Skagerrak)2016Konferensbidrag (Refereegranskat)
  • 216.
    Grantcharova, Nina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Teknisk-naturvetenskapliga fakulteten, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Developmental Control of Cell Division in Streptomyces coelicolor2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Cell division in the Gram-positive bacterium Streptomyces coelicolor starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of S. coelicolor exploit two types of cell division with strikingly different outcomes depending on the developmental stage.

    The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery.

    By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The ftsZ17(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation.

    An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings.

    The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of ftsZ expression and the turnover of the FtsZ protein.

    S. coelicolor is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like Escherichia coli. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves.

    Delarbeten
    1. Dynamics of FtsZ assembly during sporulation in Streptomyces coelicolor A3(2)
    Öppna denna publikation i ny flik eller fönster >>Dynamics of FtsZ assembly during sporulation in Streptomyces coelicolor A3(2)
    2005 Ingår i: Journal of Bacteriology, Vol. 187, s. 3227-37Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-94044 (URN)
    Tillgänglig från: 2006-02-24 Skapad: 2006-02-24Bibliografiskt granskad
    2. Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor
    Öppna denna publikation i ny flik eller fönster >>Influence of CrgA on assembly of the cell division protein FtsZ during development of Streptomyces coelicolor
    Visa övriga...
    2006 Ingår i: Journal of Bacteriology, Vol. 188, nr 4, s. 1540-50Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-94045 (URN)
    Tillgänglig från: 2006-02-24 Skapad: 2006-02-24Bibliografiskt granskad
    3. A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor
    Öppna denna publikation i ny flik eller fönster >>A missense mutation in ftsZ differentially affects vegetative and developmentally controlled cell division in Streptomyces coelicolor
    Visa övriga...
    2003 Ingår i: Molecular Microbiology, Vol. 47, s. 645-656Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-94046 (URN)
    Tillgänglig från: 2006-02-24 Skapad: 2006-02-24 Senast uppdaterad: 2015-04-02Bibliografiskt granskad
    4. MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores
    Öppna denna publikation i ny flik eller fönster >>MreB of Streptomyces coelicolor is not essential for vegetative growth but is required for the integrity of aerial hyphae and spores
    Visa övriga...
    2006 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, nr 4, s. 838-852Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    MreB forms a cytoskeleton in many rod-shaped bacteria which is involved in cell shape determination and chromosome segregation. PCR-based and Southern analysis of various actinomycetes, supported by analysis of genome sequences, revealed mreB homologues only in genera that form an aerial mycelium and sporulate. We analysed MreB in one such organism, Streptomyces coelicolor. Ectopic overexpression of mreB impaired growth, and caused swellings and lysis of hyphae. A null mutant with apparently normal vegetative growth was generated. However, aerial hyphae of this mutant were swelling and lysing; spores doubled their volume and lost their characteristic resistance to stress conditions. Loss of cell wall consistency was observed in MreB-depleted spores by transmission electron microscopy. An MreB-EGFP fusion was constructed to localize MreB in the mycelium. No clearly localized signal was seen in vegetative mycelium. However, strong fluorescence was observed at the septa of sporulating aerial hyphae, then as bipolar foci in young spores, and finally in a ring- or shell-like pattern inside the spores. Immunogold electron microscopy using MreB-specific antibodies revealed that MreB is located immediately underneath the internal spore wall. Thus, MreB is not essential for vegetative growth of S. coelicolor, but exerts its function in the formation of environmentally stable spores, and appears to primarily influence the assembly of the spore cell wall.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-94047 (URN)10.1111/j.1365-2958.2006.05134.x (DOI)16677297 (PubMedID)
    Tillgänglig från: 2006-02-24 Skapad: 2006-02-24 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
  • 217. Griekspoor, Petra
    et al.
    Engvall, Eva Olsson
    Akerlind, Britt
    Olsen, Björn
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Infektionssjukdomar.
    Waldenstrom, Jonas
    Genetic diversity and host associations in Campylobacter jejuni from human cases and broilers in 2000 and 20082015Ingår i: Veterinary Microbiology, ISSN 0378-1135, E-ISSN 1873-2542, Vol. 178, nr 1-2, s. 94-98Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Campylobacter jejuni is an important food-borne pathogen, with a global distribution. It can colonize numerous host species, including both domestic and wild animals, but is particularly associated with birds (poultry and wild birds). For human campylobacteriosis, poultry products are deemed the most significant risk factor for acquiring infection. We conducted a genotyping and host attribution study of a large representative collection of C jejuni isolated from humans and broilers in Sweden in the years 2000 and 2008. In total 673 broiler and human isolates from 10 different abattoirs and 6 different hospitals were genotyped with multilocus sequence typing. Source attribution analyses confirmed the strong linkage between broiler C jejuni and domestic human cases, but also indicated a significant association to genotypes more commonly found in wild birds. Genotype distributions did not change dramatically between the two study years, suggesting a stable population of infecting bacteria.

  • 218.
    Grubisic, Lorena M.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Bertilsson, Stefan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Eiler, Alexander
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Heinrich, Friederike
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Brutemark, Andreas
    Tvarminne Zool Stn, Hango, Finland.;Novia Univ Appl Sci, ARONIA Coastal Zone Res Team, Ekenas, Finland.;Abo Akad Univ, Ekenas, Finland..
    Alonso-Sáez, Laura
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Andersson, Anders F.
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. KTH Royal Inst Technol & Sci Life Lab, Sch Biotechnol, Stockholm, Sweden.
    Gantner, Stephan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Leibniz Inst Sci & Math Educ, Dept Educ Sci Biol, Kiel, Germany..
    Riemann, Lasse
    Univ Copenhagen, Dept Biol, Marine Biol Sect, Helsingor, Denmark..
    Beier, Sara
    Uppsala universitet, Science for Life Laboratory, SciLifeLab. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Leibniz Inst Balt Sea Res Warnemunde IOW, Rostock, Germany..
    Lake bacterioplankton dynamics over diurnal timescales2017Ingår i: Freshwater Biology, ISSN 0046-5070, E-ISSN 1365-2427, Vol. 62, nr 1, s. 191-204Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    1. Planktonic bacterial community dynamics over short timescales can be of great importance for food webs and ecosystem functioning but are rarely described when microbial community and composition are assessed. To study the significance of such dynamics we sampled the surface water at the deepest point of a mesotrophic lake (Lake Erken, Sweden) every third hour over two days. 2. By combining 454 pyrosequencing of 16S rRNA genes with bromodeoxyuridine immunocapturing of DNA, replicating populations were identified and compared to the community retrieved from total DNA samples. This comparison revealed a significant difference between the actively replicating and total community. 3. The high-frequency diurnal sampling was compared to a year-long survey conducted in the same lake in order to compare the diurnal and seasonal variation in bacterioplankton community composition. At the diurnal-scale, the variation was significantly higher in the replicating than in the total community. However, variation in both active and total diurnal community was significantly lower than the variation in the seasonal total community. 4. Our analysis revealed pronounced short-term dynamics of individual bacterial populations uncoupled from the diurnal light cycle. For example, the proliferating fraction of the most abundant bacterial tribe (LD12) followed a cyclic pattern that covaried with viral abundance. This implies that environmental factors other than light may act as important drivers of microbial community composition, at least in mesotrophic Lake Erken.

  • 219.
    Gräsberg, Sofia
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Recombinant production of the Giardia intestinalis cysteine protease CP10217 in Pichia pastoris2019Självständigt arbete på avancerad nivå (yrkesexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Giardia intestinalis is one of the leading causes of diarrheal diseases, affecting about 280 million people every year. By characterizing the virulence factors of G. intestinalis, new drug targets can be found to treat giardiasis. In addition to the adhesive disc and the variant surface proteins, cysteine proteases are some of the most interesting virulence factors in Giardia. In this project, one of the major secreted cysteine proteases, CP10217, was studied. The intent was to study the structure by modelling and to characterize CP10217 by expressing it in yeast cells and purifying the supernatant by using Immobilized Metal Affinity Chromatography (IMAC). The molecular modelling showed that the Giardia CP can be modelled on the structures of human and Trypanosoma brucei CPs. The process of expressing and purifying CP10217 in this manner proved difficult. The protease seemed to be very active when expressed, probably resulting in self-cleavage into its active form and later digestion of the whole protein, leading to a low protein yield from the purification. Two approaches were tested in order to increase the protein yield. First, expression at different pH ranges, and secondly, by re-cloning CP10217 with an extra 108 bp sequence at the 5’ end. While changes in pH did not seem to affect the yield, sequencing results of the new vector showed that the cloning worked. More work on this new vector is needed to further analyse, and possibly characterize CP10217.

  • 220.
    Gumpert, Heidi
    et al.
    Tech Univ Denmark, Dept Syst Biol, Lyngby, Denmark.;Univ Copenhagen, Hvidovre Hosp, Dept Clin Microbiol, Hvidovre, Denmark..
    Kubicek-Sutherland, Jessica Z.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Porse, Andreas
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Karami, Nahid
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Munck, Christian
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Linkevicius, Marius
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Adlerberth, Ingegerd
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Wold, Agnes E.
    Univ Gothenburg, Sahlgrenska Acad, Inst Biomed, Dept Infect Dis, Gothenburg, Sweden..
    Andersson, Dan I.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Sommer, Morten O. A.
    Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Lyngby, Denmark..
    Transfer and Persistence of a Multi-Drug Resistance Plasmid in situ of the Infant Gut Microbiotain the Absence of Antibiotic Treatment2017Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, artikel-id 1852Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The microbial ecosystem residing in the human gut is believed to play an important role in horizontal exchange of virulence and antibiotic resistance genes that threatens human health. While the diversity of gut-microorganisms and their genetic content has been studied extensively, high-resolution insight into the plasticity, and selective forces shaping individual genomes is scarce. In a longitudinal study, we followed the dynamics of co-existing Escherichia coli lineages in an infant not receiving antibiotics. Using whole genome sequencing, we observed large genomic deletions, bacteriophage infections, as well as the loss and acquisition of plasmids in these lineages during their colonization of the human gut. In particular, we captured the exchange of multidrug resistance genes, and identified a clinically relevant conjugative plasmid mediating the transfer. This resistant transconjugant lineage was maintained for months, demonstrating that antibiotic resistance genes can disseminate and persist in the gut microbiome; even in absence of antibiotic selection. Furthermore, through in vivo competition assays, we suggest that the resistant transconjugant can persist through a fitness advantage in the mouse gut in spite of a fitness cost in vitro. Our findings highlight the dynamic nature of the human gut microbiota and provide the first genomic description of antibiotic resistance gene transfer between bacteria in the unperturbed human gut. These results exemplify that conjugative plasmids, harboring resistance determinants, can transfer and persists in the gut in the absence of antibiotic treatment.

  • 221.
    Guschanski, Katerina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Zooekologi.
    Warnefors, Maria
    Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance.
    Kaessmann, Henrik
    Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance.
    The evolution of duplicate gene expression in mammalian organs2017Ingår i: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 27, nr 9, s. 1461-1474Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gene duplications generate genomic raw material that allows the emergence of novel functions, likely facilitating adaptive evolutionary innovations. However, global assessments of the functional and evolutionary relevance of duplicate genes in mammals were until recently limited by the lack of appropriate comparative data. Here, we report a large-scale study of the expression evolution of DNA-based functional gene duplicates in three major mammalian lineages (placental mammals, marsupials, egg-laying monotremes) and birds, on the basis of RNA sequencing (RNA-seq) data from nine species and eight organs. We observe dynamic changes in tissue expression preference of paralogs with different duplication ages, suggesting differential contribution of paralogs to specific organ functions during vertebrate evolution. Specifically, we show that paralogs that emerged in the common ancestor of bony vertebrates are enriched for genes with brain-specific expression and provide evidence for differential forces underlying the preferential emergence of young testis-and liver-specific expressed genes. Further analyses uncovered that the overall spatial expression profiles of gene families tend to be conserved, with several exceptions of pronounced tissue specificity shifts among lineage-specific gene family expansions. Finally, we trace new lineage-specific genes that may have contributed to the specific biology of mammalian organs, including the little-studied placenta. Overall, our study provides novel and taxonomically broad evidence for the differential contribution of duplicate genes to tissue-specific transcriptomes and for their importance for the phenotypic evolution of vertebrates.

  • 222. Gustafson, Rolf
    et al.
    Jaenson, Thomas G.T.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Zoologiska institutionen. Uppsala universitet, Enheten för musik och museer, Evolutionsmuseet.
    Gardulf, Ann
    Mejlon, Hans
    Uppsala universitet, Enheten för musik och museer, Evolutionsmuseet. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Zoologiska institutionen.
    Svenungsson, Bo
    Prevalence of Borrelia burgdorferi sensu lato infection in Ixodes ricinus in Sweden1995Ingår i: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 27, nr 6, s. 597-601Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Between 1988-1991, a total of 3,141 Ixodes ricinus ticks, 2,740 adults and 401 nymphs, was collected from different localities in 23 of the 25 provinces of Sweden. The ticks were identified, dissected and examined for the presence of Borrelia spirochetes. Indirect immunofluorescence was performed, using an antiserum obtained from rabbits, immunized with sonicated, whole Borrelia burgdorferi spirochetes isolated from Swedish Ixodes ricinus ticks. Borrelia-positive I. ricinus were found in all 23 provinces. The prevalence of infection in adults ranged from 3% in Jämtland to 23% in Södermanland. In nymphs, the infection prevalence ranged from 0% in 9 provinces to 15% in Södermanland. A significantly greater proportion of the adult ticks were found to be positive for Borrelia in the southern and central parts of Sweden as compared to the northern part (Norrland). No significant difference in prevalence could be demonstrated between the western and eastern parts of Sweden. On average, 10% of the nymphs and 15% of the adult I. ricinus were positive for Borrelia. Of 41 non-I. ricinus ticks, none was positive for Borrelia. This study shows that Borrelia burgdorferi sensu lato is present throughout the distributional area of I. ricinus in Sweden. This should lead to increased awareness of signs and symptoms compatible with Lyme borreliosis in persons living in or visiting areas where I. ricinus is present.

  • 223.
    Gustafsson, Ingegerd
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper.
    Biological and Pharmacological Factor that Influence the Selection of Antibiotic Resistance2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Antibiotic treatment causes an ecological disturbance on the human microflora. Four commensal bacteria: E. coli, enterococci, a-streptococci and coagulase-negative staphylococci, from patients with extensive, high antibiotic usage were investigated with regard to resistance pattern and mutation frequency. Among 193 investigated strains it was found that high antibiotic usage selected for resistant bacteria and enriched for bacteria with a small but significantly increased mutation frequency.

    The relative biological fitness cost of resistance in Staphylococcus epidermidis was assessed in a human in vivo model where the indigenous flora was present. In vitro data of the bacterial growth rate correlated well to in vivo fitness assayed in the competition experiments on skin.

    An in vitro kinetic model was shown to be a useful tool to establish the pharmacokinetic and pharmacodynamic (PK/PD) indices for efficacy of antibiotics. It was confirmed that the time, when the concentration exceeds the minimal inhibitory concentration (MIC), correlates with efficacy for b-lactam antibiotics. To achieve maximal killing for penicillin-resistant pneumococci, with an MIC of 2 mg/L, the peak concentration was also of importance.

    Suboptimal dosing regimen facilitates selection of resistance. Penicillin-resistant pneumococci were easily selected in a mixed population with penicillin-sensitive, -intermediate and -resistant pneumococci in an in vitro kinetic model. The selection of the resistant strain was prevented when the benzylpenicillin concentration exceeded the MIC for approximately 50% of 24 h.

    Delarbeten
    1. Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage
    Öppna denna publikation i ny flik eller fönster >>Bacteria with increased mutation frequency and antibiotic resistance are enriched in the commensal flora of patients with high antibiotic usage
    Visa övriga...
    2003 (Engelska)Ingår i: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 52, nr 4, s. 645-650Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    BACKGROUND: We examined how prolonged antibiotic treatment affected the resistance and mutation frequency of human microflora isolated from intestine (Escherichia coli, enterococci spp.), pharynx (alpha-streptococci) and nostril (coagulase-negative staphylococci, CoNS).

    METHODS: Samples were collected from patients at the Center of Cystic Fibrosis (n=18) and the haematology ward (n=18) of the University Hospital, Uppsala, Sweden. The individually used amount of antibiotics for 1 year was recorded as the defined daily dose (DDD). Primary health care patients (n=30), with no antibiotic treatment for 1 year before sampling, were used as controls. Three isolates of each bacterium from each patient were examined. Antibiotic susceptibilities were determined by disc diffusion. Mutation frequencies to rifampicin resistance were measured on 30 independent cultures of each bacterial species from each individual by plating on rifampicin agar plates. For alpha-streptococci the mutation frequency to streptomycin resistance was also determined.

    RESULTS: Isolates from patients with high antibiotic use showed a pronounced shift towards increased resistance and a small but significant increase in the mutation frequency compared with isolates from the controls. For E. coli, enterococci and CoNS the increase in geometric mean mutation frequency in the patient group was 3-, 1.8- and 1.5-fold, respectively (P values 0.0001, 0.016 and 0.012). For alpha-streptococci there was a significant difference in geometric mean mutation frequency between patient and control groups for streptomycin resistance (P=0.024) but not for rifampicin resistance (P=0.74).

    CONCLUSIONS: High antibiotic use selected for commensals with highly increased resistance and a slight increase in mutation frequency.

    Nationell ämneskategori
    Medicin och hälsovetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-92063 (URN)10.1093/jac/dkg427 (DOI)12972454 (PubMedID)
    Tillgänglig från: 2004-09-15 Skapad: 2004-09-15 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
    2. Fitness of antibiotic resistant Staphylococcus epidermidis assessed by competition on skin of human volunteers
    Öppna denna publikation i ny flik eller fönster >>Fitness of antibiotic resistant Staphylococcus epidermidis assessed by competition on skin of human volunteers
    (Engelska)Manuskript (Övrigt vetenskapligt)
    Identifikatorer
    urn:nbn:se:uu:diva-90205 (URN)
    Tillgänglig från: 2003-03-19 Skapad: 2003-03-19 Senast uppdaterad: 2011-06-30Bibliografiskt granskad
    3. Pharmacokinetic and pharmacodynamic parameters for antimicrobial effects of cefotaxime and amoxicillin in an in vitro kinetic model
    Öppna denna publikation i ny flik eller fönster >>Pharmacokinetic and pharmacodynamic parameters for antimicrobial effects of cefotaxime and amoxicillin in an in vitro kinetic model
    2001 Ingår i: Antimicrobial Agents and Chemotherapy, Vol. 45, nr 9, s. 2436-2440Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-90206 (URN)
    Tillgänglig från: 2003-03-19 Skapad: 2003-03-19Bibliografiskt granskad
    4. Suboptimal antibiotic dosage as a risk factor for selection of penicillin-resistant Streptococcus pneumoniae: In vitro kinetic model
    Öppna denna publikation i ny flik eller fönster >>Suboptimal antibiotic dosage as a risk factor for selection of penicillin-resistant Streptococcus pneumoniae: In vitro kinetic model
    2003 Ingår i: Antimicrobial Agents and Chemotherapy, Vol. 47, nr 2, s. 518-523Artikel i tidskrift (Refereegranskat) Published
    Identifikatorer
    urn:nbn:se:uu:diva-90207 (URN)
    Tillgänglig från: 2003-03-19 Skapad: 2003-03-19Bibliografiskt granskad
  • 224.
    Guy, Lionel
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    Spang, Anja
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    Saw, Jimmy Hser Wah
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    Ettema, Thijs J. G.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution.
    'Geoarchaeote NAG1' is a deeply rooting lineage of the archaeal order Thermoproteales rather than a new phylum2014Ingår i: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 8, nr 7, s. 1353-1357Artikel i tidskrift (Övrigt vetenskapligt)
  • 225.
    Hagag, Naglaa M.
    et al.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Erfan, Ahmed M.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    El-Husseiny, Mohamed
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Shalaby, Azhar G.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Saif, Mohamed A.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Tawakol, Maram M.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Nour, Ahmed A.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Selim, Abdullah A.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Arafa, Abdel-Satar
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Hassan, Mohamed K.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Hassan, Wafaa M. M.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Fahmy, Hanan A.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Ibraheem, Essam
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Attia, Mohamed
    Gen Org Vet Serv, Nadi El Said St, Cairo 12619, Dokki Giza, Egypt.
    Abdelhakim, Ali M. M.
    Gen Org Vet Serv, Nadi El Said St, Cairo 12619, Dokki Giza, Egypt.
    Shahein, Momtaz A.
    Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Naguib, Mahmoud
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Anim Hlth Res Inst, Natl Lab Vet Qual Control Poultry Prod, 7 Nadi El Seid St, Cairo 12618, Dokki Giza, Egypt.
    Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt2019Ingår i: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 11, nr 6, artikel-id 565Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

  • 226.
    Hagardson, Karin
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Comparison of DNA isolation methods to detect Leishmania parasites in blood samples2006Självständigt arbete på grundnivå (kandidatexamen), 10 poäng / 15 hpStudentuppsats
    Abstract [en]

    Leishmaniasis is a disease affecting more than 12 million people worldwide. It is caused by the protozoan parasite Leishmania, which is transmitted to humans and dog hosts through bites of infected sand flies belonging to genus Phlebotomine. Several studies have shown Polymerase Chain Reaction (PCR) to be effective for the diagnosis of VL in clinical samples compared to the classical methods. The aims of this study were first to compare four different sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and furthermore to find a method that is sensitive, rapid, cost benefit, simple and easy to perform. Two preparation methods were compared for the isolation of leukocytes (with Ficoll and Tris –EDTA buffer) and two DNA isolation methods (with Proteinase K and QIAgen kit). From the methods that were compared, lysis of erythrocytes with TE and the QIAgen kit seems to be the most suitable to use.

  • 227. Hagemann, Martin
    et al.
    Hasse, Dirk
    University of Rostock, Germany.
    Berg, Gabriele
    Detection of a phage genome carrying a zonula occludens like toxin gene (zot) in clinical isolates of Stenotrophomonas maltophilia.2006Ingår i: Archives of Microbiology, ISSN 0302-8933, E-ISSN 1432-072X, Vol. 185, nr 6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called phiSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage phiLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on phiSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.

  • 228. Hagemann, Martin
    et al.
    Ribbeck-Busch, Kathrin
    Klähn, Stephan
    Hasse, Dirk
    University of Rostock, Germany.
    Steinbruch, Robert
    Berg, Gabriele
    The plant-associated bacterium Stenotrophomonas rhizophila expresses a new enzyme for the synthesis of the compatible solute glucosylglycerol.2008Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, nr 17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.

  • 229. Hagglund, Sara
    et al.
    Hu, Kefei
    Blodorn, Krister
    Makabi-Panzu, Boby
    Gaillard, Anne-Laure
    Ellencrona, Karin
    Chevret, Didier
    Hellman, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Kemisk biologi.
    Bengtsson, Karin Lovgren
    Riffault, Sabine
    Taylor, Geraldine
    Valarcher, Jean Francois
    Eleouet, Jean-Francois
    Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus2014Ingår i: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 21, nr 7, s. 997-1004Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins alpha(V)beta(1), alpha(V)beta(3), and alpha(3)beta(1). The quantity of the major protein F was 4- to 5-fold greater than that of N (similar to 77 mu g versus similar to 17 mu g/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.

  • 230.
    Hammar, Frank
    Uppsala universitet, Medicinska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Development of a DNA-extraction method from cereal samples for PCR-detection and identification of potentially thricothecene-producing Fusarium species.2005Självständigt arbete på avancerad nivå (magisterexamen), 20 poäng / 30 hpStudentuppsats
    Abstract [sv]

    Mögelsvampsinfektioner av spannmålsprodukter är ett av de vanligaste problemen inom mat- och jordbruksindustrierna runt om i världen. Enligt FNs Food and Agriculture Organization beräknas att cirka 25 procent av världens spannmålsgrödor är infekterade med mykotoxinbildande mögelsvampar vilket kan leda till stora hälsorisker för konsumenterna och ekonomiska förluster för mat- och jord-bruksindustrierna. Mykotoxiner är sekundära produkter från svampens ämnesomsättning som troligen har betydelse för svampens överlevnad, men kan ge toxiska effekter hos människa och djur. I Europa är mögelsvampsläktet Fusarium den vanligaste och viktigaste av mykotoxinbildande svampar och producerar de för jordbruksnäringen två viktigaste mykotoxingrupperna trichotechener och fumonisi-ner.

    På grund av den breda förekomsten av dessa Fusarium-svampar finns det idag ett behov av att utveckla snabba och pålitliga metoder för att detektera och identifiera mögelsvamparna redan direkt på de färska spannmålen. Under hösten 2002 startades projektet Bestämning av potentiella mykotoxin-producerande Fusariumsvampar med PCR-metodik vid Mikrobiologiska enheten på Livsmedelsver-ket i Uppsala. Projektet syftar till att utveckla en molekylärbiologisk bestämningsmetod där identifie-ringen av svamparna sker med hjälp av dess DNA istället för via mikroskopiska undersökningar. Det-ta examensarbete har varit en del av det projektet och har i huvudsak inriktats på att utveckla en stabil metod för själva utvinningen av svamp-DNA från de färska spannmålen. De slutsatser som nåtts är att de utvinningsmetoder som examensarbetet omfattade inte kan anses stabila i nuläget utan behöver utvecklas och stabiliseras ytterligare. Vad gäller de molekylärbiologiska metoderna har de kunnat visas stabila även på färskt spannmål.

  • 231.
    Hammarlöf, Disa L.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    EF-Tu and RNase E: Essential and Functionally Connected Proteins2011Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme.

    We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode.

    RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

    Delarbeten
    1. Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
    Öppna denna publikation i ny flik eller fönster >>Mutants of the RNA-processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu
    2008 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, nr 5, s. 1194-1209Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Salmonella enterica with mutant EF-Tu (Gln125Arg) has a low level of EF-Tu, a reduced rate of protein synthesis and an extremely slow growth rate. Eighty independent suppressor mutations were selected that restored normal growth. In some cases (n = 7) suppression was due to mutations in tufA but, surprisingly, in most cases (n = 73) to mutations in rne, the gene coding for RNase E. These rne mutations alone had only modest effects on growth rate. Fifty different suppressor mutations were isolated in rne, all located in or close to the N-terminal endonucleolytic half of RNase E. Steady state levels of several mRNAs were lower in the mutant tuf strain but restored to wild-type levels in the tuf-rne double mutant. In contrast, the half-lives of mRNAs were unaffected by the tuf mutation. We propose a model where the tuf mutation causes the ribosome following RNA polymerase to pause, possibly in a codon-specific manner, exposing unshielded nascent message to RNase E cleavage. Normal growth rate can be restored by increasing EF-Tu activity or by reducing RNase E activity. Accordingly, RNase E is suggested to act at two distinct stages in the life of mRNA: early, on the nascent transcript; late, on the complete mRNA.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:uu:diva-107026 (URN)10.1111/j.1365-2958.2008.06472.x (DOI)000261070300012 ()18990188 (PubMedID)
    Tillgänglig från: 2009-07-15 Skapad: 2009-07-15 Senast uppdaterad: 2017-12-13Bibliografiskt granskad
    2. Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    Öppna denna publikation i ny flik eller fönster >>Reducing ppGpp Level Rescues an Extreme Growth Defect Caused by Mutant EF-Tu
    2014 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 2, s. e90486-Artikel i tidskrift (Övrigt vetenskapligt) Published
    Abstract [en]

    Salmonella enterica grows extremely slowly when it depends on tufA499 (encoding the Gln125Arg mutant form of EF-Tu) to drive protein synthesis. We screened a plasmid library for multi-copy suppressors of the slow growth phenotype and identified spoT as a candidate. The spoT gene encodes a dual function enzyme with both ppGpp synthetase and hydrolase activities. When spoT was cloned behind an arabinose-inducible promoter the growth rate of the mutant strain increased in response to arabinose addition. We found that the slow-growing mutant strain had a relatively high basal level of ppGpp during exponential growth in rich medium. Overexpression of spoT significantly reduced this level of ppGpp suggesting that inappropriately high ppGpp levels might cause the slow growth rate associated with tufA499. We tested this hypothesis by inactivating relA (codes for RelA, a ribosome-associated ppGpp synthetase) in the mutant strain. This inactivation decreased the level of ppGpp in the mutant strain and increased its growth rate. Based on these data we propose that ribosomes depending on tufA499 for their supply of ternary complex (EF-Tu•GTP•aa-tRNA) experience amino acid starvation and that RelA on these starving ribosomes produces an excess of the alarmone ppGpp. This results in a suboptimal partitioning of transcription activity between genes important for fast growth in rich medium and genes important for growth in a poor medium. Accordingly, mutant bacteria growing in a rich medium act physiologically as though they were growing in a nutrient-poor environment. We propose that this generates a vicious circle and contributes to the extreme slow-growth phenotype associated with mutant EF-Tu. Reducing the level of ppGpp increases the growth rate of the mutant because it breaks this circle and reduces the wasteful misdirection of resources in the cell.

    Nyckelord
    tufA; ppGpp; RelA; Salmonella enterica; growth regulation
    Nationell ämneskategori
    Mikrobiologi
    Forskningsämne
    Mikrobiologi; Molekylär cellbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-159663 (URN)10.1371/journal.pone.0090486 (DOI)000332396200210 ()
    Anmärkning

    Jessica M. Bergman and Disa L. Hammarlöf contributed equally to this work.

    Tillgänglig från: 2011-10-06 Skapad: 2011-10-05 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
    3. Temperature-sensitive mutants of RNase E in Salmonella enterica
    Öppna denna publikation i ny flik eller fönster >>Temperature-sensitive mutants of RNase E in Salmonella enterica
    2011 (Engelska)Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6639-6650Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

    Nationell ämneskategori
    Naturvetenskap Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-159661 (URN)10.1128/JB.05868-11 (DOI)000296795600023 ()21949072 (PubMedID)
    Tillgänglig från: 2011-10-05 Skapad: 2011-10-05 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
    4. Extragenic suppressors of RNase E ts mutants
    Öppna denna publikation i ny flik eller fönster >>Extragenic suppressors of RNase E ts mutants
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    RNase E is an essential endoribonuclease and plays a central role in regulating mRNA levels and stable RNA activity in the bacterial cell. Previous studies of RNA half-life and processing in strains carrying rne mutations have shown that it is the catalytic half of RNase E that is essential for bacterial growth, but have not identified a specific reason for this essentiality. In this study we have used two ts mutations in the catalytic region of RNase E (rne-6 and rne-9) from Salmonella as tools to select and screen for extragenic suppressors of the temperature-sensitive phenotype. We reasoned that identifying extragenic suppressors might give information on the essential function of RNase E. 15 independent extragenic suppressors were isolated and mapped to three different loci on the Salmonella chromosome: rpsA (encoding ribosomal protein S1); vacB (encoding RNase R); and within and neighbouring the ORFs STM1551/1550, putatively encoding a toxin-antitoxin system similar to RelBE from E. coli. Each suppressor mutation could cross-suppress the ts phenotypes of rne-6 and rne-9 and each suppressor mutation alone was viable in a wild-type background. We discuss a model where at the non-permissive temperature an excess of mRNA (including defective species) may trap ribosomes non-productively, reducing the rate of protein synthesis and growth. Accordingly the rpsA mutation may suppress the ts phenotype by reducing the rate of translation initiation, and by default increasing the probability that residual RNase E activity turns over mRNA. The vacB mutations may expand the substrate range of RNase R allowing it to more efficiently substitute for poorly active RNase E in degrading mRNA. Finally, the mutations in the STM1551 region may increase the amount of RelE-like toxin and thereby increase the rate of mRNA turnover. This model makes predictions which can be experimentally tested.

    Nyckelord
    rpsA; vacB; RNase R; RelBE; RNase E; mRNA turnover
    Nationell ämneskategori
    Mikrobiologi
    Forskningsämne
    Mikrobiologi; Molekylär cellbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-159662 (URN)
    Tillgänglig från: 2011-10-06 Skapad: 2011-10-05 Senast uppdaterad: 2011-11-10
  • 232.
    Hammarlöf, Disa L
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Bergman, Jessica
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Extragenic suppressors of RNase E ts mutantsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    RNase E is an essential endoribonuclease and plays a central role in regulating mRNA levels and stable RNA activity in the bacterial cell. Previous studies of RNA half-life and processing in strains carrying rne mutations have shown that it is the catalytic half of RNase E that is essential for bacterial growth, but have not identified a specific reason for this essentiality. In this study we have used two ts mutations in the catalytic region of RNase E (rne-6 and rne-9) from Salmonella as tools to select and screen for extragenic suppressors of the temperature-sensitive phenotype. We reasoned that identifying extragenic suppressors might give information on the essential function of RNase E. 15 independent extragenic suppressors were isolated and mapped to three different loci on the Salmonella chromosome: rpsA (encoding ribosomal protein S1); vacB (encoding RNase R); and within and neighbouring the ORFs STM1551/1550, putatively encoding a toxin-antitoxin system similar to RelBE from E. coli. Each suppressor mutation could cross-suppress the ts phenotypes of rne-6 and rne-9 and each suppressor mutation alone was viable in a wild-type background. We discuss a model where at the non-permissive temperature an excess of mRNA (including defective species) may trap ribosomes non-productively, reducing the rate of protein synthesis and growth. Accordingly the rpsA mutation may suppress the ts phenotype by reducing the rate of translation initiation, and by default increasing the probability that residual RNase E activity turns over mRNA. The vacB mutations may expand the substrate range of RNase R allowing it to more efficiently substitute for poorly active RNase E in degrading mRNA. Finally, the mutations in the STM1551 region may increase the amount of RelE-like toxin and thereby increase the rate of mRNA turnover. This model makes predictions which can be experimentally tested.

  • 233.
    Hammarlöf, Disa L.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Liljas, Lars
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
    Hughes, Diarmaid
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Temperature-sensitive mutants of RNase E in Salmonella enterica2011Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, s. 6639-6650Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    RNase E has an important role in mRNA turnover and stable RNA processing although the reason for its essentiality is unknown. We isolated conditional mutants of RNase E to provide genetic tools to probe its essential function. In Salmonella enterica serovar Typhimurium an extreme slow-growth phenotype caused by mutant EF-Tu (Gln125Arg, tufA499) can be rescued by mutants of RNase E that have reduced activity. We exploited this phenotype to select mutations in RNase E and screened these for temperature sensitivity (ts) for growth. Four different ts mutations were identified, all in the N-terminal domain of RNase E: Gly66→Cys; Ile207→Ser; Ile207→Asn; Ala327→Pro. We also selected second-site mutations in RNase E that reversed temperature-sensitivity. The complete set of RNase E mutations (53 primary mutations including the ts mutations, and 23 double mutations) were analyzed for their possible effects on the structure and function of RNase E using the available 3-D structures. Most single mutations were predicted to destabilize the structure while second-site mutations that reversed the ts phenotype were predicted to restore stability to the structure. Three isogenic strain pairs carrying single or double mutations in RNase E (ts, and ts plus second-site mutation) were tested for their effects on the degradation, accumulation and processing of mRNA, rRNA and tRNA. The greatest defect was observed on rne mRNA autoregulation and this correlated with ability to rescue the tufA499-associated slow growth phenotype. This is consistent with the RNase E mutants being defective in initial binding or subsequent cleavage of an mRNA critical for fast growth.

  • 234.
    Hammond, Maria
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Royal Inst Technol KTH, Div Prote & Nanobiotechnol, Sci Life Lab, Stockholm, Sweden..
    Homa, Felix
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Andersson-Svahn, Helene
    Royal Inst Technol KTH, Div Prote & Nanobiotechnol, Sci Life Lab, Stockholm, Sweden..
    Ettema, Thijs J. G.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Joensson, Haakan N.
    Royal Inst Technol KTH, Div Prote & Nanobiotechnol, Sci Life Lab, Stockholm, Sweden..
    Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis2016Ingår i: Microbiome, ISSN 0026-2633, E-ISSN 2049-2618, Vol. 4, artikel-id 52Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Whole genome amplification (WGA) is a challenging, key step in metagenomic studies of samples containing minute amounts of DNA, such as samples from low biomass environments. It is well known that multiple displacement amplification (MDA), the most commonly used WGA method for microbial samples, skews the genomic representation in the sample. We have combined MDA with droplet microfluidics to perform the reaction in a homogeneous emulsion. Each droplet in this emulsion can be considered an individual reaction chamber, allowing partitioning of the MDA reaction into millions of parallel reactions with only one or very few template molecules per droplet. Results: As a proof-of-concept, we amplified genomic DNA from a synthetic metagenome by MDA either in one bulk reaction or in emulsion and found that after sequencing, the species distribution was better preserved and the coverage depth was more evenly distributed across the genomes when the MDA reaction had been performed in emulsion. Conclusions: Partitioning MDA reactions into millions of reactions by droplet microfluidics is a straightforward way to improve the uniformity of MDA reactions for amplifying complex samples with limited amounts of DNA.

  • 235.
    Harding-Esch, Emma M.
    et al.
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Holland, Martin J.
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England;MRC Labs, POB 273, Fajara, Banjul, Gambia.
    Schemann, Jean-Francois
    IRD, Dakar, Senegal.
    Sillah, Ansumana
    Minist Hlth & Social Welf, Natl Eye Hlth Programme, Kanifing, Gambia.
    Sarr, Boubacar
    Minist Sante, Programme Natl Lutte Cecite, BP 3817, Dakar, Senegal.
    Christerson, Linus
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi.
    Pickering, Harry
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Molina-Gonzalez, Sandra
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Sarr, Isatou
    MRC Labs, POB 273, Fajara, Banjul, Gambia.
    Andreasen, Aura A.
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Jeffries, David
    MRC Labs, POB 273, Fajara, Banjul, Gambia.
    Grundy, Chris
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Mabey, David C. W.
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Herrmann, Björn
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi.
    Bailey, Robin L.
    London Sch Hyg & Trop Med, Keppel St, London WC1E 7HT, England.
    Impact of a single round of mass drug administration with azithromycin on active trachoma and ocular Chlamydia trachomatis prevalence and circulating strains in The Gambia and Senegal2019Ingår i: Parasites & Vectors, ISSN 1756-3305, E-ISSN 1756-3305, Vol. 12, artikel-id 497Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Mass drug administration (MDA) with azithromycin is a cornerstone of the trachoma elimination strategy. Although the global prevalence of active trachoma has declined considerably, prevalence persists or even increases in some communities and districts. To increase understanding of MDA impact, we investigated the prevalence of active trachoma and ocular C. trachomatis prevalence, organism load, and circulating strains at baseline and one-year post-MDA in The Gambia and Senegal.

    Methods: Pre- and one-year post-MDA, children aged 0-9 years were examined for clinical signs of trachoma in six Gambian and 12 Senegalese villages. Ocular swabs from each child's right conjunctiva were tested for evidence of ocular C. trachomatis infection and organism load (ompA copy number), and ompA and multi-locus sequence typing (MLST) was performed.

    Results: A total of 1171 children were examined at baseline and follow-up in The Gambia. Active trachoma prevalence decreased from 23.9% to 17.7%, whereas ocular C. trachomatis prevalence increased from 3.0% to 3.8%. In Senegal, 1613 and 1771 children were examined at baseline and follow-up, respectively. Active trachoma prevalence decreased from 14.9% to 8.0%, whereas ocular C. trachomatis prevalence increased from 1.8% to 3.6%. Higher organism load was associated with having active trachoma and severe inflammation. Sequence typing demonstrated that all Senegalese samples were genovar A, whereas Gambian samples were a mix of genovars A and B. MLST provided evidence of clustering at village and household levels and demonstrated differences of strain variant frequencies in Senegal, indicative of an "outbreak". MLST, including partial ompA typing, provided greater discriminatory power than complete ompA typing.

    Conclusions: We found that one round of MDA led to an overall decline in active trachoma prevalence but no impact on ocular C. trachomatis infection, with heterogeneity observed between villages studied. This could not be explained by MDA coverage or number of different circulating strains pre- and post-MDA. The poor correlation between active trachoma and infection prevalence supports the need for further work on alternative indicators to clinical signs for diagnosing ocular C. trachomatis infection. MLST typing has potential molecular epidemiology utility, including better understanding of transmission dynamics, although relationship to whole-genome sequence variability requires further exploration.

  • 236.
    Hasse, Dirk
    et al.
    University of Rostock, Germany.
    Mikkat, Stefan
    Thrun, Hans-Albrecht
    Hagemann, Martin
    Bauwe, Hermann
    Properties of recombinant glycine decarboxylase P- and H-protein subunits from the cyanobacterium Synechocystis sp. strain PCC 6803.2007Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, nr 7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The multi-enzyme complex glycine decarboxylase is important for one-carbon metabolism, essential for the photorespiratory glycolate cycle of plants, and comprises four different polypeptides, P-, H-, T-, and L-protein. We report on the production and properties of recombinant P-protein from the cyanobacterium Synechocystis and also describe features of recombinant H-protein from the same organism. The P-protein shows enzymatic activity with lipoylated H-protein and very low activity with H-apoprotein or lipoate as artificial cofactors. Its affinity towards glycine is unaffected by the presence and nature of the methyleneamine acceptor molecule. The cyanobacterial H-protein apparently forms stable dimers.

  • 237.
    Hayward, Alexander
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Cornwallis, Charlie K.
    Jern, Patric
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Pan-vertebrate comparative genomics unmasks retrovirus macroevolution2015Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, nr 2, s. 464-469Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Although extensive research has demonstrated host-retrovirus microevolutionary dynamics, it has been difficult to gain a deeper understanding of the macroevolutionary patterns of host-retrovirus interactions. Here we use recent technological advances to infer broad patterns in retroviral diversity, evolution, and host-virus relationships by using a large-scale phylogenomic approach using endogenous retroviruses (ERVs). Retroviruses insert a proviral DNA copy into the host cell genome to produce new viruses. ERVs are provirus insertions in germline cells that are inherited down the host lineage and consequently present a record of past host-viral associations. By mining ERVs from 65 host genomes sampled across vertebrate diversity, we uncover a great diversity of ERVs, indicating that retroviral sequences are much more prevalent and widespread across vertebrates than previously appreciated. The majority of ERV clades that we recover do not contain known retroviruses, implying either that retroviral lineages are highly transient over evolutionary time or that a considerable number of retroviruses remain to be identified. By characterizing the distribution of ERVs, we show that no major vertebrate lineage has escaped retroviral activity and that retroviruses are extreme host generalists, having an unprecedented ability for rampant host switching among distantly related vertebrates. In addition, we examine whether the distribution of ERVs can be explained by host factors predicted to influence viral transmission and find that internal fertilization has a pronounced effect on retroviral colonization of host genomes. By capturing the mode and pattern of retroviral evolution and contrasting ERV diversity with known retroviral diversity, our study provides a cohesive framework to understand host-virus coevolution better.

  • 238. He, Lin
    et al.
    Söderbom, Fredrik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart
    Binnie, Uta
    Binns, Nigel
    Masters, Millicent
    PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids1993Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 9, nr 6, s. 1131-1142Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

  • 239.
    He, Shaomei
    et al.
    Univ Wisconsin, Dept Bacteriol, Madison, WI 53706 USA.;Univ Wisconsin, Dept Geosci, Madison, WI USA..
    Stevens, Sarah L. R.
    Univ Wisconsin, Dept Bacteriol, Madison, WI 53706 USA..
    Chan, Leong-Keat
    DOE Joint Genome Inst, Walnut Creek, CA USA..
    Bertilsson, Stefan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    del Rio, Tijana Glavina
    DOE Joint Genome Inst, Walnut Creek, CA USA..
    Tringe, Susannah G.
    DOE Joint Genome Inst, Walnut Creek, CA USA..
    Malmstrom, Rex R.
    DOE Joint Genome Inst, Walnut Creek, CA USA..
    McMahon, Katherine D.
    Univ Wisconsin, Dept Bacteriol, Madison, WI 53706 USA.;Univ Wisconsin, Dept Civil & Environm Engn, Madison, WI 53706 USA..
    Ecophysiology of Freshwater Verrucomicrobia Inferred from Metagenome-Assembled Genomes2017Ingår i: MSPHERE, ISSN 2379-5042, Vol. 2, nr 5, artikel-id e00277-17Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Microbes are critical in carbon and nutrient cycling in freshwater ecosystems. Members of the Verrucomicrobia are ubiquitous in such systems, and yet their roles and ecophysiology are not well understood. In this study, we recovered 19 Verrucomicrobia draft genomes by sequencing 184 time-series metagenomes from a eutrophic lake and a humic bog that differ in carbon source and nutrient availabilities. These genomes span four of the seven previously defined Verrucomicrobia subdivisions and greatly expand knowledge of the genomic diversity of freshwater Verrucomicrobia. Genome analysis revealed their potential role as (poly) saccharide degraders in freshwater, uncovered interesting genomic features for this lifestyle, and suggested their adaptation to nutrient availabilities in their environments. Verrucomicrobia populations differ significantly between the two lakes in glycoside hydrolase gene abundance and functional profiles, reflecting the autochthonous and terrestrially derived allochthonous carbon sources of the two ecosystems, respectively. Interestingly, a number of genomes recovered from the bog contained gene clusters that potentially encode a novel porin-multiheme cytochrome c complex and might be involved in extracellular electron transfer in the anoxic humus-rich environment. Notably, most epilimnion genomes have large numbers of so-called "Planctomycete-specific" cytochrome c-encoding genes, which exhibited distribution patterns nearly opposite to those seen with glycoside hydrolase genes, probably associated with the different levels of environmental oxygen availability and carbohydrate complexity between lakes/layers. Overall, the recovered genomes represent a major step toward understanding the role, ecophysiology, and distribution of Verrucomicrobia in freshwater. IMPORTANCE Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly) saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called " Planctomycete-specific" cytochrome c-encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia, while highlighting their potential role in freshwater carbon cycling.

  • 240.
    Hechard, Tifaine
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinsk biokemi och mikrobiologi.
    Barriers to Dissemination of Antibiotic Resistance Genes from Wastewater2017Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Antibiotic resistance genes can spread between environments and eventually reach pathogenic bacteria through horizontal gene transfer. Wastewater treatment plants (WWTPs) receive water from various sources and contain a mixture of human pathogens and antibiotic waste. Therefore, WWTPs are believed to be a major source of antibiotic resistant gene dissemination. However, it has been demonstrated that even if resistance genes are abundant in WWTPs, only a few are disseminated to other environments. In this study, we expressed 58 genes from a WWTP in Denmark. The genes are known to give resistance towards different antibiotics in Escherichia coli. The level of resistance to antibiotics as well as the fitness cost was analysed to evaluate if these factors could constitute barriers to gene dissemination. We found that both the level of resistance and the fitness cost conferred by the antibiotic resistance WWTP genes were highly variable. Moreover, an increased level of resistance was not necessarily correlated with a fitness cost. This study could not conclude that the level of resistance or the fitness cost could explain the barrier to dissemination of these genes. 

  • 241.
    Hedman, Asa K
    et al.
    Imperial College London, UK.
    Li, Ming-Shi
    Langford, Paul R
    Kroll, J Simon
    Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 6, s. e39718-Artikel i tidskrift (Refereegranskat)
  • 242. Heidorn, Thorsten
    et al.
    Camsund, Daniel
    Huang, Hsin-Ho
    Lindberg, Pia
    Oliveira, Paulo
    Stensjö, Karin
    Lindblad, Peter
    Synthetic biology in cyanobacteria: engineering and analyzing novel functions2011Ingår i: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988Artikel i tidskrift (Refereegranskat)
  • 243.
    Heilmann, Beate
    et al.
    Heinrich Heine Univ Dusseldorf, Cluster Excellence Plant Sci CEPLAS, Inst Synthet Microbiol, Univ Str 1, D-40225 Dusseldorf, Germany.;Beuth Univ Appl Sci, Dept Biotechnol, Seestr 64, D-13347 Berlin, Germany..
    Hakkila, Kaisa
    Univ Turku, Dept Biochem, FI-20014 Turku, Finland..
    Georg, Jens
    Univ Freiburg, Fac Biol, Genet & Expt Bioinformat, Schanzlestr 1, D-79104 Freiburg, Germany..
    Tyystjarvi, Taina
    Univ Turku, Dept Biochem, FI-20014 Turku, Finland..
    Hess, Wolfgang R.
    Univ Freiburg, Fac Biol, Genet & Expt Bioinformat, Schanzlestr 1, D-79104 Freiburg, Germany..
    Axmann, Ilka M.
    Heinrich Heine Univ Dusseldorf, Cluster Excellence Plant Sci CEPLAS, Inst Synthet Microbiol, Univ Str 1, D-40225 Dusseldorf, Germany..
    Dienst, Dennis
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik. Heinrich Heine Univ Dusseldorf, Cluster Excellence Plant Sci CEPLAS, Inst Synthet Microbiol, Univ Str 1, D-40225 Dusseldorf, Germany..
    6S RNA plays a role in recovery from nitrogen depletion in Synechocystis sp PCC 68032017Ingår i: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 17, artikel-id 229Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: The 6S RNA is a global transcriptional riboregulator, which is exceptionally widespread among most bacterial phyla. While its role is well-characterized in some heterotrophic bacteria, we subjected a cyanobacterial homolog to functional analysis, thereby extending the scope of 6S RNA action to the special challenges of photoautotrophic lifestyles.

    Results: Physiological characterization of a 6S RNA deletion strain (Delta ssaA) demonstrates a delay in the recovery from nitrogen starvation. Significantly decelerated phycobilisome reassembly and glycogen degradation are accompanied with reduced photosynthetic activity compared to the wild type. Transcriptome profiling further revealed that predominantly genes encoding photosystem components, ATP synthase, phycobilisomes and ribosomal proteins were negatively affected in Delta ssaA. In vivo pull-down studies of the RNA polymerase complex indicated that the presence of 6S RNA promotes the recruitment of the cyanobacterial housekeeping s factor SigA, concurrently supporting dissociation of group 2 s factors during recovery from nitrogen starvation.

    Conclusions: The combination of genetic, physiological and biochemical studies reveals the homologue of 6S RNA as an integral part of the cellular response of Synechocystis sp. PCC 6803 to changing nitrogen availability. According to these results, 6S RNA supports a rapid acclimation to changing nitrogen supply by accelerating the switch from group 2 s factors SigB, SigC and SigE to SigA-dependent transcription. We therefore introduce the cyanobacterial 6S RNA as a novel candidate regulator of RNA polymerase sigma factor recruitment in Synechocystis sp. PCC 6803. Further studies on mechanistic features of the postulated interaction should shed additional light on the complexity of transcriptional regulation in cyanobacteria.

  • 244.
    Heinrich, Kristina
    et al.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Sci Life Lab, Stockholm, Sweden.
    Leslie, David J.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Sci Life Lab, Stockholm, Sweden.
    Morlock, Michaela
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Sci Life Lab, Stockholm, Sweden.
    Bertilsson, Stefan
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi. Uppsala universitet, Science for Life Laboratory, SciLifeLab.
    Jonas, Kristina
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Sci Life Lab, Stockholm, Sweden.
    Molecular Basis and Ecological Relevance of Caulobacter Cell Filamentation in Freshwater Habitats2019Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, nr 4, artikel-id e01557-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    All living cells are characterized by certain cell shapes and sizes. Many bacteria can change these properties depending on the growth conditions. The underlying mechanisms and the ecological relevance of changing cell shape and size remain unclear in most cases. One bacterium that undergoes extensive shape-shifting in response to changing growth conditions is the freshwater bacterium Caulobacter crescentus. When incubated for an extended time in stationary phase, a subpopulation of C. crescentus forms viable filamentous cells with a helical shape. Here, we demonstrated that this stationary-phase-induced filamentation results from downregulation of most critical cell cycle regulators and a consequent block of DNA replication and cell division while cell growth and metabolism continue. Our data indicate that this response is triggered by a combination of three stresses caused by prolonged growth in complex medium, namely, the depletion of phosphate, alkaline pH, and an excess of ammonium. We found that these conditions are experienced in the summer months during algal blooms near the surface in freshwater lakes, a natural habitat of C. crescentus, suggesting that filamentous growth is a common response of C. crescentus to its environment. Finally, we demonstrate that when grown in a biofilm, the filamentous cells can reach beyond the surface of the biofilm and potentially access nutrients or release progeny. Altogether, our work highlights the ability of bacteria to alter their morphology and suggests how this behavior might enable adaptation to changing environments.

    IMPORTANCE Many bacteria drastically change their cell size and morphology in response to changing environmental conditions. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus and related species transform into filamentous cells in response to conditions that commonly occur in their natural habitat as a result of algal blooms during the warm summer months. These filamentous cells may be better able to scavenge nutrients when they grow in biofilms and to escape from protist predation during planktonic growth. Our findings suggest that seasonal changes and variations in the microbial composition of the natural habitat can have profound impact on the cell biology of individual organisms. Furthermore, our work highlights that bacteria exist in morphological and physiological states in nature that can strongly differ from those commonly studied in the laboratory.

  • 245. Heldtander Königsson, Malin
    et al.
    Ballagi, Andras
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för ytbioteknik med Centrum för ytbioteknik. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för fysikalisk och analytisk kemi.
    Jansson, Eva
    Johansson, Karl-Erik
    Detection of Renibacterium salmoninarum in tissue samples by sequence capture and fluorescent PCR based on the 16S rRNA gene2005Ingår i: Veterinary Microbiology, Vol. 105, nr 3-4, s. 235-243Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The 16S rRNA genes from eight isolates of Renibacterium salmoninarum with different origins and dates of isolation were

    sequenced to evaluate the possibility to construct a diagnostic PCR system with target sites within this gene. The sequences were

    found to be identical but for one single position in one of the isolates, and two regions with an adequate number of nucleotide

    differences as compared to closely related species were identified. Species-specific fluorescent PCR primers complementary to

    these regions were constructed as well as oligonucleotides for DNA preparation by sequence capture. A mimic molecule was

    constructed to be used as an internal control. The PCR was specific and allowed the detection of DNA equivalent to 1–10 R.

    salmoninarum genomes per reaction. The DNA preparation with sequence capture and analysis by PCR with a mimic was found

    to be a reliable method for analysis of kidneys from fish with BKD. The amount of PCR inhibiting substances present in the

    tissue was reduced, and the relevant DNAwas concentrated in the capture step. Furthermore, the use of the mimic molecule in

    the system assured that false negative results could be identified.

  • 246.
    Hellman, Lars T.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Akula, Srinivas
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Thorpe, Michael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fu, Zhirong
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Tracing the Origins of IgE, Mast Cells, and Allergies by Studies of Wild Animals2017Ingår i: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, artikel-id 1749Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In most industrialized countries, allergies have increased in frequency quite dramatically during the past 50 years. Estimates show that 20-30% of the populations are affected. Allergies have thereby become one of the major medical challenges of the twenty-first century. Despite several theories including the hygiene hypothesis, there are still very few solid clues concerning the causes of this increase. To trace the origins of allergies, we have studied cells and molecules of importance for the development of IgE-mediated allergies, including the repertoire of immunoglobulin genes. These studies have shown that IgE and IgG most likely appeared by a gene duplication of IgY in an early mammal, possibly 220-300 million years ago. Receptors specific for IgE and IgG subsequently appeared in parallel with the increase in Ig isotypes from a subfamily of the recently identified Fc receptor-like molecules. Circulating IgE levels are generally very low in humans and laboratory rodents. However, when dogs and Scandinavian wolfs were analyzed, IgE levels were found to be 100-200 times higher compared to humans, indicating a generally much more active IgE synthesis in free-living animals, most likely connected to intestinal parasite infections. One of the major effector molecules released upon IgEmediated activation by mast cells are serine proteases. These proteases, which belong to the large family of hematopoietic serine proteases, are extremely abundant and can account for up to 35% of the total cellular protein. Recent studies show that several of these enzymes, including the chymases and tryptases, are old. Ancestors for these enzymes were most likely present in an early mammal more than 200 million years ago before the separation of the three extant mammalian lineages; monotremes, marsupials, and placental mammals. The aim is now to continue these studies of mast cell biology and IgE to obtain additional clues to their evolutionary conserved functions. A focus concerns why the humoral immune response involving IgE and mast cells have become so dysregulated in humans as well as several of our domestic companion animals.

  • 247. Henche, Anna-Lena
    et al.
    Ghosh, Abhrajyoti
    Yu, Xiong
    Jeske, Torsten
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för ekologi och genetik, Limnologi.
    Egelman, Edward
    Albers, Sonja-Verena
    Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius2012Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 14, nr 12, s. 3188-3202Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (archaeal adhesive pilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross-talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ∼ 138° and a rise per subunit of ∼ 5.7 Å. The filaments have a diameter of ∼ 110 Å and the resolution was judged to be ∼ 9 Å. We concluded that small changes in sequence might be amplified by large changes in higher-order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.

  • 248.
    Herrmann, Björn
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin, Klinisk bakteriologi.
    Isaksson, Jenny
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin, Klinisk bakteriologi.
    Ryberg, Martin
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Systematisk biologi.
    Tangrot, Jeanette
    Saleh, Isam
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin, Klinisk bakteriologi.
    Versteeg, Bart
    Gravningen, Kirsten
    Bruisten, Sylvia
    Global Multilocus Sequence Type Analysis of Chlamydia trachomatis Strains from 16 Countries2015Ingår i: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 53, nr 7, s. 2172-2179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Uppsala University Chlamydia trachomatis multilocus sequence type (MLST) database (http://mlstdb.bmc.uu.se) is based on five target regions (non-housekeeping genes) and the ompA gene. Each target has various numbers of alleles-hctB, 89; CT058, 51; CT144, 30; CT172, 38; and pbpB, 35-derived from 13 studies. Our aims were to perform an overall analysis of all C. trachomatis MLST sequence types (STs) in the database, examine STs with global spread, and evaluate the phylogenetic capability by using the five targets. A total of 415 STs were recognized from 2,089 specimens. The addition of 49 ompA gene variants created 459 profiles. ST variation and their geographical distribution were characterized using eBURST and minimum spanning tree analyses. There were 609 samples from men having sex with men (MSM), with 4 predominating STs detected in this group, comprising 63% of MSM cases. Four other STs predominated among 1,383 heterosexual cases comprising, 31% of this group. The diversity index in ocular trachoma cases was significantly lower than in sexually transmitted chlamydia infections. Predominating STs were identified in 12 available C. trachomatis whole genomes which were compared to 22 C. trachomatis full genomes without predominating STs. No specific gene in the 12 genomes with predominating STs could be linked to successful spread of certain STs. Phylogenetic analysis showed that MLST targets provide a tree similar to trees based on whole-genome analysis. The presented MLST scheme identified C. trachomatis strains with global spread. It provides a tool for epidemiological investigations and is useful for phylogenetic analyses.

  • 249.
    Herrmann, Björn
    et al.
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin.
    Stolt, Pelle
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Abdeldaim, Guma
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin.
    Rubin, Carl-Johan
    Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för medicinska vetenskaper, Klinisk mikrobiologi och infektionsmedicin.
    Kirsebom, Leif A.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Thollesson, Mikael
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för organismbiologi, Systematisk biologi.
    Differentiation and Phylogenetic Relationships in Mycobacterium spp with Special Reference to the RNase P RNA Gene rnpB2014Ingår i: Current Microbiology, ISSN 0343-8651, E-ISSN 1432-0991, Vol. 69, nr 5, s. 634-639Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The rnpB gene encodes for the RNA subunit of the catalytic ribonuclease RNase P and is present in all bacteria and has both conserved and highly variable sequence regions. Determination of rnpB in 35 Mycobacterium spp. showed species specific sequences for all species except the Mycobacterium tuberculosis complex (four species). High sequence variation was seen in the P3, P15 and P19 regions of suggested secondary structures of the corresponding RNase P RNA molecules. Phylogenetic analysis showed that rnpB gave similar tree topologies as 16S rRNA and hsp65 genes. A combined analysis of the three genes increased the number of nodes with significant support from 10 to 19. The results indicate that rnpB is useful for phylogenetic studies and is a possible target for identification and detection of Mycobacterium spp.

  • 250.
    Hess, Sebastian
    et al.
    Dalhousie Univ, Dept Biochem & Mol Biol, Ctr Comparat Genom & Evolutionary Bioinformat, Halifax, NS, Canada;Dalhousie Univ, Ctr Comparat Genom & Evolutionary Bioinformat, Dept Biol, Halifax, NS, Canada;Univ Cologne, Inst Zool, Cologne, Germany.
    Eme, Laura
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Molekylär evolution. Uppsala universitet, Science for Life Laboratory, SciLifeLab. Dalhousie Univ, Dept Biochem & Mol Biol, Ctr Comparat Genom & Evolutionary Bioinformat, Halifax, NS, Canada.
    Roger, Andrew J.
    Dalhousie Univ, Dept Biochem & Mol Biol, Ctr Comparat Genom & Evolutionary Bioinformat, Halifax, NS, Canada.
    Simpson, Alastair G. B.
    Dalhousie Univ, Ctr Comparat Genom & Evolutionary Bioinformat, Dept Biol, Halifax, NS, Canada.
    A natural toroidal microswimmer with a rotary eukaryotic flagellum2019Ingår i: Nature Microbiology, E-ISSN 2058-5276, Vol. 4, nr 10, s. 1620-1626Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We describe Idionectes vortex gen. nov., sp. nov., a unicellular microeukaryote that swims by continuous inversion of its surface, similar to a vortex ring. This previously unreported mode of motility approximates a hypothetical concept called the 'toroidal swimmer', in which a doughnut-shaped object rotates around its circular axis and travels in the opposite direction to its outer surface motion. During swimming, the flagellum of Idionectes rotates relative to its cell body, which is normally a hallmark of prokaryotic rather than eukaryotic flagella.

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