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  • 1.
    Abdeldaim, Guma M. K.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Strålin, Kristoffer
    Department of Infectious Diseases, Örebro University Hospital.
    Kirsebom, Leif A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Olcén, Per
    Department of Clinical Microbiology, Örebro University Hospital.
    Blomberg, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Herrmann, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Bacteriology.
    Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction2009In: Diagnostic microbiology and infectious disease, ISSN 0732-8893, E-ISSN 1879-0070, Vol. 64, no 4, p. 366-373Article in journal (Refereed)
    Abstract [en]

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 104 DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  • 2.
    Adler, Sara
    et al.
    Umea Univ, Unit Clin Res Ctr Ostersund, Dept Publ Hlth & Clin Med, Umea, Sweden..
    Widerstrom, Micael
    Umea Univ, Unit Communicable Dis Control & Prevent Ostersund, Dept Clin Microbiol, Umea, Sweden..
    Lindh, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lilja, Mikael
    Umea Univ, Unit Clin Res Ctr Ostersund, Dept Publ Hlth & Clin Med, Umea, Sweden..
    Symptoms and risk factors of Cryptosporidium hominis infection in children: data from a large waterborne outbreak in Sweden2017In: Parasitology Research, ISSN 0932-0113, E-ISSN 1432-1955, Vol. 116, no 10, p. 2613-2618Article in journal (Refereed)
    Abstract [en]

    Cryptosporidium is a major cause of diarrheal disease worldwide. In developing countries, this infection is endemic and in children, associated with growth faltering and cognitive function deficits, with the most severe impact on those aged <2 years. Little has been reported about symptoms and risk factors for children in industrialized countries, although the disease incidence is increasing in such regions. In November 2010, a large waterborne outbreak of C. hominis occurred in the city of Ostersund in Sweden. Approximately 27,000 of the 60,000 inhabitants were symptomatic. We aimed to describe duration of symptoms and the risk factors for infection with C. hominis in children aged <15 years in a Western setting. Within 2 months after a boil water advisory, a questionnaire was sent to randomly selected inhabitants of all ages, including 753 children aged <15 years. Those with >= 3 loose stools/day were defined as cases of diarrhoea. The response rate was 70.3%, and 211 children (39.9%) fulfilled the case definition. Mean duration of diarrhoea was 7.5 days (median 6, range 1-80 days). Recurrence, defined as a new episode of diarrhoea after >= 2 days of normal stools, occurred in 52.5% of the cases. Significant risk factors for infection, besides living within the distribution area of the contaminated water plant, included a high level of water consumption, male sex, and a previous history of loose stools. The outbreak was characterized by high attack and recurrence rates, emphasizing the necessity of water surveillance to prevent future outbreaks.

  • 3.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The Appearance and Diversification of Receptors for IgM During Vertebrate Evolution2017In: IGM AND ITS RECEPTORS AND BINDING PROTEINS / [ed] Kubagawa, H Burrows, PD, SPRINGER-VERLAG BERLIN , 2017, p. 1-23Chapter in book (Refereed)
    Abstract [en]

    Three different receptors that interact with the constant domains of IgM have been identified: the polymeric immunoglobulin (Ig) receptor (PIGR), the dual receptor for IgA/IgM (Fc alpha mu R) and the IgM receptor (Fc mu R). All of them are related in structure and located in the same chromosomal region in mammals. The functions of the PIGRs are to transport IgM and IgA into the intestinal lumen and to saliva and tears, whereas the Fc alpha mu Rs enhance uptake of immune complexes and antibody coated bacteria and viruses by B220+ B cells and phagocytes, as well as dampening the Ig response to thymus-independent antigens. The Fc mu Rs have broad-spectrum effects on B-cell development including effects on IgM homeostasis, B-cell survival, humoral immune responses and also in autoantibody formation. The PIGR is the first of these receptors to appear during vertebrate evolution and is found in bony fish and all tetrapods but not in cartilaginous fish. The Fc mu R is present in all extant mammalian lineages and also in the Chinese and American alligators, suggesting its appearance with early reptiles. Currently the Fc alpha mu R has only been found in mammals and is most likely the evolutionary youngest of the three receptors. In bony fish, the PIGR has either 2, 3, 4, 5 or 6 extracellular Ig-like domains, whereas in amphibians, reptiles and birds it has 4 domains, and 5 in all mammals. The increase in domain number from 4 to 5 in mammals has been proposed to enhance the interaction with IgA. Both the Fc alpha mu Rs and the Fc mu Rs contain only one Ig domain; the domain that confers Ig binding. In both of these receptors this domain shows the highest degree of sequence similarity to domain 1 of the PIGR. All Ig domains of these three receptors are V type domains, indicating they all have the same origin although they have diversified extensively in function during vertebrate evolution by changing expression patterns and cytoplasmic signaling motifs.

  • 4.
    Amlinger, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.

    CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.

    Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter.

    The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system. 

    List of papers
    1. Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    Open this publication in new window or tab >>Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312230 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    2. Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Open this publication in new window or tab >>Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312231 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    3. Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    Open this publication in new window or tab >>Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312233 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    4. Efficient programmable gene silencing by Cascade
    Open this publication in new window or tab >>Efficient programmable gene silencing by Cascade
    Show others...
    2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 1, p. 237-246Article in journal (Refereed) Published
    Abstract [en]

    Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-249042 (URN)10.1093/nar/gku1257 (DOI)000350207100026 ()25435544 (PubMedID)
    Available from: 2015-04-23 Created: 2015-04-10 Last updated: 2018-02-28Bibliographically approved
  • 5.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, E. Gerhart H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Quantification of CRISPR-Cas spacer integration using a fluorescent reporterManuscript (preprint) (Other academic)
  • 6.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 7.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 8.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Larsson, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas systemManuscript (preprint) (Other academic)
  • 9.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Saunders, Sita J.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Backofen, Rolf
    Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systemsManuscript (preprint) (Other academic)
  • 10.
    Andersson, Anders F.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Pelve, Erik A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Lindeberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Nilsson, Peter
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Replication-biased genome organisation in the crenarchaeon Sulfolobus2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, p. 454-Article in journal (Refereed)
    Abstract [en]

    Background: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. Results: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. Conclusion: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

  • 11.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Antibiotic resistance and its cost: is it possible to reverse resistance?2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 4, p. 260-271Article, review/survey (Refereed)
    Abstract [en]

    Most antibiotic resistance mechanisms are associated with a fitness cost that is typically observed as a reduced bacterial growth rate. The magnitude of this cost is the main biological parameter that influences the rate of development of resistance, the stability of the resistance and the rate at which the resistance might decrease if antibiotic use were reduced. These findings suggest that the fitness costs of resistance will allow susceptible bacteria to outcompete resistant bacteria if the selective pressure from antibiotics is reduced. Unfortunately, the available data suggest that the rate of reversibility will be slow at the community level. Here, we review the factors that influence the fitness costs of antibiotic resistance, the ways by which bacteria can reduce these costs and the possibility of exploiting them.

  • 12.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gene amplification and adaptive evolution in bacteria2009In: Annual Review of Genetics, ISSN 0066-4197, E-ISSN 1545-2948, Vol. 43, p. 167-195Article, review/survey (Refereed)
    Abstract [en]

    Gene duplication-amplification (GDA) processes are highly relevant biologically because they generate extensive and reversible genetic variation on which adaptive evolution can act. Whenever cellular growth is restricted, escape from these growth restrictions often occurs by GDA events that resolve the selective problem. In addition, GDA may facilitate subsequent genetic change by allowing a population to grow and increase in number, thereby increasing the probability for subsequent adaptive mutations to occur in the amplified genes or in unrelated genes. Mathematical modeling of the effect of GDA on the rate of adaptive evolution shows that GDA will facilitate adaptation, especially when the supply of mutations in the population is rate-limiting. GDA can form via several mechanisms, both RecA-dependent and RecA-independent, including rolling-circle amplification and nonequal crossing over between sister chromatids. Due to the high intrinsic instability and fitness costs associated with GDAs, they are generally transient in nature, and consequently their evolutionary and medical importance is often underestimated.

  • 13.
    Andersson, Dan I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Muller's ratchet decreases fitness of a DNA-based microbe1996In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 93, no 2, p. 906-907Article in journal (Refereed)
    Abstract [en]

    Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.

  • 14.
    Andersson, Dan I.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Biological roles of translesion synthesis DNA polymerases in eubacteria2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 3, p. 540-548Article, review/survey (Refereed)
    Abstract [en]

    Biological systems are strongly selected to maintain the integrity of their genomes by prevention and repair of external and internal DNA damages. However, some types of DNA lesions persist and might block the replication apparatus. The universal existence of specialized translesion synthesis DNA polymerases (TLS polymerases) that can bypass such lesions in DNA implies that replication blockage is a general biological problem. We suggest that the primary function for which translesion synthesis polymerases are selected is to rescue cells from replication arrest at lesions in DNA, a situation that, if not amended, is likely to cause an immediate and severe reduction in cell fitness and survival. We will argue that the mutagenesis observed during translesion synthesis is an unavoidable secondary consequence of this primary function and not, as has been suggested, an evolved mechanism to increase mutation rates in response to various stresses. Finally, we will discuss recent data on additional roles for translesion synthesis polymerases in the formation of spontaneous deletions and in transcription-coupled TLS, where the coupling of transcription to TLS is proposed to allow the rescue of the transcription machinery arrested at DNA lesions.

  • 15. Andersson, Evalena
    et al.
    Lagerbäck, Pernilla
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Carlson, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 4, p. 1003-1016Article in journal (Refereed)
    Abstract [en]

    Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16Da, 136aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P21 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.

  • 16.
    Andersson, Jan O
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Lateral gene transfer in eukaryotes.2005In: Cell Mol Life Sci, ISSN 1420-682X, Vol. 62, no 11, p. 1182-97Article in journal (Refereed)
    Abstract [en]

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular.

  • 17.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organism Biology, Molecular Evolution.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The genome of Giardia and other diplomonads2010In: Anaerobic Parasitic Protozoa: Genomics and Molecular Biology / [ed] C. Graham Clark, Patricia J. Johnson, Rodney D. Adam, Caister Academic Press , 2010, p. 23-44Chapter in book (Other academic)
  • 18.
    Andersson, Jan O
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Sarchfield, Stewart W
    Roger, Andrew J
    Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids.2005In: Mol Biol Evol, ISSN 0737-4038, Vol. 22, no 1, p. 85-90Article in journal (Refereed)
    Abstract [en]

    Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence of split genes in the Nanoarchaeum genome is a derived feature.

  • 19.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Sjögren, Åsa M.
    Horner, David S.
    Murphy, Colleen A.
    Dyal, Patricia L.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Logsdon, Jr., John M.
    Ragan, Mark A.
    Hirt, Robert P.
    Roger, Andrew J.
    A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution2007In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 8, p. 51-Article, review/survey (Refereed)
    Abstract [en]

    Background: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). Results: The analyses revealed a compact genome with few, if any, introns and very short 3′ untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes - mostly encoding metabolic proteins - that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. Conclusion: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

  • 20.
    Ankarklev, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization2012In: PLoS Neglected Tropical Diseases, ISSN 1935-2735Article in journal (Other academic)
    Abstract [en]

    Background

    The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts world-wide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, little data is available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

    Methodology/Principal findings

    Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. No significant association was found in the studied population with regards to the presence of Giardia and gender, type of toilet, source of drinking-water or type of housing. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage A, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable and a significant association was found between Giardia assemblage B and H. pylori infection, OR=5.0 (1.9- 16).

    Conclusions/Significance

    This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that Giardia-infected children have a 3-fold higher risk of being infected by H. pylori. The data also suggests that assemblage B Giardia may be more closely associated with H. pylori infection.

  • 21.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Franzen, Oscar
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden. KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jerlstrom-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lebbad, Marianne
    Publ Hlth Agcy Sweden, Dept Microbiol, SE-17182 Solna, Sweden..
    Andersson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Bjorn
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates2015In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, article id 697Article in journal (Refereed)
    Abstract [en]

    Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

  • 22.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hestvik, Elin
    Lebbad, Marianne
    Lindh, Johan
    Kaddu-Mulindwa, Deogratias H.
    Andersson, Jan O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Tylleskar, Thorkild
    Tumwine, James K.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children2012In: PLOS Neglected Tropical Diseases, ISSN 1935-2735, Vol. 6, no 8, p. e1780-Article in journal (Refereed)
    Abstract [en]

    Background: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

    Methodology/Principal Findings: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection.

    Conclusions/Significance: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.

  • 23.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ringqvist, Emma
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Troell, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Behind the smile: cell biology and disease mechanisms of Giardia species2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 6, p. 413-422Article, review/survey (Refereed)
    Abstract [en]

    The eukaryotic intestinal parasite Giardia intestinalis was first described in 1681, when Antonie van Leeuwenhoek undertook a microscopic examination of his own diarrhoeal stool. Nowadays, although G. intestinalis is recognized as a major worldwide contributor to diarrhoeal disease in humans and other mammals, the disease mechanisms are still poorly understood. Owing to its reduced complexity and proposed early evolutionary divergence, G. intestinalis is used as a model eukaryotic system for studying many basic cellular processes. In this Review we discuss recent discoveries in the molecular cell biology and pathogenesis of G. intestinalis.

  • 24.
    Ankarklev, Johan
    et al.
    Stockholm Univ, Dept Mol Biosci, SE-10691 Stockholm, Sweden.
    Lebbad, Marianne
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Franzen, Oscar
    Karolinska Inst, Integrated Cardio Metab Ctr, Novum, Box 285, SE-14157 Stockholm, Sweden.
    Ahola, Harri
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Troell, Karin
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination2018In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 60, p. 7-16Article in journal (Refereed)
    Abstract [en]

    Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

  • 25.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lebbad, Marianne
    Allelic sequence heterozygosity in single Giardia parasites2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, article id 65Article in journal (Refereed)
    Abstract [en]

    Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

  • 26.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Baker, Louise
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Melbourne, Vic, Australia..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 398Article in journal (Refereed)
    Abstract [en]

    Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate: ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID(10), 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid a alpha-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID(10) diverged from those in 713-r and WB-r (r <= 0.2), which were more similar to each other (r = 0.47). In 106-2ID(10), a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

  • 27.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Bio21 Mol Sci & Biotechnol Inst, Melbourne, Vic, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis2016In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 10, p. 6034-6045Article in journal (Refereed)
    Abstract [en]

    Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and alpha-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.

  • 28.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Parkville, Vic 3052, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Young, Neil D.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Hall, Ross S.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Rojas, Cristian A. A.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites2015In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 9, no 12, article id e0004261Article in journal (Refereed)
    Abstract [en]

    Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Although G. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. The present study employed RNA sequencing to characterize the mRNA transcriptome of G. duodenalis trophozoites in axenic culture, at log (48 h of growth), stationary (60 h), and declining (96 h) growth phases. Using similar to 400-times coverage of the transcriptome, we identified 754 differentially transcribed genes (DTGs), mainly representing two large DTG groups: 438 that were down-regulated in the declining phase relative to log and stationary phases, and 281 that were up-regulated. Differential transcription of prominent antioxidant and glycolytic enzymes implicated oxygen tension as a key factor influencing the transcriptional program of axenic trophozoites. Systematic bioinformatic characterization of numerous DTGs encoding hypothetical proteins of unknown function was achieved using structural homology searching. This powerful approach greatly informed the differential transcription analysis and revealed putative novel antioxidant-coding genes, and the presence of a nearcomplete two-component-like signaling system that may link cytosolic redox or metabolite sensing to the observed transcriptional changes. Motif searching applied to promoter regions of the two large DTG groups identified different putative transcription factor-binding motifs that may underpin global transcriptional regulation. This study provides new insights into the drivers and potential mediators of transcriptional variation in axenic G. duodenalis and provides context for static transcriptional studies.

  • 29.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dagley, Michael J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Drug resistance in Giardia duodenalis2015In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 6, p. 888-901Article, review/survey (Refereed)
    Abstract [en]

    Giardia duodenalis is a microaerophilic parasite of the human gastrointestinal tract and a major contributor to diarrheal and post-infectious chronic gastrointestinal disease world-wide. Treatment of G. duodenalis infection currently relies on a small number of drug classes. Nitroheterocyclics, in particular metronidazole, have represented the front line treatment for the last 40 years. Nitroheterocyclic-resistant G. duodenalis have been isolated from patients and created in vitro, prompting considerable research into the biomolecular mechanisms of resistance. These compounds are redox-active and are believed to damage proteins and DNA after being activated by oxidoreductase enzymes in metabolically active cells. In this review, we explore the molecular phenotypes of nitroheterocyclic-resistant G. duodenalis described to date in the context of the protisfs unusual glycolytic and antioxidant systems. We propose that resistance mechanisms are likely to extend well beyond currently described resistance-associated enzymes (i.e., pyruvate ferredoxin oxidoreductases and nitroreductases), to include NAD(P)H- and flavin-generating pathways, and possibly redox-sensitive epigenetic regulation. Mechanisms that allow G. duodenalis to tolerate oxidative stress may lead to resistance against both oxygen and nitroheterocyclics, with implications for clinical control. The present review highlights the potential for systems biology tools and advanced bioinformatics to further investigate the multifaceted mechanisms of nitroheterocyclic resistance in this important pathogen.

  • 30.
    Ardell, David H
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The genomic pattern of tDNA operon expression in E. coli2005In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 1, no 1, p. e12-Article in journal (Refereed)
    Abstract [en]

    In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

  • 31.
    Ausmees, Nora
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Intermediate filament-like cytoskeleton of Caulobacter crescentus2006In: Journal of Molecular Microbiology and Biotechnology, ISSN 1464-1801, Vol. 11, no 3-5, p. 152-158Article in journal (Refereed)
    Abstract [en]

    Eukaryotic cytoskeleton consists of three main types of filaments: actin microfilaments, microtubules and intermediate filaments (IFs). Actin and tubulin-like proteins are also found in bacteria where they perform diverse cytoskeletal functions. IFs, however, are considered to be a characteristic constituent of metazoan cells only, where they (among other functions) are involved in determination and maintenance of cell shape and cellular integrity. Surprisingly, a coiled coil-rich protein called crescentin was recently shown to play a key role in determining the complex curved and helical cell shapes of the bacterium Caulobacter crescentus, and to exhibit several characteristic properties of animal IF proteins. First, the arrangement of the coiled coil domains of crescentin closely resembles the tripartite molecular architecture of IF proteins. Second, crescentin also possesses the defining biochemical property of IF proteins to assemble into 10-nm-wide filaments in vitro without cofactors. Furthermore, crescentin forms a higher-order helical structure in vivo, which is localized asymmetrically along the concave side of the cell. In close association with the cell membrane, the crescentin structure promotes the helical growth of the cell and thereby determines a curved or a helical shape, depending on the length of the cell. The unexpected finding of an IF-like element in a bacterium raises several interesting questions concerning, for example, the molecular mechanisms whereby complex and asymmetric cell shapes are generated by different bacteria, or the functional and evolutionary relatedness of crescentin to animal IF proteins.

  • 32.
    Ausmees, Nora
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wahlstedt, Helene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Bagchi, Sonchita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elliot, Marie A.
    Buttner, Mark J.
    Flärdh, Klas
    SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa2007In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 65, no 6, p. 1458-1473Article in journal (Refereed)
    Abstract [en]

    Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.

  • 33.
    Bagchi, Sonchita
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Coiled coil Cytoskeleton in Bacterial Cell Architecture: Studies of Growth and Development in Streptomyces2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Bacterial cytoskeleton is an exciting and relatively new field of research. Recent findings have proven that microbes are well-organized and neatly structured organisms. In this study we have shown that intermediate filament-like proteins with a characteristic rod domain architecture of coiled coil segments separated by non-coiled coil linkers, are widely spread among bacteria.

    We identified and characterized an intermediate filament-like protein (named FilP after filamentous protein) in Streptomyces coelicolor. It shares the characteristic biochemical property of eukaryotic intermediate filaments of formation of spontaneous filaments in vitro without requiring any energy or co-factor. We have provided here a preliminary model of its assembly in vitro. FilP also forms in vivo filaments in S. coelicolor hyphae, which are strongest at the sub-apical location of growing vegetative hyphae. We have proposed that FilP cytoskeletal network provides rigidity to the hyphae, especially at the growing tips, by interacting with an essential coiled coil protein DivIVA and possibly other partner elements, yet to be found.

    S. coelicolor is a well-studied model organism with a complicated life cycle. It germinates from a spore and spreads by forming branched vegetative hyphae. Lack of nutrients in the environment initiates formation of aerial hyphae in the air, perpendicular to the vegetative ones. The aerial hyphae differentiate into spore chains and eventually grey-pigmented dispersed individual spores are released. The signals involved in sporulation including cell division and chromosome segregation are not clear yet. We characterized here a novel locus consisting of two genes: a small putative membrane protein with no defined function, named SmeA and a member of the SpoIIIE/FtsK family, called SffA. The expression of this locus appears to be dependent on whiA and whiG-whiH-whiI pathways. This finding is intriguing as it can provide insight to the relationship between two apparently unrelated pathways, both leading to the same function of septation and maturation during sporulation.

    List of papers
    1. Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces
    Open this publication in new window or tab >>Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces
    2008 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 70, no 4, p. 1037-1050Article in journal (Refereed) Published
    Abstract [en]

    Actin and tubulin cytoskeletons are conserved and widespread in bacteria. A strikingly intermediate filament (IF)-like cytoskeleton, composed of crescentin, is also present in Caulobacter crescentus and determines its specific cell shape. However, the broader significance of this finding remained obscure, because crescentin appeared to be unique to Caulobacter. Here we demonstrate that IF-like function is probably a more widespread phenomenon in bacteria. First, we show that 21 genomes of 26 phylogenetically diverse species encoded uncharacterized proteins with a central segmented coiled coil rod domain, which we regarded as a key structural feature of IF proteins and crescentin. Experimental studies of three in silico predicted candidates from Mycobacterium and other actinomycetes revealed a common IF-like property to spontaneously assemble into filaments in vitro. Furthermore, the IF-like protein FilP formed cytoskeletal structures in the model actinomycete Streptomyces coelicolor and was needed for normal growth and morphogenesis. Atomic force microscopy of living cells revealed that the FilP cytoskeleton contributed to mechanical fitness of the hyphae, thus closely resembling the function of metazoan IF. Together, the bioinformatic and experimental data suggest that an IF-like protein architecture is a versatile design that is generally present in bacteria and utilized to perform diverse cytoskeletal tasks.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-107121 (URN)10.1111/j.1365-2958.2008.06473.x (DOI)000260194300022 ()
    Available from: 2009-07-16 Created: 2009-07-16 Last updated: 2017-12-13Bibliographically approved
    2. Co‐operation between two coiled coil cytoskeletons in polar growth in Streptomyces
    Open this publication in new window or tab >>Co‐operation between two coiled coil cytoskeletons in polar growth in Streptomyces
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-158269 (URN)
    Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2011-11-03
    3. A preliminary model of in vitro assembly of a bacterial coiled coil cytoskeletal protein FilP
    Open this publication in new window or tab >>A preliminary model of in vitro assembly of a bacterial coiled coil cytoskeletal protein FilP
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-158268 (URN)
    Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2011-11-03
    4. SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa
    Open this publication in new window or tab >>SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa
    Show others...
    2007 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 65, no 6, p. 1458-1473Article in journal (Refereed) Published
    Abstract [en]

    Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-13077 (URN)10.1111/j.1365-2958.2007.05877.x (DOI)000249425600009 ()17824926 (PubMedID)
    Available from: 2008-01-21 Created: 2008-01-21 Last updated: 2017-12-11Bibliographically approved
  • 34.
    Bagchi, Sonchita
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fuchino, Katsuya
    Department of Biology, Lund University.
    Cantlay, Stuart
    Department of Biology, Lund University.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Bergman, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kamali‐Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Flardh, Klas
    Department of Biology, Lund University.
    Ausmees, Nora
    Department of Biology, Lund University.
    Co‐operation between two coiled coil cytoskeletons in polar growth in StreptomycesManuscript (preprint) (Other academic)
  • 35.
    Bagchi, Sonchita
    et al.