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  • 201. Lind, Peter A
    et al.
    Tobin, Christina
    Berg, Otto G
    Kurland, Charles G
    Andersson, Dan I
    Compensatory gene amplification restores fitness after inter-species gene replacements.2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 75, no 5Article in journal (Refereed)
    Abstract [en]

    Genes introduced by gene replacements and other types of horizontal gene transfer (HGT) represent a significant presence in many archaeal and eubacterial genomes. Most alien genes are likely to be neutral or deleterious upon arrival and their long-term persistence may require a mechanism that improves their selective contribution. To examine the fate of inter-species gene replacements, we exchanged three native S. typhimurium genes encoding ribosomal proteins with orthologues from various other microbes. The results show that replacement of each of these three genes reduces fitness to such an extent that it would provide an effective barrier against inter-species gene replacements in eubacterial populations. However, these fitness defects could be partially ameliorated by gene amplification that augmented the dosage of the heterologous proteins. This suggests that suboptimal expression is a common fitness constraint for inter-species gene replacements, with fitness costs conferred by either a lower expression level of the alien protein compared with the native protein or a requirement for an increased amount of the alien protein to maintain proper function. Our findings can explain the observation that duplicated genes are over-represented among horizontally transferred genes, and suggest a potential coupling between compensatory gene amplification after HGT and the evolution of new genes.

  • 202.
    Lindell, Kristoffer
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cell-to-cell communication and virulence in Vibrio anguillarum2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small molecules to coordinate activities such as growth, biofilm formation, virulence, and stress response as a population. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population.

    In Vibrio anguillarum, three parallel quorum-sensing phosphorelay systems channels information via three hybrid sensor kinases VanN, VanQ, and CqsS that function as receptors for signal molecules produced by the synthases VanM, VanS, and CqsA, respectively. The phosphorelay systems converge onto a single regulatory pathway via the phosphotransferase VanU, which phosphorylates the response regulator VanO. Together with the alternative sigma factor RpoN, VanO activates the expression of a small RNA, Qrr1 (Quorum regulatory RNA), which in conjunction with the small RNA chaperone Hfq, destabilizes vanT mRNA, which encode the major quorum-sensing regulator in V. anguillarum. This thesis furthers the knowledge on the quorum-sensing phosphorelay systems in V. anguillarum.

    In this study, three additional qrr genes were identified, which were expressed during late logarithmic growth phase. The signal synthase VanM activated the expression of the Qrr1-4, which stands in contrast to Qrr regulation in other vibrios. Moreover, in addition to VanO, we predict the presence of a second response regulator which can be phosphorylated by VanU and repress Qrr1-4 expression. Thus, VanU functions as a branch point that can regulate the quorum-sensing regulon by activating or repressing VanT expression. Furthermore, VanT was shown to directly activate VanM expression and thus forming a negative regulatory loop, in which VanM represses VanT expression indirectly via Qrr1-4. In addition, VanM expression was negatively regulated post-transcriptionally by Hfq. Furthermore, a universal stress protein UspA repressed VanM expression via the repression of VanT expression. We showed that UspA binds Hfq, thus we suggest that UspA plays a role in sequestering Hfq and indirectly affect gene expression.

    This thesis also investigated the mechanism by which V. anguillarum can attach to and colonize fish skin tissue. We show unequivocally that fish skin epithelial cells can internalize bacteria, thus keeping the skin clear from pathogens. In turn, V. anguillarum utilized the lipopolysaccharide O-antigen to evade internalization by the fish skin epithelial cells. This study provides new insights into the molecular mechanism by which pathogen interacts with marine animals to cause disease.

  • 203.
    Lindgren, Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Meyer, Lena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection2013In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, article id 20Article in journal (Refereed)
    Abstract [en]

    Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

    Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

    Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

  • 204.
    Lindgren, Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Eneslätt, Kjell
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Bröms, Jeanette
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Importance of PdpC, IglC, IglI, and IglG for modulation of a host cell death pathway induced by Francisella tularensis LVSManuscript (preprint) (Other academic)
    Abstract [en]

    Modulation of host cell death pathways appears to be a prerequisite for the successful life styles of many intracellular pathogens. The facultative intracellular bacterium Francisella tularensis is highly pathogenic and effective proliferation in the macrophage cytosol leading to host cell death is a requirement for its virulence. To better understand how this is achieved, macrophages were infected with the F. tularensis live vaccine strain (LVS) and the effects were compared to those resulting from infections with deletion mutants lacking expression of either of the pdpC, iglC, iglG, or iglI genes. All of these genes encode components that together with a dozen other proteins form the Francisella pathogenicity island (FPI), a type VI secretion system. Within 12 h, a majority of the J774 cells infected with the LVS strain showed production of mitochondrial superoxide and after 24 h, marked signs of mitochondrial damage, caspase-9 and caspase-3 activation, phosphatidylserine expression, nucleosome formation, and membrane leakage. In contrast, neither of these events occurred after infection with the ∆iglI or ∆iglC mutant, although the former strain replicated. The ∆iglG mutant replicated effectively but induced only marginal cytopathogenic effects after 24 h and intermediate effects after 48 h. In contrast, the ∆pdpC mutant showed no replication, but induced marked mitochondrial superoxide production and mitochondrial damage, caspase-3 activation, nucleosome formation, and phosphatidylserine expression, although the effects were delayed compared to LVS. The unique phenotypes of the mutants provide novel insights regarding the roles of individual FPI components for the modulation of the cytopathogenic effects resulting from the F. tularensis infection.

  • 205. Lindh, Markus V.
    et al.
    Figueroa, Daniela
    Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF). Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Sjostedt, Johanna
    Baltar, Federico
    Lundin, Daniel
    Andersson, Agneta
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Legrand, Catherine
    Pinhassi, Jarone
    Transplant experiments uncover Baltic Sea basin-specific responses in bacterioplankton community composition and metabolic activities2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 223Article in journal (Refereed)
    Abstract [en]

    Anthropogenically induced changes in precipitation are projected to generate increased river runoff to semi enclosed seas, increasing loads of terrestrial dissolved organic matter and decreasing salinity. To determine how bacterial community structure and functioning adjust to such changes, we designed microcosm transplant experiments with Baltic Proper (salinity 7.2) and Bothnian Sea (salinity 3.6) water. Baltic Proper bacteria generally reached higher abundances than Bothnian Sea bacteria in both Baltic Proper and Bothnian Sea water, indicating higher adaptability. Moreover, Baltic Proper bacteria growing in Bothnian Sea water consistently showed highest bacterial production and beta-glucosidase activity. These metabolic responses were accompanied by basin specific changes in bacterial community structure. For example, Baltic Proper Pseudomonas and Limnobacter populations increased markedly in relative abundance in Bothnian Sea water, indicating a replacement effect. In contrast, Roseobacter and Rheinheknera populations were stable or increased in abundance when challenged by either of the waters, indicating an adjustment effect. Transplants to Bothnian Sea water triggered the initial emergence of particular Burkholderiaceae populations, and transplants to Baltic Proper water triggered Alteromonadaceae populations. Notably, in the subsequent re transplant experiment, a priming effect resulted in further increases to dominance of these populations. Correlated changes in community composition and metabolic activity were observed only in the transplant experiment and only at relatively high phylogenetic resolution. This suggested an importance of successional progression for interpreting relationships between bacterial community composition and functioning. We infer that priming effects on bacterial community structure by natural episodic events or climate change induced forcing could translate into long-term changes in bacterial ecosystem process rates.

  • 206. Lindh, Markus V.
    et al.
    Sjöstedt, Johanna
    Casini, Michele
    Andersson, Agneta
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Legrand, Catherine
    Pinhassi, Jarone
    Local Environmental Conditions Shape Generalist But Not Specialist Components of Microbial Metacommunities in the Baltic Sea2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 2078Article in journal (Refereed)
    Abstract [en]

    Marine microbes exhibit biogeographical patterns linked with fluxes of matter and energy. Yet, knowledge of the mechanisms shaping bacterioplankton community assembly across temporal scales remains poor. We examined bacterioplankton 16S rRNA gene fragments obtained from Baltic Sea transects to determine phylogenetic relatedness and assembly processes coupled with niche breadth. Communities were phylogenetically more related over time than expected by chance, albeit with considerable temporal variation. Hence, habitat filtering, i.e., local environmental conditions, rather than competition structured bacterioplankton communities in summer but not in spring or autumn. Species sorting (SS) was the dominant assembly process, but temporal and taxonomical variation in mechanisms was observed. For May communities, Cyanobacteria, Actinobacteria, Alpha- and Betaproteobacteria exhibited SS while Bacteroidetes and Verrucomicrobia were assembled by SS and mass effect. Concomitantly, Gammaproteobacteria were assembled by the neutral model and patch dynamics. Temporal variation in habitat filtering and dispersal highlights the impact of seasonally driven reorganization of microbial communities. Typically abundant Baltic Sea populations such as the NS3a marine group (Bacteroidetes) and the SAR86 and SAR11 clade had the highest niche breadth. The verrucomicrobial Spartobacteria population also exhibited high niche breadth. Surprisingly, variation in bacterioplankton community composition was regulated by environmental factors for generalist taxa but not specialists. Our results suggest that generalists such as NS3a, SAR86, and SAR11 are reorganized to a greater extent by changes in the environment compared to specialists and contribute more strongly to determining overall biogeographical patterns of marine bacterial communities.

  • 207.
    Lindqvist, Richard
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Kurhade, Chaitanya
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Gilthorpe, Jonathan
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Viperin restrict neurotropic flavivirus infection in cell type and region-specific mannerManuscript (preprint) (Other academic)
  • 208. Lindström, Stafva
    et al.
    Rowe, Owen F.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF). Department of Microbiology, University of Helsinki, Helsinki, Finland.
    Timonen, Sari
    Sundström, Liselotte
    Johansson, Helena
    Trends in bacterial and fungal communities in ant nests observed with Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Next Generation Sequencing (NGS) techniques-validity and compatibility in ecological studies2018In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, Vol. 6, article id e5289Article in journal (Refereed)
    Abstract [en]

    Microbes are ubiquitous and often occur in functionally and taxonomically complex communities. Unveiling these community dynamics is one of the main challenges of microbial research. Combining a robust, cost effective and widely used method such as Terminal Restriction Fragment Length Polymorphism (T-RFLP) with a Next Generation Sequencing (NGS) method (Illumina MiSeq), offers a solid alternative for comprehensive assessment of microbial communities. Here, these two methods were combined in a study of complex bacterial and fungal communities in the nest mounds of the ant Formica exsecta, with the aim to assess the degree to which these methods can be used to complement each other. The results show that these methodologies capture similar spatiotemporal variations, as well as corresponding functional and taxonomical detail, of the microbial communities in a challenging medium consisting of soil, decomposing plant litter and an insect inhabitant. Both methods are suitable for the analysis of complex environmental microbial communities, but when combined, they complement each other well and can provide even more robust results. T-RFLP can be trusted to show similar general community patterns as Illumina MiSeq and remains a good option if resources for NGS methods are lacking.

  • 209.
    Liu, Junfa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thanikkal, Edvin
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Obi, Ikenna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis2012In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 163, no 8, p. 518-530Article in journal (Refereed)
    Abstract [en]

    One way that Gram-negative bacteria respond to extracytoplasmic stress is through the CpxA-CpxR system. An activated CpxA sensor kinase phosphorylates the CpxR response regulator to instigate positive auto-amplification of Cpx pathway activation, as well as synthesis of various bacterial survival factors. In the absence of CpxA, human enteropathogenic Yersinia pseudotuberculosis accumulates high CpxR~P levels aided by the action of low molecular weight phosphodonors such as acetyl~P. Critically, these bacteria are also defective for plasmid encoded Ysc-Yop-dependent type III synthesis and secretion, an essential determinant of virulence. Herein, we investigated whether elevated CpxR~P levels account for lost Ysc-Yop function. Decisively, reducing CpxR~P in Yersinia defective for CpxA phosphatase activity - through incorporating second-site suppressor mutations in ackA-pta or cpxR - dramatically restored Ysc-Yop T3S function. Moreover, the repressive effect of accumulated CpxR~P is a direct consequence of binding to the promoter regions of the T3S genes. Thus, Cpx pathway activation has two consequences in Yersinia; one, to maintain quality control in the bacterial envelope, and the second, to restrict ysc-yop gene expression to those occasions where it will have maximal effect.

  • 210. Logares, Ramiro
    et al.
    Tesson, Sylvie V. M.
    Canback, Björn
    Pontarp, Mikael
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Biology, Lund University, Lund, Sweden; Department of Evolutionary Biology and EnvironmentalStudies, University of Zurich, Zurich, Switzerland.
    Hedlund, Katarina
    Rengefors, Karin
    Contrasting prevalence of selection and drift in the community structuring of bacteria and microbial eukaryotes2018In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 6, p. 2231-2240Article in journal (Refereed)
    Abstract [en]

    Whether or not communities of microbial eukaryotes are structured in the same way as bacteria is a general and poorly explored question in ecology. Here, we investigated this question in a set of planktonic lake microbiotas in Eastern Antarctica that represent a natural community ecology experiment. Most of the analysed lakes emerged from the sea during the last 6000 years, giving rise to waterbodies that originally contained marine microbiotas and that subsequently evolved into habitats ranging from freshwater to hypersaline. We show that habitat diversification has promoted selection driven by the salinity gradient in bacterial communities (explaining approximate to 72% of taxa turnover), while microeukaryotic counterparts were predominantly structured by ecological drift (approximate to 72% of the turnover). Nevertheless, we also detected a number of microeukaryotes with specific responses to salinity, indicating that albeit minor, selection has had a role in the structuring of specific members of their communities. In sum, we conclude that microeukaryotes and bacteria inhabiting the same communities can be structured predominantly by different processes. This should be considered in future studies aiming to understand the mechanisms that shape microbial assemblages.

  • 211.
    Lu, Pei
    et al.
    Chinese Academy of Sciences, Wuhan, China.
    Zhang, Yong
    Chinese Academy of Sciences, Wuhan, China.
    Hu, Yangbo
    Chinese Academy of Sciences, Wuhan, China.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Chen, Shiyun
    Chinese Academy of Sciences, Wuhan, China.
    A cis-encoded sRNA controls the expression of fabH2 in Yersinia2014In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 10, p. 1961-1966Article in journal (Refereed)
    Abstract [en]

    YsrH is a novel cis-encoded sRNA located on the opposite strand to fabH2, which is essential for fatty acid biosynthesis in bacteria. In this study, YsrH-mediated regulation of fabH2 expression was investigated in Yersinia pseudotuberculosis. Constitutive and inducible over-expression of YsrH decreased the mRNA level of fabH2, while expression of downstream fabD and fabG remained unaffected. Polynucleotide phosphorylase (PNPase) also played an important role in this regulation process by mediating YsrH decay in the exponential phase. Thus, our data defines a cis-encoded sRNA that regulates fatty acid synthesis via a regulatory mechanism also involving PNPase.

  • 212.
    Lwande, Olivia Wesula
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bucht, Goran
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Ahlm, Kristoffer
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Naslund, Jonas
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Mosquito-borne Inkoo virus in northern Sweden - isolation and whole genome sequencing2017In: Virology Journal, ISSN 1743-422X, E-ISSN 1743-422X, Vol. 14, article id 61Article in journal (Refereed)
    Abstract [en]

    Background: Inkoo virus (INKV) is a less known mosquito-borne virus belonging to Bunyaviridae, genus Orthobunyavirus, California serogroup. Studies indicate that INKV infection is mainly asymptomatic, but can cause mild encephalitis in humans. In northern Europe, the sero-prevalence against INKV is high, 41% in Sweden and 51% in Finland. Previously, INKV RNA has been detected in adult Aedes (Ae.) communis, Ae. hexodontus and Ae. punctor mosquitoes and Ae. communis larvae, but there are still gaps of knowledge regarding mosquito vectors and genetic diversity. Therefore, we aimed to determine the occurrence of INKV in its mosquito vector and characterize the isolates.

    Methods: About 125,000 mosquitoes were collected during a mosquito-borne virus surveillance in northern Sweden during the summer period of 2015. Of these, 10,000 mosquitoes were processed for virus isolation and detection using cell culture and RT-PCR. Virus isolates were further characterized by whole genome sequencing. Genetic typing of mosquito species was conducted by cytochrome oxidase subunit I (COI) gene amplification and sequencing (genetic barcoding).

    Results: Several Ae. communis mosquitoes were found positive for INKV RNA and two isolates were obtained. The first complete sequences of the small (S), medium (M), and large (L) segments of INKV in Sweden were obtained. Phylogenetic analysis showed that the INKV genome was most closely related to other INKV isolates from Sweden and Finland. Of the three INKV genome segments, the INKV M segment had the highest frequency of non-synonymous mutations. The overall G/C-content of INKV genes was low for the N/NSs genes (43.8–45.5%), polyprotein (Gn/Gc/NSm) gene (35.6%) and the RNA polymerase gene (33.8%) This may be due to the fact that INKV in most instances utilized A or T in the third codon position.

    Conclusions: INKV is frequently circulating in northern Sweden and Ae. communis is the key vector. The high mutation rate of the INKV M segment may have consequences on virulence

  • 213.
    Lécrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Post-transcriptional regulation by RNases in Streptococcus pyogenes2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribonucleases (RNases) are proteins that adjust cellular RNA levels by processing RNA transcripts, leading to their stabilization or degradation. RNases are grouped based on their ability to cleave the transcript internally (endoRNases) or degrade the transcript starting from the ends (exoRNases). Specificities of RNA degradation vary among bacterial species, attributable to different sets of endo- and exoRNases. Most of the current knowledge gathered about the roles of RNases and their targets relies on the study of a few model bacteria, such as Escherichia coli and Bacillus subtilis. The aim of this thesis was to understand how Streptococcus pyogenes, a strict human pathogen, controls and adjusts gene expression by characterizing in vivo RNase activities.

    The transcriptome of S. pyogenes was inspected to identify cleavages in vivo performed by RNases of interest using RNA sequencing. For this purpose, we developed a method to compare transcript 5′ and 3′ ends in RNase deletion mutants with those in the parental strain. We first applied our method for the study of endoRNase III, which cleaves ds RNA, and endoRNase Y, which is specific for ss RNA. We accurately retrieved RNase III cleavage positions in structured regions, characterized by 2 nucleotide (nt) 3′ overhangs, and we showed RNase III nicking activity in vivo. We observed that RNase Y processed transcripts after a guanosine. The upstream and downstream fragments generated by a single cleavage event were never both identified, indicating that RNase Y processing always led to the degradation of one of the two fragments. To investigate further the degradation of the upstream fragment subsequent to RNase Y processing, we characterized the 3′-to-5′ exoRNases R, YhaM, and PNPase. RNase R did not have any detectable activity in standard laboratory conditions. YhaM is an intriguing enzyme that removed on average 3 nt of the majority of cellular transcripts. PNPase fully degraded fragments originating from endoRNase processing and is the main 3′-to-5′ exoRNase involved in RNA decay in S. pyogenes.

    To conclude, in this work, we developed a novel method to analyze RNA sequencing data. This method was successfully applied to the study of both endo- and exoRNases. Most importantly, we identified the targetomes of RNases III, Y, R, YhaM, and PNPase and we highlighted the distinctive features of these enzymes.

  • 214.
    Lécrivain, Anne-Laure
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, D-10117 Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany..
    Broglia, Laura
    Renault, Thibaud
    Hahnke, Karin
    Ahmed-Begrich, Rina
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes2018Manuscript (preprint) (Other academic)
  • 215.
    Lécrivain, Anne-Laure
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Le Rhun, Anaïs
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Renault, Thibaud T.
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany .
    Ahmed-Begrich, Rina
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Hahnke, Karin
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Charpentier, Emmanuelle
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany.
    In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 46, p. 11814-11819Article in journal (Refereed)
    Abstract [en]

    mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5′ or 3′ end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3′-to-5′ exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes. We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3′-to-5′ exoRNase for RNA turnover in S. pyogenes. In particular, PNPase is responsible for the degradation of regulatory elements from 5′ untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3′-to-5′ exoRNases.

  • 216. Maciejewska, B
    et al.
    Roszniowski, B
    Espaillat, Akbar
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kęsik-Szeloch, A
    Majkowska-Skrobek, G
    Kropinski, AM
    Briers, Y
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lavigne, R
    Drulis-Kawa, Z
    Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes2017In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, no 2, p. 673-684Article in journal (Refereed)
    Abstract [en]

    Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus BKp15virus^ within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between L-alanine and D-glutamic acid.

  • 217. Maguire, Casey A.
    et al.
    Balaj, Leonora
    Sivaraman, Sarada
    Crommentuijn, Matheus H. W.
    Ericsson, Maria
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Gianni, Davide
    Tannous, Bakhos A.
    Sena-Esteves, Miguel
    Breakefield, Xandra O.
    Skog, Johan
    Microvesicle-associated AAV Vector as a Novel Gene Delivery System2012In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 20, no 5, p. 960-971Article in journal (Refereed)
    Abstract [en]

    Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

  • 218. Makarova, Kira S
    et al.
    Wolf, Yuri I
    Alkhnbashi, Omer S
    Costa, Fabrizio
    Shah, Shiraz A
    Saunders, Sita J
    Barrangou, Rodolphe
    Brouns, Stan J J
    Charpentier, Emmanuelle
    Department of Regulation in Infection, Max Planck Institute for Infection Biology.
    Haft, Daniel H
    Horvath, Philippe
    Moineau, Sylvain
    Mojica, Francisco J M
    Terns, Rebecca M
    Terns, Michael P
    White, Malcolm F
    Yakunin, Alexander F
    Garrett, Roger A
    van der Oost, John
    Backofen, Rolf
    Koonin, Eugene V
    An updated evolutionary classification of CRISPR-Cas systems2015In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 13, no 11, p. 722-736Article in journal (Refereed)
    Abstract [en]

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.

  • 219.
    Mangold, Stefanie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Growth and survival of Acidithiobacilli in Acidic, metal rich environments2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Acidithiobacilli are acidophilic microorganisms that play important roles in many natural processes such as acidification of the environment, influencing metal mobility, and impacting on global sulfur and iron cycles. Due to their distinct metabolic properties they can be applied in the industrial extraction of valuable metals. Acidithiobacilli thrive in an environment which is extremely acidic and usually low in organic carbon but highly polluted with metals. In the quest to gain insight into how these microorganisms can thrive in their extreme environment, relevant facets of metabolism, metal resistance, and pH homeostasis were exploredwith the focus on two model organisms,

    Acidithiobacillus caldus and Acidithiobacillus ferrooxidans. Understanding these fundamental aspects of an acidophilic lifestyle will help to eventually control detrimental effects on the environment due to acidification and metal pollution as well as improving metal extraction utilizing acidophilic microorganisms.

    Bioinformatics can give information about the genetic capacity of an organism. Likewise, ‘omics’ techniques, such as transcriptomics and proteomics to study gene transcription profiles and differentially expressed proteins canyield insights into general responses as well as giving clues regarding specific mechanisms for adaptation to life in extreme environments. This approach was used to investigate the sulfur metabolism of

    At. caldus which is an important sulfur oxidizer for industrial metal extraction. It was found that sulfur oxidation pathways were diverse within acidithiobacilli and a model of At. caldus sulfur oxidation was proposed. Furthermore, At. ferrooxidans anaerobic sulfur oxidation coupled to ferric iron reduction was studied which can be of importance for industrial processes. It was shown that anaerobic sulfur oxidation was, at least in part, indirectly coupled to ferric iron reduction via sulfide generation. Moreover, metal toxicity and resistance mechanisms in acidophiles are of major interest. Thus, zinc toxicity in three model organisms, At. caldus, Acidimicrobium ferrooxidans, and ‘Ferroplasma acidarmanus’, was explored. An important finding was that the speciation of metals and other chemical influences were of great importance for zinc toxicity in acidophiles. Additionally, the three organisms showed distinct responses to elevated zinc levels. Finally, the response of At. caldus to various suboptimal growth pH was evaluated to gain insights into pH homeostasis mechanisms. The results indicated that At. caldus used acid resistance mechanisms similar to those described for neutrophilic microorganisms. Analysis of fatty acid profiles demonstrated an active modulation of the cyctoplasmic membrane in response to proton concentration, likely resulting in a more rigid membrane at lower pH.

  • 220.
    Mangold, Stefanie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Potrykus, Joanna
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Aberdeen Fungal Group, University of Aberdeen, Scotland, UK.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lövgren, Lars
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Centre for Ecology and Evolution in Microbial Model Systems, School of Natural Sciences, Linnaeus University, Kalmar, Sweden.
    Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains2013In: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 17, no 1, p. 75-85Article in journal (Refereed)
    Abstract [en]

    Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon 'Ferroplasma acidarmanus', the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of 'F. acidarmanus' ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, 'F. acidarmanus' kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.

  • 221.
    Mangold, Stefanie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    rao Jonna, Venkateswara
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Response of Acidithiobacillus caldus towards suboptimal pH conditionsManuscript (preprint) (Other academic)
  • 222. Martin, Oceane C. B.
    et al.
    Bergonzini, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    D'Amico, Federica
    Chen, Puran
    Shay, Jerry W.
    Dupuy, Jacques
    Svensson, Mattias
    Masucci, Maria G.
    Frisan, Teresa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Infection with genotoxin-producing Salmonella enterica synergises with loss of the tumour suppressor APC in promoting genomic instability via the PI3K pathway in colonic epithelial cells2019In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 21, no 12, article id e13099Article in journal (Refereed)
    Abstract [en]

    Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.

  • 223.
    Mazur-Marzec, Hanna
    et al.
    University of Gdansk, Poland.
    Bertos-Fortis, Mireia
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    Torunska-Sitarz, Anna
    University of Gdansk, Poland.
    Fidor, Anna
    University of Gdansk, Poland.
    Legrand, Catherine
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    Chemical and Genetic Diversity of Nodularia spumigena from the Baltic Sea2016In: Marine Drugs, ISSN 1660-3397, E-ISSN 1660-3397, Vol. 14, no 11, article id 209Article in journal (Refereed)
    Abstract [en]

    Nodularia spumigena is a toxic, filamentous cyanobacterium occurring in brackish waters worldwide, yet forms extensive recurrent blooms in the Baltic Sea. N. spumigena produces several classes of non-ribosomal peptides (NRPs) that are active against several key metabolic enzymes. Previously, strains from geographically distant regions showed distinct NRP metabolic profiles. In this work, conspecific diversity in N. spumigena was studied using chemical and genetic approaches. NRP profiles were determined in 25 N. spumigena strains isolated in different years and from different locations in the Baltic Sea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genetic diversity was assessed by targeting the phycocyanin intergenic spacer and flanking regions (cpcBA-IGS). Overall, 14 spumigins, 5 aeruginosins, 2 pseudaeruginosins, 2 nodularins, 36 anabaenopeptins, and one new cyanopeptolin-like peptide were identified among the strains. Seven anabaenopeptins were new structures; one cyanopeptolin-like peptide was discovered in N. spumigena for the first time. Based on NRP profiles and cpcBA-IGS sequences, the strains were grouped into two main clusters without apparent influence of year and location, indicating persistent presence of these two subpopulations in the Baltic Sea. This study is a major step in using chemical profiling to explore conspecific diversity with a higher resolution than with a sole genetic approach.

  • 224.
    McGee, Karen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hörstedt, Per
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Milton, Debra L.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Identification and characterization of additional flagellin genes from Vibrio anguillarum1996In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, no 17, p. 5188-5198Article in journal (Refereed)
    Abstract [en]

    Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.

  • 225.
    Milton, Debra L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Chalker, V. J.
    Kirke, D.
    Hardman, A.
    Cámara, M.
    Williams, P.
    The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone: 2001In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 183, no 12, p. 3537-3547Article in journal (Refereed)
    Abstract [en]

    Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).

  • 226.
    Milton, Debra L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hardman, A.
    Camara, M.
    Chhabra, S. R.
    Bycroft, B. W.
    Stewart, G. S.
    Williams, P.
    Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone1997In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 179, no 9, p. 3004-3012Article in journal (Refereed)
    Abstract [en]

    Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.

  • 227.
    Milton, Debra L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Norqvist, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum1992In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 174, no 22, p. 7235-7244Article in journal (Refereed)
    Abstract [en]

    Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.

  • 228.
    Milton, Debra L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Norqvist, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sequence of a novel virulence-mediating gene, virC, from Vibrio anguillarum1995In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 164, no 1, p. 95-100Article in journal (Refereed)
    Abstract [en]

    Previously, the double-transposon (Tn) mutant VAN20 of Vibrio anguillarum (Va) 775.17B was isolated. This mutant lacked a major surface antigen (MSA) suggested to be a lipopolysaccharide (LPS) and showed a 10(5)-fold increase in the 50% lethal dose (LD50) when fish were infected intraperitoneally. In this study, the two Tn insertion sites within the chromosome were identified, a plasmid insertion mutation was made at each locus in a more virulent strain of Va, NB10, and the virulence was analyzed. One mutant displayed a 10(4)-fold increase in LD50, whereas the second mutant showed the wild-type (wt) phenotype. However, both mutants still expressed the MSA, suggesting that there may be more than two Tn insertions in VAN20 or that a double mutation is required to prevent production of the MSA. The DNA locus for the virulent phenotype was cloned and sequenced. A potential transcriptional unit consisting of three putative open reading frames (ORFs) was identified. The Tn was located in the second ORF, virC (virulence). The first ORF (34.8 kDa) showed 30% homology to the Escherichia coli and Salmonella typhimurium cysG (cysteine) genes. The virC gene (51.4 kDa) and the third ORF (24 kDa) showed no homology to other proteins in GenBank. Plasmid insertion mutants were made within each of these ORFs and the virulence was assayed. Only the virC mutant showed a loss in virulence, indicating that virC is a novel gene that is essential for the virulence of Va.

  • 229.
    Milton, Debra L.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    O'Toole, Ronan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hörstedt, Per
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Flagellin A is essential for the virulence of Vibrio anguillarum1996In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 178, no 5, p. 1310-1319Article in journal (Refereed)
    Abstract [en]

    A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

  • 230.
    Mojica, Sergio A.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Salin, Olli
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bastidas, Robert J.
    Sunduru, Naresh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, C. David
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Núñez-Otero, Carlos
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Engström, Patrik
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Valdivia, Raphael H.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    N-acylated derivatives of sulfamethoxazole block Chlamydia fatty acid synthesis and interact with FabF2017In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 61, no 10, article id e00716-17Article in journal (Refereed)
    Abstract [en]

    The type II fatty acid synthesis (FASII) pathway is essential for bacterial lipid biosynthesis and continues to be a promising target for novel antibacterial compounds. Recently, it has been demonstrated that Chlamydia is capable of FASII and this pathway is indispensable for Chlamydia growth. Previously, a high-content screen with Chlamydia trachomatis-infected cells was performed, and acylated sulfonamides were identified to be potent growth inhibitors of the bacteria. C. trachomatis strains resistant to acylated sulfonamides were isolated by serial passage of a wild-type strain in the presence of low compound concentrations. Results from whole-genome sequencing of 10 isolates from two independent drug-resistant populations revealed that mutations that accumulated in fabF were predominant. Studies of the interaction between the FabF protein and small molecules showed that acylated sulfonamides directly bind to recombinant FabF in vitro and treatment of C. trachomatis-infected HeLa cells with the compounds leads to a decrease in the synthesis of Chlamydia fatty acids. This work demonstrates the importance of FASII for Chlamydia development and may lead to the development of new antimicrobials.

  • 231.
    Mojica, Sergio
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Eriksson, Anna U.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Davis, Rohan A.
    Bahnan, Wael
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Red Fluorescent Chlamydia trachomatis Applied to Live Cell Imaging and Screening for Antibacterial Agents2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 3151Article in journal (Refereed)
    Abstract [en]

    In this study, we describe the application of a transformed Chlamydia trachomatis strain constitutively expressing the red fluorescent protein mCherry, to allow real-time monitoring of the infection cycle and screening for agents that block replication of C. trachomatis. The red fluorescent C. trachomatis strain was detected autonomously without antibody staining and was equally susceptible to doxycycline as the wild type strain. A high-throughput screening assay was developed using the transformed strain and automated fluorescence microscopy. The assay was used in a pilot screen of a 349 compound library containing natural products from Australian flora and fauna. Compounds with anti-chlamydial activity were tested for dose response and toxicity to host cells and two non-toxic compounds had 50% effective concentration (EC50) values in the low micromolar range. Natural products are valuable sources for drug discovery and the identified Chlamydia growth inhibition may be starting points for future drug development. Live cell imaging was used to visualize growth of the red fluorescent C. trachomatis strain over time. The screening assay reduced workload and reagents compared to an assay requiring immunostaining and could further be used to monitor the development of Chlamydia inclusions and anti-chlamydial effect in real time.

  • 232.
    Monteux, Sylvain
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    A song of ice and mud: Interactions of microbes with roots, fauna and carbon in warming permafrost-affected soils2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Permafrost-affected soils store a large quantity of soil organic matter (SOM) – ca. half of worldwide soil carbon – and currently undergo rapid and severe warming due to climate change. Increased SOM decomposition by microorganisms and soil fauna due to climate change, poses the risk of a positive climate feedback through the release of greenhouse gases. Direct effects of climate change on SOM decomposition, through such mechanisms as deepening of the seasonally-thawing active layer and increasing soil temperatures, have gathered considerable scientific attention in the last two decades. Yet, indirect effects mediated by changes in plant, microbial, and fauna communities, remain poorly understood. Microbial communities, which may be affected by climate change-induced changes in vegetation composition or rooting patterns, and may in turn affect SOM decomposition, are the primary focus of the work described in this thesis.

    We used (I) a field-scale permafrost thaw experiment in a palsa peatland, (II) a laboratory incubation of Yedoma permafrost with inoculation by exotic microorganisms, (III) a microcosm experiment with five plant species grown either in Sphagnum peat or in newly-thawed permafrost peat, and (IV) a field-scale cold season warming experiment in cryoturbated tundra to address the indirect effects of climate change on microbial drivers of SOM decomposition. Community composition data for bacteria and fungi were obtained by amplicon sequencing and phospholipid fatty acid extraction, and for collembola by Tullgren extraction, alongside measurements of soil chemistry, CO2 emissions and root density.

    We showed that in situ thawing of a palsa peatland caused colonization of permafrost soil by overlying soil microbes. Further, we observed that functional limitations of permafrost microbial communities can hamper microbial metabolism in vitro. Relieving these functional limitations in vitro increased cumulative CO2 emissions by 32% over 161 days and introduced nitrification. In addition, we found that different plant species did not harbour different rhizosphere bacterial communities in Sphagnum peat topsoil, but did when grown in newly-thawed permafrost peat. Plant species may thus differ in how they affect functional limitations in thawing permafrost soil. Therefore, climate change-induced changes in vegetation composition might alter functioning in the newly-thawed, subsoil permafrost layer of northern peatlands, but less likely so in the topsoil. Finally, we observed that vegetation encroachment in barren cryoturbated soil, due to reduced cryogenic activity with higher temperatures, change both bacterial and collembola community composition, which may in turn affect soil functioning.

    This thesis shows that microbial community dynamics and plant-decomposer interactions play an important role in the functioning of warming permafrost-affected soils. More specifically, it demonstrates that the effects of climate change on plants can trickle down on microbial communities, in turn affecting SOM decomposition in thawing permafrost.

  • 233.
    Moodie, Lindon W. K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway.
    Cervin, Gunnar
    Trepos, Rozenn
    Labriere, Christophe
    Hellio, Claire
    Pavia, Henrik
    Svenson, Johan
    Design and Biological Evaluation of Antifouling Dihydrostilbene Oxime Hybrids2018In: Marine Biotechnology, ISSN 1436-2228, E-ISSN 1436-2236, Vol. 20, no 2, p. 257-267Article in journal (Refereed)
    Abstract [en]

    By combining the recently reported repelling natural dihydrostilbene scaffold with an oxime moiety found in many marine antifoulants, a library of nine antifouling hybrid compounds was developed and biologically evaluated. The prepared compounds were shown to display a low antifouling effect against marine bacteria but a high potency against the attachment and growth of microalgae down to MIC values of 0.01 μg/mL for the most potent hybrid. The mode of action can be characterized as repelling via a reversible non-toxic biostatic mechanism. Barnacle cyprid larval settlement was also inhibited at low μg/mL concentrations with low levels or no toxicity observed. Several of the prepared compounds performed better than many reported antifouling marine natural products. While several of the prepared compounds are highly active as antifoulants, no apparent synergy is observed by incorporating the oxime functionality into the dihydrostilbene scaffold. This observation is discussed in light of recently reported literature data on related marine natural antifoulants and antifouling hybrids as a potentially general strategy for generation of improved antifoulants.

  • 234.
    Moreno, Renata
    et al.
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Hidalgo, Aurelio
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Cava, Felipe
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Fernández-Lafuente, Roberto
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Guisán, José Manuel
    Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica-CSIC, Campus de Cantoblanco, Madrid, Spain.
    Berenguer, José
    Centro de Biología Molecular 'Severo Ochoa' CSIC-UAM, Campus de Cantoblanco, Madrid, Spain.
    Use of an antisense RNA strategy to investigate the functional significance of Mn-catalase in the extreme thermophile Thermus thermophilus2004In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 22, p. 7804-7806Article in journal (Refereed)
    Abstract [en]

    The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target.

  • 235.
    Moreno-Garcia, Jaime
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Microbiology, University of Córdoba, Córdoba, Spain; Department of Chemistry, Umeå University, Umeå, Sweden.
    Jose Martin-Garcia, Francisco
    Ogawa, Minami
    Garcia-Martinez, Teresa
    Moreno, Juan
    Mauricio, Juan C.
    Bisson, Linda F.
    FLO1, FLO5 and FLO11 flocculation gene Expression impacts saccharomyces cerevisiae attachment to penicillium chrysogenum in a Co-immobilization technique2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2586Article in journal (Refereed)
    Abstract [en]

    A reoccurring flaw of most yeast immobilization systems that limits the potential of the technique is leakage of the cells from the matrix. Leakage may be due to weakly adherent cells, deterioration of the matrix, or to new growth and loss of non-adherent daughter cells. Yeast biocapsules are a spontaneous, cost effective system of immobilization whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum, creating hollow spheres that allow recovery and reutilization. This attachment is based on naturally occurring adherent properties of the yeast cell surface. We hypothesized that proteins associated with flocculation might play a role in adherence to fungal hyphae. To test this hypothesis, yeast strains with overexpressed and deleted flocculation genes (FLO1, FLO5, and FLO11) were evaluated for biocapsule formation to observe the impact of gene expression on biocapsule diameter, number, volume, dry mass, and percent immobilized versus non-immobilized cells. Overexpression of all three genes enhanced immobilization and resulted in larger diameter biocapsules. In particular, overexpression of FLO11 resulted in a five fold increase of absorbed cells versus the wild type isogenic strain. In addition, deletion of FLO1 and FLO11 significantly decreased the number of immobilized yeast cells compared to the wild type BY4742. These results confirm the role of natural adherent properties of yeast cells in attachment to fungal hyphae and offer the potential to create strongly adherent cells that will produce adherent progeny thereby reducing the potential for cell leakage from the matrix.

  • 236.
    Morin, Dominique
    et al.
    BRGM, Bureau de Recherches Géologiques et Minières.
    Lips, Andor
    BRGM, Bureau de Recherches Géologiques et Minières.
    Pinches, Tony
    Mintek, Randburg, South Africa.
    Huisman, Jacco
    Paques, El Balk, The Netherlands.
    Frías Gomez, Carlos
    Técnicas Reunidas, Madrid, Spain.
    Norberg, Anders
    Skeria, Skellefteå.
    Eric, Forssberg
    Luleå tekniska universitet.
    BioMinE: integrated project for the development of biotechnology for metal-bearing materials in Europe2006In: Hydrometallurgy, ISSN 0304-386X, E-ISSN 1879-1158, Vol. 83, no 1-4, p. 69-76Article in journal (Refereed)
    Abstract [en]

    Biohydrometallurgy is the offspring of the unexpected union of biotechnology and metallurgy. From specific properties of some extreme biotopes, active principles of interactions between microbial metabolisms and minerals have been extracted to be used as efficient metallurgical processes.

    Many profitable industrial operations based on these bioprocesses have been running to recover copper, gold, uranium or cobalt for instance and many other applications have been designed.

    Europe was quite active in this area in the past, but currently the leadership is in South Africa, America and Australia.

    BioMinE (Biotechnology for Metal-bearing material In Europe) is a large integrated project launched with the support of the European Commission. It is aimed at stimulating synergies between the most relevant universities, research and industrial organisations to develop new concepts in this technical field that allow a better exploitation of the mineral resources in the future.

    The main technical subject is the investigation of the opportunities to apply bioleach processes to primary and secondary resources of metal-bearing materials. The second technical area of the project in terms of effort is the study of the recovery of metals from pregnant bioleach solution using biological reagents. All along the project duration, these investigations are focussed on the relevant resources in Europe screened according to an iterative process. The integration of the innovative pathways of processing will be evaluated up to the pilot scale whenever it is appropriate.

    The Consortium of BioMinE comprises 35 partners from industry (12 including 5 SMEs) research organisations (9) universities (14) and government (2). The participants are from 12 EU member states, from 1 candidate country (Romania), and from South Africa (INCO Country).

    The overall budget of the project is 17.9 million Euros, with a contribution from the European Commission of 11.6 million Euros. Started on November 1, 2004, the project will last 4 years.

    An overview of BioMinE in the general context of the biohydrometallurgy development is the subject of this presentation.

  • 237.
    Morin, Dominique
    et al.
    BRGM, Bureau de Recherches Géologiques et Minières.
    Pinches, Tony
    Mintek, Randburg, South Africa.
    Huisman, Jacco
    Paques, AB Balk, The Netherlands.
    Frías, Carlos
    Técnicas Reunidas, Madrid, Spain.
    Norberg, Anders
    Skeria, Skellefteå.
    Forssberg, Eric
    Luleå tekniska universitet.
    Progress after three years of BioMinE-Research and Technological Development project for a global assessment of biohydrometallurgical processes applied to European non-ferrous metal resources2008In: Hydrometallurgy, ISSN 0304-386X, E-ISSN 1879-1158, Vol. 94, no 1-4, p. 58-68Article in journal (Refereed)
    Abstract [en]

    BioMinE is an integrated project under the sixth framework programme of research supported by the European Commission, which started in November 2004 and will last until October 2008 (Ref. NMP2-CT-2005-500329). It is dedicated to the evaluation of biohydrometallurgy to improve the exploitation of the European non-ferrous metal resources in a sustainable way. At the end of 2007, the Consortium of BioMinE comprised 37 partners from industry (13 including 6 Small or Medium Enterprises), research organisations (8), universities (15), and government (1). The participants are from 13 EU member states and from Serbia and South Africa (INCO Countries). For more details see http://biomine.brgm.fr.

    The three main kinds of resources considered for bioleaching studies are:

    - Copper polymetallics (concentrates and tailings),

    - Zinc polymetallics (zinc and zinc polymetallic concentrates)

    - Secondary wastes (tailings, rock and metallurgical wastes, etc.)

    For each of these resources, amenability studies of application of bioleaching technologies by various approaches have been undertaken or still ongoing. Further processing assessment will be conducted up to the demonstration scale. Technological improvements have been made to apply bioleaching in the context of the European resources in terms of complexity and sustainability requirements. The relevant fundamental studies covering bio-prospecting, molecular ecology, biochemistry, and genetics areas aimed at improving the understanding and the control of the selected technologies have given original results.

    Much progress has also been obtained in the use of the microbial sulfate-reducing process to polish effluents and to recover metals from leachates containing low concentrations of metals. The finding of micro-organisms thriving at low and high temperature, respectively 8 and 65 °C, leads to an extension of the application range of the process. It has been also observed that this process could be pushed down to pH 4.5 and 4 creating opportunities of selective metal recovery as metal sulphides. It has also been demonstrated that sulphate can be removed at high concentrations, as well as arsenic or selenium. The next step in this work is pilot testing. This will allow to determine scale-up criteria and to assess the residual metal concentration under actual conditions.

    The pilot-scale demonstration operations, as well as the techno-economic and comparative sustainability assessments will be achieved during 2008, the last year of the project.

    The prototypes of the learning objects for training about biohydrometallurgy accessible by internet have been elaborated. A public output of this work is accessible at http://wiki.biomine.skelleftea.se/wiki. The basic knowledge thus delivered is aimed at disseminating the understanding of the origins and use of biohydrometallurgy.

    Contacts with mining operators in Europe have been taken and collaboration schemes have been established in various ways according to the respective contexts. When a high potential of technical involvement could be foreseen, a direct participation of the mining operators in the project was favoured, this led to integrate KGHM (Pol), Boliden (Sw) and Copper Institute of Bor (Serbia) into the consortium of partners.

    When no direct technical commitment was conceivable at the first stage, collaboration was established with companies with the most urgent requirement to have access to the relevant resource.

  • 238.
    Mortezaei, Narges
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Singh, Bhupender
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bullitt, Esther
    Boston University School of Medicine.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    P-fimbriae in the presence of anti-PapA antibodies: new insight of antibodies action against pathogens2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, article id 3393Article in journal (Refereed)
    Abstract [en]

    Uropathogenic strains of Escherichia coli establish urinary tract infections by attaching to host epithelial cells using adhesive organelles called fimbriae. Fimbriae are helix-like structures with a remarkable adaptability, offering safeguarding for bacteria exposed to changing fluid forces in the urinary tract. We challenged this property of P-fimbriae by cross-linking their subunits with shaft-specific antibodies and measuring the corresponding force response at a single organelle level. Our data show compromised extension and rewinding of P-fimbriae in the presence of antibodies and reduced fimbrial elasticity, which are important properties of fimbriae contributing to the ability of bacteria to cause urinary tract infections. The reduced elasticity found by cross-linking fimbrial subunits could thus be another assignment for antibodies; in addition to marking bacteria as foreign, antibodies physically compromise fimbrial function. We suggest that our assay and results will be a starting point for further investigations aimed at inhibiting sustained bacterial adhesion by antibodies.

  • 239.
    Muthusamy, Sarala Devi
    et al.
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    Baltar, Federico
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    González, José M.
    Univ La Laguna, Dept Microbiol, Tenerife, Spain.
    Pinhassi, Jarone
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    Dynamics of metabolic activities and gene expression in the Roseobacter clade bacterium Phaeobacter sp. MED193 during growth with thiosulfate2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 22, p. 6933-6942Article in journal (Refereed)
    Abstract [en]

    Metagenomic analyses of surface seawater reveal that genes for sulfur oxidation are widespread in bacterioplankton communities. However, little is known about the metabolic processes used to exploit the energy potentially gained from inorganic sulfur oxidation in oxic seawater. We therefore studied the sox gene system containing Roseobacter clade isolate Phaeobacter sp. strain MED193 in acetate minimal medium with and without thiosulfate. The addition of thiosulfate enhanced the bacterial growth yields up to 40% in this strain. Concomitantly, soxB and soxY gene expression increased about 8-fold with thiosulfate and remained 11-fold higher than that in controls through stationary phase. At stationary phase, thiosulfate stimulated protein synthesis and anaplerotic CO2 fixation rates up to 5- and 35-fold, respectively. Several genes involved in anaplerotic CO2 fixation (i.e., pyruvate carboxylase, propionyl coenzyme A [CoA], and crotonyl-CoA carboxylase) were highly expressed during active growth, coinciding with high CO2 fixation rates. The high expression of key genes in the ethylmalonyl-CoA pathway suggests that this is an important pathway for the utilization of two-carbon compounds in Phaeobacter sp. MED193. Overall, our findings imply that Roseobacter clade bacteria carrying sox genes can use their lithotrophic potential to gain additional energy from sulfur oxidation for both increasing their growth capacity and improving their long-term survival.

  • 240.
    Muthusamy, Saraladevi
    Linnéuniversitetet, Institutionen för biologi och miljö (BOM).
    Functional Profiling Of Metabolic Regulation In Marine Bacteria2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Oceans are powered by active, metabolically diverse microorganisms, which are important in regulating biogeochemical cycles on Earth. Most of the ocean surface is often limited by nutrients, influencing bacterial growth and activities. Bacterial adaptation to fluctuating environmental conditions involves extensive reprogramming, and redirection of bacterial metabolism and physiology. In this thesis, I investigated the molecular mechanisms of bacterial adaptation strategies to sustain their growth and survival, focusing on the regulation of gene and protein expression in heterotrophic marine bacteria.

    Comparative proteomics analyses of the growth and non-growth conditions, uncovered central adaptations that marine bacteria employ to allow them to change their metabolism to support exponential growth in response to nutrients and to readjust to stationary phase under nutrient limitation. Our results highlight that during nutrient rich conditions three distinct bacteria lineages have great similarities in their proteome. On the other hand, we observed pronounced differences in behavior between taxa during stationary phase.

    Analyses of the proteorhodopsin containing bacterium Vibrio sp. AND4 during starvation showed that significantly improved survival in the light compared to darkness. Notably, proteins involved in promoting cell vitality and survival had higher relative abundance under light. In contrast, cells in the dark need to degrade their endogenous resources to support their basic cellular demands under starvation. Thus, light strongly influences how PR-containing bacteria organize their molecular composition in response to starvation.

    Study of alternative energy generation metabolisms in the Alphaproteobacteria Phaeobacter sp. MED193 showed that the addition of thiosulfate enhanced the bacterial growth yields. Concomitantly, inorganic sulfur oxidation gene expression increased with thiosulfate compared to controls. Moreover, thiosulfate stimulated protein synthesis and anaplerotic CO2 fixation. These findings imply that this bacterium could use their lithotrophic potential to gain additional energy from sulfur oxidation for both improving their growth and survival.

    This thesis concludes that analyses in model organisms under defined growth conditions gives invaluable knowledge about the regulatory networks and physiological strategies that ensure the growth and survival of heterotrophic bacteria. This is critically important for interpreting bacterial responses to dynamic environmental changes.

    Moreover, these analyses are crucial for understanding genetic and proteomic responses in microbial communities or uncultivated organisms in terms of defining ecological niches of planktonic bacteria

  • 241.
    Nakao, Ryoma
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan.
    Myint, Si Lhyam
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Enhanced Biofilm Formation and Membrane Vesicle Release by Escherichia coli Expressing a Commonly Occurring Plasmid Gene, kil2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2605Article in journal (Refereed)
    Abstract [en]

    Escherichia coli is one of the most prevalent microorganisms forming biofilms on indwelling medical devices, as well as a representative model to study the biology and ecology of biofilms. Here, we report that a small plasmid gene, kil, enhances biofilm formation of E coli. The kil gene is widely conserved among naturally occurring colicinogenic plasmids such as ColE1 plasmid, and is also present in some plasmid derivatives used as cloning vectors. First, we found that overexpression of the kil gene product dramatically increased biofilm mass enriched with extracellular DNA in the outer membrane-compromised strain RN102, a deep rough LPS mutant E. coli K-12 derivative. We also found that the kil-enhanced biofilm formation was further promoted by addition of physiologically relevant concentrations of Mg2+, not only in the case of RN102, but also with the parental strain BW25113, which retains intact core-oligosaccharide LPS. Biofilm formation by kil-expressing BW25113 strain (BW25113 kil+) was significantly inhibited by protease but not DNase I. In addition, a large amount of proteinous materials were released from the BW25113 kil+ cells. These materials contained soluble cytoplasmic and periplasmic proteins, and insoluble membrane vesicles (MVs). The kil-induced MVs were composed of not only outer membrane/periplasmic proteins, but also inner membrane/cytoplasmic proteins, indicating that MVs from both of the outer and inner membranes could be released into the extracellular milieu. Subcellular fractionation analysis revealed that the Kil proteins translocated to both the outer and inner membranes in whole cells of BW25113 kil+. Furthermore, the BW25113 kil+ showed not only reduced viability in the stationary growth phase, but also increased susceptibility to killing by predator bacteria, Vibrio cholerae expressing the type VI secretion system, despite no obvious change in morphology and physiology of the bacterial membrane under regular culture conditions. Taken together, our findings suggest that there is risk of increasing biofilm formation and spreading of numerous MVs releasing various cellular components due to kil gene expression. From another point of view, our findings could also offer efficient MV production strategies using a conditional kil vector in biotechnological applications.

  • 242. Nancucheo, Ivan
    et al.
    Rowe, Owen F.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, Viikki Biocenter 1, University of Helsinki, Helsinki, Finland.
    Hedrich, Sabrina
    Johnson, D. Barrie
    Solid and liquid media for isolating and cultivating acidophilic and acid-tolerant sulfate-reducing bacteria2016In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 363, no 10, article id fnw083Article in journal (Refereed)
    Abstract [en]

    Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro. The main features of the 'standard' solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors.

  • 243. Netterling, Sakura
    et al.
    Bäreclev, Caroline
    Vaitkevicius, Karolis
    Johansson, Jörgen
    RNA Helicase Important for Listeria monocytogenes Hemolytic Activity and Virulence Factor Expression.2015In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 84, no 1Article in journal (Refereed)
    Abstract [en]

    RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain.

  • 244.
    Nilsson, Kristina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Jäger, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    An unmodified wobble uridine in tRNAs specific for Glutamine, Lysine, and Glutamic acid from Salmonella enterica Serovar Typhimurium results in nonviability-Due to increased missense errors?2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 4, article id e0175092Article in journal (Refereed)
    Abstract [en]

    In the wobble position of tRNAs specific for Gln, Lys, and Glu a universally conserved 5-methylene- 2-thiouridine derivative (xm(5) s(2) U34, x denotes any of several chemical substituents and 34 denotes the wobble position) is present, which is 5-(carboxy) methylaminomethyl- 2-thiouridine ((c) mnm(5) s(2) U34) in Bacteria and 5-methylcarboxymethyl-2-thiouridine (mcm(5) s(2) U34) in Eukarya. Here we show that mutants of the bacterium Salmonella enterica Serovar Typhimurium LT2 lacking either the s(2) - or the (c) mnm(5) -group of (c) mnm(5) s(2) U34 grow poorly especially at low temperature and do not grow at all at 15 degrees C in both rich and glucose minimal media. A double mutant of S. enterica lacking both the s(2)- and the (c) mnm(5)-groups, and that thus has an unmodified uridine as wobble nucleoside, is nonviable at different temperatures. Overexpression of tRN(cmnm5s2UUG)(AGln) lacking either the s(2) - or the (c) mnm(5)-group and of tRNA(mnm5s2UUU)(Lys) lacking the s(2) -group exaggerated the reduced growth induced by the modification deficiency, whereas overexpression of tRNA(mnm5s2UUU)(Lys) lacking the mnm(5)-group did not. From these results we suggest that the primary function of cmnm(5) s(2) U34 in bacterial tRNA(cmnm5s2UUG)(Gln) and mnm(5) s(2) U34 in tRNA(Lys) (mnm5s2UUU) is to prevent missense errors, but the mnm(5) -group of tRNA(Lys) (mnm5s2UUU) does not. However, other translational errors causing the growth defect cannot be excluded. These results are in contrast to what is found in yeast, since overexpression of the corresponding hypomodified yeast tRNAs instead counteracts the modification deficient induced phenotypes. Accordingly, it was suggested that the primary function of mcm(5) s(2) U34 in these yeast tRNAs is to improve cognate codon reading rather than prevents missense errors. Thus, although the xm(5) s(2) U34 derivatives are universally conserved, their major functional impact on bacterial and eukaryotic tRNAs may be different.

  • 245.
    Nilsson, Kristina
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lundgren, Hans K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hagervall, Tord G.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The cysteine desulfurase IscS is required for synthesis of all five thiolated nucleosides present in tRNA from Salmonella enterica serovar typhimurium2002In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 184, no 24, p. 6830-6835Article in journal (Refereed)
  • 246.
    Normark, Monica
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Winestrand, Sandra
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lestander, Torbjörn A.
    Department of Forest Biomaterials and Technology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine2014In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 14, article id 20Article in journal (Refereed)
    Abstract [en]

    Background: Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results: All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions: Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass.

  • 247. Nuding, Sabine
    et al.
    Gersemann, Michael
    Hosaka, Yoshio
    Konietzny, Sabrina
    Schaefer, Christian
    Beisner, Julia
    Schröder, Björn O.
    Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Germany.
    Ostaff, Maureen J
    Saigenji, Katunori
    Ott, German
    Schaller, Martin
    Stange, Eduard F
    Wehkamp, Jan
    Gastric Antimicrobial Peptides Fail to Eradicate Helicobacter pylori Infection Due to Selective Induction and Resistance2013In: PLoS ONE, E-ISSN 1932-6203, Vol. 8, no 9, article id e73867Article in journal (Refereed)
    Abstract [en]

    Background: Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection.

    Materials and Methods: We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy.

    Results: H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples.

    Conclusion: These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.

  • 248.
    Nydahl, Anna
    et al.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Panigrahi, Satya
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Environment & Safety Division, Indira Gandhi Centre for Atomic Research, Kalpakkam, India.
    Wikner, Johan
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Science and Technology, Umeå Marine Sciences Centre (UMF).
    Increased microbial activity in a warmer and wetter climate enhances the risk of coastal hypoxia2013In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 85, no 2, p. 338-347Article in journal (Refereed)
    Abstract [en]

    The coastal zone is the most productive area of the marine environment and the area that is most exposed to environmental drivers associated with human pressures in a watershed. In dark bottle incubation experiments, we investigated the short-term interactive effects of changes in salinity, temperature and riverine dissolved organic matter (rDOM) on microbial respiration, growth and abundance in an estuarine community. An interaction effect was found for bacterial growth, where the assimilation of rDOM increased at higher salinities. A 3 °C rise in the temperature had a positive effect on microbial respiration. A higher concentration of DOM consistently enhanced respiration and bacterial abundance, while an increase in temperature reduced bacterial abundance. The latter result was most likely caused by a positive interaction effect of temperature, salinity and rDOM on the abundance of bacterivorous flagellates. Elevated temperature and precipitation, causing increased discharges of rDOM and an associated lowered salinity, will therefore primarily promote bacterial respiration, growth and bacterivore abundance. Our results suggest a positive net outcome for microbial activity under the projected climate change, driven by different, partially interacting environmental factors. Thus, hypoxia in coastal zones may increase due to enhanced respiration caused by higher temperatures and rDOM discharge acting synergistically.

  • 249.
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pathogen entry mechanisms and endocytic responses to plasma membrane damage2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair.

    During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways.

    Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension.

    In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair. 

  • 250.
    Nygård Skalman, Lars
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore2018In: Biology Open, ISSN 2046-6390, Vol. 7, no 10Article in journal (Refereed)
    Abstract [en]

    Endocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.

2345678 201 - 250 of 383
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