| 1. |
- Dyrhage, Karl, et al.
(författare)
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Genome Evolution of a Symbiont Population for Pathogen Defence in Honeybees
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Ingår i: Genome Biology and Evolution. - 1759-6653.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The honeybee gut microbiome is thought to be important for bee health, but the role of the individual members is poorly understood. Here, we present closed genomes and associated mobilomes of 102 Apilactobacillus kunkeei isolates obtained from the honey crop (foregut) of honeybees sampled from beehives in Helsingborg in the south of Sweden and from the islands Gotland and Åland in the Baltic Sea. Each beehive contained a unique composition of isolates and repeated sampling of similar isolates from two beehives in Helsingborg suggests that the bacterial community is stably maintained across bee generations during the summer months. The sampled bacterial population contained an open pan- genome structure with a high genomic density of transposons. A subset of strains affiliated with phylogroup A inhibited growth of the bee pathogen Melisococcus plutonius, all of which contained a 19.5 kb plasmid for the synthesis of the antimicrobial compound kunkecin A, while a subset of phylogroups B and C strains contained a 32.9 kb plasmid for the synthesis of a putative polyketide antibiotic. This study suggests that the mobile gene pool of A. kunkeei plays a key role in pathogen defence in honeybees, providing new insights into the evolutionary dynamics of defensive symbiont populations.
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| 2. |
- Dyrhage, Karl, et al.
(författare)
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Genome Evolution of a Symbiont Population for Pathogen Defense in Honeybees
- 2022
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Ingår i: Genome Biology and Evolution. - : Oxford University Press (OUP). - 1759-6653. ; 14:11
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Tidskriftsartikel (refereegranskat)abstract
- The honeybee gut microbiome is thought to be important for bee health, but the role of the individual members is poorly understood. Here, we present closed genomes and associated mobilomes of 102 Apilactobacillus kunkeei isolates obtained from the honey crop (foregut) of honeybees sampled from beehives in Helsingborg in the south of Sweden and from the islands Gotland and angstrom land in the Baltic Sea. Each beehive contained a unique composition of isolates and repeated sampling of similar isolates from two beehives in Helsingborg suggests that the bacterial community is stably maintained across bee generations during the summer months. The sampled bacterial population contained an open pan-genome structure with a high genomic density of transposons. A subset of strains affiliated with phylogroup A inhibited growth of the bee pathogen Melissococcus plutonius, all of which contained a 19.5 kb plasmid for the synthesis of the antimicrobial compound kunkecin A, while a subset of phylogroups B and C strains contained a 32.9 kb plasmid for the synthesis of a putative polyketide antibiotic. This study suggests that the mobile gene pool of A. kunkeei plays a key role in pathogen defense in honeybees, providing new insights into the evolutionary dynamics of defensive symbiont populations.
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| 3. |
- Dyrhage, Karl, et al.
(författare)
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Mapping transcriptomics and proteomics data onto a metabolic pathway model of fructophilic lactic acid bacteria
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Fructophilic lactic acid bacteria have been isolated from fructose-rich habitats, such as fruits and fermented food derived from fruit products. Despite their unique biochemical characteristics, no studies of the expression patterns of enzymes involved in the fermentation of fructose have been performed. Here, we report a genome-wide study of expression profiles in Apilactobacillus kunkeei, an obligate fructophilic bacterium isolated from honeybees. Transcriptomics, proteomics and metabolomics data were collected from A. kunkeei strain A1401 at early exponential and early stationary growth in MRS medium supplemented with fructose and mapped onto a metabolic pathway model. The results confirmed high expression levels of enzymes involved in the fermentation of fructose to lactate and acetate during exponential growth. The transcription levels of genes for enzymes involved in the conversion of fructose to glucose-6-phosphate increased about 40-fold during the stationary phase. Likewise, the transcription levels of two operons for enzymes involved in de novo biosynthesis of UMP were upregulated about 30-fold during the shift to stationary phase. Moreover, genes coding for proteins involved in oxidative stress, protein degradation, heat shock and acid shock were highly upregulated during stationary growth. The results serve as an excellent basis for future genetic engineering efforts to exploit the unique biotechnological, ecological and dietary potential of Apilactobacillus kunkeei.
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| 4. |
- Huang, Hsin-Ho, et al.
(författare)
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Analysis of the leakage of gene repression by an artificial TetR-regulated promoter in cyanobacteria
- 2015
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Ingår i: BMC Research Notes. - : Springer Science and Business Media LLC. - 1756-0500. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA-protein interactions.RESULTS: DNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard-Bishop-Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10(-4) cannot lead to detectable variations in the DNA-protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region.CONCLUSIONS: Both theoretical and experimental analyses of the L09 promoter's leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.
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| 5. |
- Mahajan, Mayank, et al.
(författare)
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Evolutionary Remodeling of the Cell Envelope in Bacteria of the Planctomycetes Phylum
- 2020
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Ingår i: Genome Biology and Evolution. - : Oxford University Press (OUP). - 1759-6653. ; 12:9, s. 1528-1548
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.
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| 6. |
- Pandya, Nikhil J, et al.
(författare)
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Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors
- 2018
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 24:5, s. 1218-1230
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Tidskriftsartikel (refereegranskat)abstract
- Lateral diffusion on the neuronal plasma membrane of the AMPA-type glutamate receptor (AMPAR) serves an important role in synaptic plasticity. We investigated the role of the secreted glycoprotein Noelin1 (Olfactomedin-1 or Pancortin) in AMPAR lateral mobility and its dependence on the extracellular matrix (ECM). We found that Noelin1 interacts with the AMPAR with high affinity, however, without affecting rise- and decay time and desensitization properties. Noelin1 co-localizes with synaptic and extra-synaptic AMPARs and is expressed at synapses in an activity-dependent manner. Single-particle tracking shows that Noelin1 reduces lateral mobility of both synaptic and extra-synaptic GluA1-containing receptors and affects short-term plasticity. While the ECM does not constrain the synaptic pool of AMPARs and acts only extrasynaptically, Noelin1 contributes to synaptic potentiation by limiting AMPAR mobility at synaptic sites. This is the first evidence for the role of a secreted AMPAR-interacting protein on mobility of GluA1-containing receptors and synaptic plasticity.
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| 7. |
- Seeger, Christian, 1982-, et al.
(författare)
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Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II
- 2017
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Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 30, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM-binding region of Ca2+/calmodulin-dependent kinase II (CaMKII290-309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity (KDSPR=3M). Calmodulin-CaMKII290-309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity (KDSPR7.1nM). The CaM-Ng interaction had higher affinity under Ca2+-depleted (KDSPR480nM,3.4x105M-1s-1 and k(-1) = 1.6 x 10(-1)s(-1)) than Ca2+-saturated conditions (KDSPR19M). The IQ motif of Ng (Ng(27-50)) had similar affinity for CaM as Ng under Ca2+-saturated conditions (KDSPR=14M), but no interaction was seen under Ca2+-depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng (KDMST890nM) and CaMKII290-309(KDMST190nM) interactions. Although CaMKII290-309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng(27-50), agree with the current model on Ng regulation of Ca2+/CaM signaling.
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| 8. |
- Seeger, Christian, 1982-, et al.
(författare)
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Characterization and composition of membrane vesicles and secreted proteins in Apilactobacillus kunkeei
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Secreted particles, including membrane vesicles, have increasingly been recognized as important for bacterial community functions and host-interaction processes, but their specific compositions and functional roles are debated. In this study, we have characterized the secreted particles of Apilactobacillus kunkeei, a defensive symbiont of honeybees. We cultivated A. kunkeei strains A1401 and A0901 and separated the secreted protein particles from the extracellular membrane vesicles using density gradient ultracentrifugation. A proteomics analysis identified more than 500 proteins in each strain, of which 27 to 45 proteins were relatively more abundant in the cell-free supernatant, including glycoside hydrolases and peptidases. The extracellular transcriptome associated with the membrane vesicles contained a relatively higher fraction of mRNAs derived from highly transcribed operons such as those coding for ribosomal proteins and ATP synthase subunits, whereas highly expressed tRNAs were relatively more abundant in the cellular fraction. Based on these results, we propose that mRNAs for highly expressed proteins are overproduced and that superfluous mRNAs are fragmented, packaged into membrane vesicles and secreted. The results have implications for the utilization of membrane vesicles in A. kunkeei as a delivery tool for small RNA molecules, while also providing more general insights into the role of membrane vesicles in bacteria.
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| 9. |
- Seeger, Christian, 1982-
(författare)
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Revealing Secrets of Synaptic Protein Interactions : A Biosensor based Strategy
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels.The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin.Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery.AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction.The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.
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| 10. |
- Seeger, Christian, 1982-, et al.
(författare)
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The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- The Planctomycetes bacteria have unique cell architectures with heavily invaginated membranes as confirmed by three-dimensional models reconstructed from FIB-SEM images of Tuwongella immobilis and Gemmata obscuriglobus. The subcellular proteome of T. immobilis was examined by differential solubilization followed by LC-MS/MS analysis, which identified 1569 proteins in total. The Tris-soluble fraction contained mostly cytoplasmic proteins, while inner and outer membrane proteins were found in the Triton X-100 and SDS-soluble fractions, respectively. For comparisons, the subcellular proteome of Escherichia coli was also examined using the same methodology. A notable difference in the overall fractionation pattern of the two species was a fivefold higher number of predicted cytoplasmic proteins in the SDS-soluble fraction in T. immobilis. One category of such proteins is represented by innovations in the Planctomycetes lineage, including unique sets of serine/threonine kinases and extracytoplasmic sigma factors with WD40 repeat domains for which no homologs are present in E. coli. Other such proteins are members of recently expanded protein families in which the newly evolved paralog with a new domain structure is recovered from the SDS-soluble fraction, while other paralogs may have similar domain structures and fractionation patterns as the single homolog in E. coli. The expanded protein families in T. immobilis include enzymes involved in replication-repair processes as well as in rRNA and tRNA modification and degradation. These results show that paralogization and domain shuffling have yielded new proteins with distinct fractionation characteristics. Understanding the molecular intricacies of these adaptive changes might aid in the development of a model for the evolution of cellular complexity.
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