| 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. |
- Dyrhage, Karl
(författare)
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Multi-omics investigation into bacterial evolution
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis is the investigation of the evolution and cellular processes of Tuwongella immobilis and Apilactobacillus kunkeei, two bacterial species with different levels of genomic and cellular complexity, using a multi-omics approach.In the first study we examined the proteome of T. immobilis with LC-MS/MS after fractionation by differential solubilisation, yielding fractions corresponding to the cytoplasm, inner membrane, and outer membrane. The experiment was repeated with Escherichia coli and the results were compared. T. immobilis had five times as many predicted cytoplasmic proteins in the most hydrophobic fraction as E. coli. Among these are innovations in the Planctomycetota lineage and protein families that have undergone recent paralogisation followed by domain shuffling, including many enzymes related to information processing.The remaining three studies dealt with honeybee symbiont A. kunkeei. In the first of these, we sequenced and compared the chromosomal and extrachromosomal content of 102 novel A. kunkeei strains. We found that A. kunkeei has an open pangenome and an active set of transposable elements. Within the population we discovered three plasmids between 19.5 and 32.9 kb, one of which codes for enzymes involved in the synthesis of the antimicrobial compound kunkecin A which inhibits growth of the bee pathogen Melisococcus plutonius.In the next study we collected transcriptomic, proteomic, and metabolomic data from two growth phases from A. kunkeei strain A1401 and mapped the results to a metabolic pathway model. Enzymes involved in fermentation of fructose were highly expressed during the exponential growth phase. Enzymes involved in UMP biosynthesis were upregulated during stationary phase, as were protein involved in stress response and detoxification.The last study concerned the secretome of A. kunkeei. We characterised two types of extracellular particles from A. kunkeei strains A1401 and A0901. One type of particle was found to be proteinaceous, while the other type constituted membrane vesicles containing RNA. Comparison of transcriptomic data from the membrane vesicles and whole cells showed that the packing of the RNA was largely untargeted, but with a bias towards highly expressed mRNAs. We suggest that the cell uses membrane vesicles as a mechanism to get rid of superfluous mRNAs after rapid-response overexpression.Together these studies provide insights into the processes driving evolution in T. immobilis and A. kunkeei, and generate several testable hypotheses for future studies.
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| 5. |
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| 6. |
- Liljeruhm, Josefine, et al.
(författare)
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Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
- 2018
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Ingår i: Journal of Biological Engineering. - : BIOMED CENTRAL LTD. - 1754-1611. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.
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| 7. |
- 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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| 8. |
- 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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| 9. |
- Tamarit, Daniel, et al.
(författare)
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Functionally structured genome architectures in Lactobacillus – insights into their variability and evolution
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial genome architectures evolve in response to selective pressures on the interplay between replication and gene expression. Several genomes contain a higher fraction of genes coding for proteins involved in information processes near the origin of replication, which is thought to be due to selection for rapid growth. We recently described a novel type of genome architecture in Lactobacillus kunkeei (Tamarit, et al. 2015). In this genome, vertically inherited genes encoding proteins with roles in translation and replication have accumulated in the chromosomal half surrounding the terminus of replication, while species-specific genes, and genes encoding proteins with metabolic and transport functions have accumulated in the chromosomal half around the origin of replication. Here, we show that this pattern is present also in the closest relatives of L. kunkeei, and similar but not identical biased genome architectures are present in other groups within the Lactobacillaceae. Thus, the biased genome structure in L. kunkeei has emerged from an ancestral clustering of vertically inherited genes around the terminus of replication, while horizontally acquired genes have been inserted near the origin of replication. The genome bias has been lost independently in several groups due to insertions of mobile elements near the terminus of replication and/or major genome rearrangements. We propose chromosomal structuring in macrodomains in the Lactobacillaceae, and suggest that further exploration of its functional consequences and generality will provide valuable insights into the forces that shape genome organization in bacteria.
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| 10. |
- Ye, Weihua, et al.
(författare)
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Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei
- 2024
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 436:6
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee -associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely -related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha -helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re -invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.
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