Bioinformatic Analysis of Genomic and Proteomic Data from Gemmata

Members of the bacterial phylum Planctomycetes have been claimed to have a compartmentalised cell plan, with cell walls lacking peptidoglycan despite being free-living. These theories have been challenged in recent years, and the nature of the planctomycete cell structure is currently under debate. Yet it remains clear that the planctomycete membranes have unique properties, and are thus likely localisations of evolutional innovation. In this study, proteomes and genomes of four planctomycete species from the Gemmata/Tuwongella clade were investigated with the aim to find candidate genes for functional characterisation. Analysis based on full genome sequencing and mass spectrometry revealed 21 proteins unique to the Gemmata/Tuwongella clade that were present in the proteomes of all four species. The gene coding for one of these was found to be organised in an operon, containing an additional four clade-specific genes, likely related to type II secretion. A planctomycete-specific cell surface signal peptide previously not seen in Gemmata was identified in all four species, with proteins found to have the motif indicating that their cell surface has a strong negative charge. Lastly, the study has revealed evidence suggesting that the planctomycetes have a traditional gram-negative cell wall, contradicting the previously proposed proteinaceous cell wall model

Multi-omics investigation into bacterial evolution

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

Multi-omics investigation into bacterial evolution

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

Bioinformatic Analysis of Genomic and Proteomic Data from Gemmata

Members of the bacterial phylum Planctomycetes have been claimed to have a compartmentalised cell plan, with cell walls lacking peptidoglycan despite being free-living. These theories have been challenged in recent years, and the nature of the planctomycete cell structure is currently under debate. Yet it remains clear that the planctomycete membranes have unique properties, and are thus likely localisations of evolutional innovation. In this study, proteomes and genomes of four planctomycete species from the Gemmata/Tuwongella clade were investigated with the aim to find candidate genes for functional characterisation. Analysis based on full genome sequencing and mass spectrometry revealed 21 proteins unique to the Gemmata/Tuwongella clade that were present in the proteomes of all four species. The gene coding for one of these was found to be organised in an operon, containing an additional four clade-specific genes, likely related to type II secretion. A planctomycete-specific cell surface signal peptide previously not seen in Gemmata was identified in all four species, with proteins found to have the motif indicating that their cell surface has a strong negative charge. Lastly, the study has revealed evidence suggesting that the planctomycetes have a traditional gram-negative cell wall, contradicting the previously proposed proteinaceous cell wall model

Inhibition study of Melissococcus plutonius

Results from inhibition assay of cell-free supernatants from Apilactobacillus kunkeei strains showing the presence/absence of inhibitory potency against Melissococcus plutonius. Experiments were performed in biological triplicates. Data is used for manuscript "Dyrhage et al. Genome Evolution of a Symbiont Population for Pathogen Defence in Honeybees, Genome Biology and Evolution, 2022." # FIGURES Summary figures in .pdf format of the inhibition experiments (EXP-21-BQ4077_results_211007.raw.pdf, EXP-21-BQ4077_results_220909.raw.pdf). # IMAGES Raw image files (.jpg from digital camera, .scn and .tif from BioRad ChemiDoc MP Imaging system). # SUMMARY Information about each experiment (strain, predicted plasmid, date, plate and image file) summarised in inhibition.analysis.summary.xlsx.</p

Genomic annotations and comparative analysis

Scripts and additional data necessary for the analyses performed in the paper "Genome Evolution of a Symbiont Population for Pathogen Defence in Honeybees". Information about specific analyses can be found in the corresponding README file, when applicable. Directories are named after the analysis in the paper: fig02_phylogeny Contains a pipeline written in Snakemake that takes a set of genome annotations in the GenBank format, groups all protein sequences with OrthoMCL, filters out genes predicted to be recombinant by Phipack, creates a trimmed concatenation of the remaining single-copy panorthologs, and reconstructs a phylogeny using IQ-TREE. The concatenated protein sequences used in the paper are included (singlecopypanorthologs.fasta). fig03_pangenome Contains a script for analysing the species pangenome, using the OrthoMCL clustering created in fig02_phylogeny. fig04_Sfig03_transposons Contains a script for plotting the location of transposons within A. kunkeei genomes. fig05_ExEs Contains scripts showing the workflow that was used to select plasmid assemblies in the study. fig06_LPxTG Contains a script for plotting the presence/absence-patterns of genes containing cell surface-binding LPxTG motifs in A. kunkeei strains.  fig07_ExEs_growth Contains a script for plotting the presence/absence of extrachromosomal elements in A. kunkeei strains. phageplasmid_classification Contains a script showing the workflow used to classify two phage-plasmids present in the A. kunkeei population, using data from Pfeifer et al. (2021). https://doi.org/10.1093/nar/gkab064 prokka_annotations Contains a script that runs prokka twice, once with the standard databases and once using a manually curated A. kunkeei annotation, then combines the result. tableS4_orthogroups Contains a script that compiles results from the analyses described above for each orthogroup (OrthoMCL results from fig02_phylogeny) and writes it as a table.  tableS5_ANI Contains a script for calculating average nucleotide identities between A. kunkeei genomes. Sfig02_growth Contains a script for plotting growth curves for A. kunkeei strain H3B2-03M. Sfig04_defence Contains a script for plotting the location of a genomic defence island in A. kunkeei, which either contains a CRISPR-CAS system of a restriction-modification system. table_16S Contains a script for calculating 16S identities between A. kunkeei strains. data Contains results from EggNOG, and Phaster, as well as some of the output from the analyses above. Specifically, it contains the annotations from prokka_annotations in GenBank format, the OrthoMCL clustering and the phylogeny in Nexus file format from fig02_phylogeny, which are used as input in other analyses.</p

The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns

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.De två första författarna delar förstaförfattarskapet.</p

Supplementary information for manuscript "<i>Apilactobacillus kunkeei</i><i> </i><i>releases RNA-associated membrane vesicles and proteinaceous nanoparticles</i>"

Supplementary information for manuscript "Apilactobacillus kunkeei releases RNA-associated membrane vesicles and proteinaceous nanoparticles".The supplementary information comprises imaging data obtained from Transmission (TEM) and Scanning Electron Microscopy (SEM) as well as negative stain TEM (nsTEM). Electron micrographs have been obtained from whole cells of two Apilactobacillus kunkeei strains, A0901 and A1401, by TEM and SEM, as well as from isolated secreted nanoparticles (nSTEM). Those particles have been described as membranous RNA-associated membrane vesicles (MVs) and proteinaceous extracellular particles (ECPs).The compressed folder contains the imaging files. Files and subdirectories are described in manifest.txt. The imaging folder contains a file, file.description.ecp.imaging.xlsx, with metainformation on the collected electron microscopy images.</p

Genome Evolution of a Symbiont Population for Pathogen Defense in Honeybees

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

Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology

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