Transposons are selfish, mobile genetic elements, moving within the genome. The transposase genemakes this possible, as it codes for the enzyme that catalyzes the movement. In the case of bacteria,they can also move horizontally between individual bacteria, and sometimes even between species.By default, they are a burden for the host organism, coding for a protein that the host does not need.They also pose the risk of disabling the host’s crucial genes by inserting themselves into it.Transposons are under some pressure to benefit the host, to help propagate themselves moreeffectively. And some transposons have indeed evolved to benefit the host. Lactobacillus kunkeei is a bacterial species known to reside in honeybee guts. It is known for itsrole in honey preservation and wine spoilage. The genome of L. kunkeei is reduced because it is asymbiont, however it contains an unusually high amount of transposons in its genome. In this study, the transposase genes (transposon enzymes) found in L. kunkeei are studied andcategorized. The L. kunkeei have been extracted from honeybees (Apis mellifera). The honeybeesthemselves have been collected from the islands Åland and Gotland. This study focuses on the transposase genes that come in pairs, one after another in the genome.Transposase genes were identified using annotation software and orthology-based methods. Theannotation software provides numbering for the genes, which allows finding paired genes. Thepaired genes were categorized based on alignments and phylogenetic software. Pseudogenizedtransposons were identified based on length and/or clustering into triplets. A total of 766 paired transposase genes were found. The transposase genes were found to take up1.9% of the genome, on average. A low level of diversity has been found when performingalignments and generating phylogenetic trees. The positions of the transposase genes are generallyconserved within phylogenetic groups. Pseudogenization has been detected for some transposasegenes – 4.5 per genome, on average. All of the studied transposons belong to the IS3 family, whichis a family of Class I transposons.

Lactobacillus kunkeei is the most common and dominant bacterium in the honey stomach of honeybees. L. kunkeei has been isolated from honeybees all over the world. Genome sequencing has identified 5 genes for exceptionally large proteins in the genome of L. kunkeei. These proteins do not show any similarity to sequences of proteins with a known structure. These giant proteins all have a conserved region of 60 amino acids in their C-terminus. This conservation led to the hypothesis that the C-terminal domains of the giant proteins are important for their function with possibly a role in the attachment to the cell wall. In this study, a total of eight different constructs were made for two of these giant proteins. The boundaries for the constructs were determined based on bioinformatic predictions. The eight constructs all have different start positions and all end at the very C-terminal end of the protein. These constructs were cloned into an expression vector. One of the full-length giant protein was cloned into an expression vector as well.  The C-terminal constructs and the full-length proteins were recombinantly produced in Escherichia coli. Expression of six C-terminal constructs was observed and an attempt was made to purify two of the C-terminal constructs. Expression of the full-length giant protein was observed as well and purification was attempted. Neither the C-terminal constructs nor the full-length giant protein could be purified at full length. The results for the C-terminal constructs show that no folded C-terminal domain has been found for the giant proteins. A purified protein construct of the N-terminal of one of the giant proteins was available. This protein was analyzed using biophysical techniques. Circular dichroism was used to test the thermal stability. The construct did not refold after being thermally denatured. Circular dichroism measurements indicated that the N-terminal construct is composed of a mixture of α-helices and ß-sheets. Small-angle X-ray scattering data indicated that the N-terminal construct had an elongated shape with knot-like parts. Protein crystals have been obtained for the N-terminal construct and these will be analyzed using X-ray diffraction.

Lactobacillus kunkeei is one of the most abundant bacteria within the honey crop of the honey bee. Genome sequencing of L. kunkeei isolated from honey bees all over the world showed several genes unique for L. kunkeei. Among these orphan genes, an array of four to five highly conserved genes coding for giant extracellular proteins were found. Cryogenic electron microscopy imaging of a giant-protein preparation from L. kunkeei A00901 showed an overall structure similar to a long string with a knot at the end. Further analysis showed high similarity between the different giants at the N-terminus, and secondary structure predictions showed that the same region was rich in β-sheets.  These results, combined with the knowledge of other large extracellular proteins, led to the hypothesis that the “knot” domain is located at the N-terminus and that these proteins are used by the cell to latch on to the intestine lining or other cells in the honey crop. In this study, predictions were made to locate the N-terminal domains of two of these giant proteins. Four different constructs were made for each protein, where three constructs were designed for expression and purification of the N-terminal domain with different end-positions, and one construct was for a predicted β-solenoid domain located downstream from the N-terminal domain. The protein constructs were recombinantly produced in E. coli, and three of the N-terminal constructs from both proteins were purified. Thermal stability was tested using nano differential scanning fluorimetry (nanoDSF), Thermofluor, and circular dichroism (CD), which all showed characteristic melting curves at low melting temperatures, ranging from 33 °C to 44 °C, for all three constructs. During CD measurements, all three constructs showed refolding after thermal denaturation and a higher abundance of antiparallel β-sheets over α-helices. Looking at the protein structure, small angle X-ray scattering data indicated that all three proteins formed elongated structures. These results indicate that a folded domain has been found for both proteins. Although, further analysis will be required to determine the boundaries of the N-terminal domains, and to elucidate if these domains have anything to do with ligand binding and the L. kunkeei ability to latch onto the honey crop.