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Despite recent advances in our understanding of the structure and function of conjugative Type 4 Secretion Systems (T4SSs), there is still only very scarce data available for the ones from Gram-positive (G+) bacteria. This is a problem, as conjugative T4SSs are main drivers for the spread of antibiotic resistance genes and virulence factors. Here, we aim to increase our understanding of G+ systems, by using bioinformatic approaches to identify proteins that are conserved in all conjugative T4SS machineries and reviewing the current knowledge available for these components. We then combine this information with the most recent advances in structure prediction technologies to propose a structural model for a G+ T4SS from the model system encoded on pCF10. By doing so, we show that conjugative G+ T4SSs likely have more in common with their Gram-negative counterparts than previously expected, and we highlight the potential of predicted structural models to serve as a starting point for experimental design.

Conjugative dissemination of mobile genetic elements (MGEs) among bacteria is initiated by assembly of the relaxosome at the MGE's origin-of-transfer (oriT) sequence. A critical but poorly defined step of relaxosome assembly involves recruitment of the catalytic relaxase to its DNA strand-specific nicking site within oriT. Here, we present evidence by AlphaFold modeling, affinity pulldowns, and in vivo site-directed photocrosslinking that the TraK Ribbon–Helix–Helix DNA-binding protein recruits TraI to oriT through a dynamic interaction in which TraI's C-terminal unstructured domain (TraICTD) wraps around TraK's C-proximal tetramerization domain. Upon relaxosome assembly, conformational changes disrupt this contact, and TraICTD instead self-associates as a prerequisite for relaxase catalytic functions or substrate engagement with the transfer channel. These findings delineate key early-stage processing reactions required for conjugative dissemination of a model MGE.

The increase of antibiotic resistance is a major threat to human health. The spread of mobile genetic elements (MGEs) via conjugation is a major contributor to this problem, especially in hospital settings. Many MGEs encode Type IV Secretion Systems (T4SSs), which are multiprotein complexes that transfer the MGE from donor to recipient cells. T4SSs are versatile systems that exist in all prokaryotes. While most research has focused on T4SSs from Gram negative (G^–) bacteria, it is important to understand the similarities and differences with T4SSs from Gram positive (G⁺) bacteria, given their different cell envelopes. Additionally, there is also variability within G^– T4SSs, which is not yet fully understood.

The aim of this thesis was to explore the diversity of T4SSs, using pKM101 from E. coli (G^–) and pCF10 from E. faecalis (G⁺) as model systems, with a focus on DNA transfer and replication (Dtr) proteins.

We biochemically characterized the relaxase TraI from pKM101, which processes plasmid DNA prior to transfer through the T4SS. We also solved the crystal structure of its transesterase domain with and without its substrate oriT DNA, highlighting its conserved mechanism of action. We further explored the relationship between TraI and the accessory protein TraK, using AlphaFold to predict an interaction involving the TraI CTD. This was confirmed experimentally using in vivo BPA-crosslinking.

Many conjugative plasmids encode single-stranded DNA-binding proteins (SSBs), which are thought to protect DNA during transfer. pCF10 encodes the protein PrgE, which was proposed to be one such SSB. However, our biochemical studies and X-ray crystallography revealed that PrgE is an OB-fold protein with unexpected DNA-binding behavior. While its benefit for the plasmid remains unclear, our functional studies have shown that it does not play a role in conjugation.

Finally, we analyzed the structural diversity of conjugative T4SSs in G^– and G⁺ bacteria, using bioinformatics and structural modelling. This revealed unknown commonalities, which indicate that G⁺ T4SS mating channels are likely more similar in structure to G^– T4SSs than expected.

In summary, this thesis provides new insights into the Dtr proteins that play an integral role in T4SS mediated conjugation, knowledge that hopefully can be used in the fight against hospital acquired infections in the future.

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

Ionophores are antibacterial compounds that affect bacterial growth by changing intracellular concentrations of the essential cations, sodium and potassium. They are extensively used in animal husbandry to increase productivity and reduce infectious diseases, but our understanding of the potential for and effects of resistance development to ionophores is poorly known. Thus, given their widespread global usage, it is important to determine the potential negative consequences of ionophore use on human and animal health. In this study, we demonstrate that exposure to the ionophore monensin can select for resistant mutants in the human and animal pathogen Staphylococcus aureus, with a majority of the resistant mutants showing increased growth rates in vitro and/or in mice. Whole-genome sequencing and proteomic analysis of the resistant mutants show that the resistance phenotype is associated with de-repression of de novo purine synthesis, which could be achieved through mutations in different transcriptional regulators including mutations in the gene purR, the repressor of the purine de novo synthesis pathway. This study shows that mutants with reduced susceptibility to the ionophore monensin can be readily selected and highlights an unexplored link between ionophore resistance, purine metabolism, and fitness in pathogenic bacteria.IMPORTANCEThis study demonstrates a novel link between ionophore resistance, purine metabolism, and virulence/fitness in the key human and animal pathogen Staphylococcus aureus. The results show that mutants with reduced susceptibility to the commonly used ionophore monensin can be readily selected and that the reduced susceptibility observed is associated with an increased expression of the de novo purine synthesis pathway. This study increases our understanding of the impact of the use of animal feed additives on both human and veterinary medicine.

Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.