Abstract
It cannot be overstated that antimicrobial resistance (AMR) is a global health threat.
Underpinning the spread of AMR is the movement of mobile genetic elements such as plasmids,
which code for genes that give rise to antibiotic resistance among human pathogens. Bacterial
conjugation is a process which plays a key role in mediating the spread of AMR, which is
accomplished via the macromolecular machinery complex, the type IV secretion system (T4SS). The
T4SS spans both the inner and outer membrane of the bacterial cell wall and also includes the
extracellular conjugative pilus. While, many aspects, such as the proteins involved which assist in
facilitating the process of conjugation, have been elucidated and their roles have been studied there
is still much to understand and discover.
In this work, we used several approaches, including: cryo-electron microscopy (cryo-EM), data
processing approaches such as helical and single particle reconstruction, fluorescent light
microscopy, cryo-electron tomography, focused ion beam milling/ scanning electron tomography, and
correlated light electron microscopy. Using cryo-EM with the helical reconstruction approach we were
able to resolve structures of three conjugation pili, two archaeal (Aeropyrum pernix and Pyrobaculum
calidifontis) and one bacterial from the model system, encoded by the Ti plasmid of Agrobacterium
tumefaciens. We show that the archaeal conjugation pili are homologous to bacterial mating pili, but
function as DNA-import apparatuses. Several published studies state that the stability of the
conjugation pili of A. tumefaciens is because of its cyclic pilin subunits. In this study we show that the
pilin subunit which comprises the A. tumefaciens pili is not cyclic and has a similar fold like the pilins
of the F-pilus and F-like pilus. Further, upon subjecting the T-pilus to extreme chemical and physical
treatment and compared the resiliency to the well-studied F-pilus we found that the F-pilus was more
resilient suggesting that the conjugative pilus is generally stable and is not related to the cyclization.
The conjugation pilus is known to facilitate the interaction between the donor and recipient cell
by bringing them into direct contact with each other allowing the two bacteria to form stable mating
pairs. In previous studies using the model organism Escherichia coli, it was discovered that one of the
essential T4SS components, TraN, from the donor cell interacts with the recipient cells outer
membrane protein A (OmpA). It was not clear how TraN and OmpA were interacting with each other
to support the stabilization of mating pairs, and thus leading to efficient conjugation. In our study we
used cryo-EM with single particle reconstruction to solve the interaction of TraN from the model
organism, carbapenem resistant Klebsiella pneumoniae encoding plasmid pKpQIL, and outer
membrane protein K36 from K. pneumoniae recipients. The structure of the two proteins, solved to
2.6 Å resolution, reveals that loop 3 which is found within the barrel of the OmpK36 porin interacts
with TraN through the insertion of the beta-hairpin tip of TraN into a monomer of the OmpK36 porin
timer. When a glycine-aspartic acid mutation is introduced to loop 3 the loop extends into the barrel of
the porin and there is an observable decrease in bacterial conjugation. The extension of the loop 3
mutation results in a more constricted pore and consequently clashes with the beta-hairpin of TraN.
This in effect causes destabilizes between the TraN and OmpK36 interaction, which lowers the
number of mating pairs, and efficient conjugation.
One question that arose from the study of stable mating pairs was, what other events are
occurring that proceed gene transfer; more specifically to what extent is the conjugative pilus
participating in ssDNA transfer? The longstanding debate is that the conjugation pilus assists in the
transfer of ssDNA, while others believe the only major role of the conjugative pilus is to facilitate the
direct contact of the donor and recipient cells. In this study, we use dam-positive donor, E. coli cells
harboring the pED208 plasmid that belongs to the IncF family isolated from Salmonella typhimurium
that constitutively expresses the tra genes, together with dam-deficient recipient cells that contain the
SeqA-YFP fusion protein that acts as a biosensor for methylated DNA. By labeling the pED208 pilus
TraA subunit with Alexa-Fluor 568 maleimide dye we were able to observe the conjugation pilus with
the fluorescent light microscope. Mixing both donor and recipient cells revealed the direct contact of
the donor conjugation pilus with a physically distant recipient cell, producing an intense fluorescent
foci from the SeqA-YFP binding to the transferred single stranded hemimethylated DNA. From our
results we concluded that the conjugation pilus is capable of acting as a conduit for ssDNA between
physically distant cells and that establishing stable mating pairs is not essential for conjugative
transfer to occur.
