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Studying the regulation of bacterial conjugative
DNA transfer by NMR spectroscopy
Bacterial conjugation describes the unidirectional transfer of single-stranded DNA of conjugative plasmids (= extra-chromosomal DNA) or chromosome-encoded conjugative elements
from a donor to a recipient cell via direct contact. This way of gene transfer is commonly
used by bacteria for exchanging genetic information, such as for example antibiotic resistance genes. It represents an important driving force for their evolution, but this also means
that conjugative DNA transfer is the main mechanism for the spread of antibiotic resistance,
a meanwhile often encountered problem in several applications, and hence a topic of ongoing interest.
Although the conjugation system is meanwhile well understood, detailed knowledge of
how this whole process is actually initiated is still lacking. Bacterial conjugation was discovered more than 60 years ago for plasmid F (= fertility) in the Escherichia coli strain K12 [1].
Conjugative DNA transfer has been found between Gram negative and Gram positive bacteria, but also between bacteria and eukaryotic plant and fungi cells [2, 3]. Conjugation is initiated, when a pilus, which is formed on the surface of the host cell, attaches to a recipient cell
[4]. This pilus then retracts and enables direct cell-to-cell contact. Subsequently, plasmid
DNA is cut, unwound and transferred through a membrane pore in the donor cell [5].
Proteins necessary for nucleic acid transfer are encoded by the donor plasmid. Most of
these proteins involved in the conjugation process are encoded in the tra (transfer) region
including proteins, which form the relaxosome. This describes a multi-component nucleoprotein complex preparing the DNA for transfer via specific interactions between auxiliary proteins and DNA. The DNA is cut and unwound by TraI [6], a protein that possesses relaxase
and helicase activity, prior to being relocated through presumably the inner membrane pore
protein TraD [7] to the transferosome, which describes a large transmembrane complex
classified as Type IV Secretion System (T4SS). Bacterial T4SS mediates the transfer of
macromolecular substrates, such as the DNA, into various target cells. The DNA-binding to
TraI is facilitated through the binding of auxiliary proteins such as TraY [8, 9], IHF [10] and
TraM [11, 12], via that the relaxosome is interacting with TraD. TraM is neither essential for
the nicking nor for the formation of the conjugative pore but it enhances the reaction preparing the DNA for transfer and is therefore thought to somehow perform the crucial signalling
function that subsequently triggers the DNA transfer.
As to date it is not completely understood how the auxiliary proteins enhance the DNAbinding specificity and bacterial conjugation is initiated, within this project structural studies of
various auxiliary proteins and their interaction with DNA are carried out to provide a more
profound understanding of their biological role.
Recently, there has been shown a connection between the bacterial conjugation (tra)
and plasmid segregation (par) system [13, 14]. This partitioning system plays an important
role during cell division and is thought to position the relaxosome to the T4SS. The role of
this partitioning system encoded by the R1 plasmid parA locus in the initiation of T4mediated nucleoprotein import and export is investigated by one of our collaborators. Particular emphasis is placed on investigating into the partitioning proteins ParR and ParM. Preliminary experiments already showed that lacking ParR and ParM negatively influences the conjugation efficiency. As this is quite likely due to protein-protein and / or protein-DNA interactions, within this project we plan to further investigate the interactions between Par and Tra
components of these two systems by NMR spectroscopy. In detail this means studying the
protein-protein interactions of ParM from the plasmid segregation system with relaxosome
proteins, as well as protein-DNA interactions of ParR with oriT DNA sequences. All these
experiments are aimed at understanding how the relaxosome is being positioned at the
T4SS prior secretion and if / how the partitioning system is mediating this process.
References
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