Evolution and Ecology
bacterial conjugation is known from more than fifty years, and
the molecular mechanism(s) have been exhaustively studied, most
experimental work has been carried out on a small number of
model plasmids. However, with the upcoming of massive DNA sequencing,
a broad range of plasmids and conjugative systems has emerged.
This diversity remains mostly unexplored, since research focused
mainly on pathogenic bacteria of clinical importance (especially
those from Proteobacteria). Therefore, the horizontal
gene pool of Phyla with crucial importance in the biosphere
such as Cyanobacteria and Actinomycetes remains
largely unknown. In this context our specifically aims are:
Classification of conjugative and mobilizable plasmids based
on their relaxase sequences. This would give us a method to
explore the diversity of transferable plasmids.
Conjugative systems of Cyanobacteria and Actinomycetes.
We aim to characterize various transfer systems in these two
phyla and compare them to those of Proteobacteria.
We hope to contribute to the development of biotechnological
tools applicable to the genetic engineering of these Phyla.
and DNA transporters across bacterial membranes
DNA transfer across membranes and between
cells by conjugation is a clear example of a rapid and natural
way to acquire new genetic information, not only between bacteria,
but also between yeast, plants and even animal cells. All conjugative
systems contain a key protein in the membrane to carry out this
process: the DNA transporter. In our system, the DNA transporter
is TrwB and its crystallographic structure has been recently
TrwB, a molecular
motor: The strong structural similarity between
TrwB and other well known molecular motors, such as the ATP
synthase or ring helicases, suggests that TrwB operates as a
motor driving a DNA strand through the transport pore, using
the energy derived from ATP hydrolysis. TrwB is the best model
in a novel group of molecular motors involved in ssDNA transport
across membranes; another example of biological molecular motors
that convert chemical energy into mechanical work.
We work with three ATPases that belong to
the type IV secretion system: TrwB, TrwD and TrwK. These three
motors are inserted in the inner membrane of the cell and are
involved in different functions: DNA transport, protein unfolding
and protein transport through the secretion channel, respectively.
The results obtained with TrwB have enabled us to propose a
common mechanism that could be shared by all members of this
family of ATPases, regardless of their role. Continuing with
this project, we will carry out structural and biochemical analyses
to determine how these proteins use conserved mechanistic principles
to accomplish specific biological actions. In addition, we will
use the new advances in bionanotecnology to study the mechanism
of these nanomachines, by using a system of optical tweezers.
A subsequent reconstitution of these engines into liposomes
will allow us to carry out DNA/protein transport experiments
through lipid vesicles. If we succeed in these goals, these
systems could become an outstanding model for future biotechnological
applications, such as DNA / protein injection into eukaryotic
cells for therapeutic purposes.
Conjugative relaxases as a target for the inhibition
of the dissemination of antibiotic resistance.
DNA transfer allows bacterial evolution and
adaptation to the changing environment. This mechanism is used
for the transfer of antibiotic resistance genes being a huge
health problem. Thus, some pathogenic bacteria are immune to
the actual drugs and this resistance is spreading in a dangerous
and alarming way. The problem is mainly important in hospitals
(also in farms and fish factories) where where the constant
use of antibiotics promotes the generation of resistant strains.
in the USA, there are 2 million cases of hospital infections
per year, 70,000 of which are lethal. The propagation of the
infections is a growing threat not just for the patients, but
for the hospital workers. The economic expense is also incredibly
high with up to ten thousand million USD only in the USA. Regarding
DNA transfer by bacterial conjugation, the protein involved
in the DNA transfer initiation (relaxase) is a key component
in the process. Thus, a detailed knowledge of the biochemistry
of the reaction and the three dimensional structure of the relaxase
could be the way for the finding of new DNA transfer inhibitors.
This work could help the design of new drugs that could prevent
that the bacteria become resistant by the inhibition of the
plasmids are key players in genetic exchange among bacterial
populations, but they are not mere tools for DNA interchange.
Broad-host-range plasmids are self-replicating DNA machines
able to establish themselves in a wide variety of bacterial
species. They are formed by the accretion of genes from a specific
repertoire: the so called Horizontal Gene Pool, and they evolve
mainly by recombination. Although probably connected to host
networks, they have regulatory circuits of their own. Since
they are able to exhibit a robust behavior in radically different
genetic backgrounds, these networks are probably highly orthogonal.
This unique combination of features makes these genetic machines
appealing models for Systems and Synthetic Biology.
We are studying the regulation network of
our model plasmid R388: the smallest conjugative plasmid of
the proteobacteria known to date. Using both in silico and in
vivo experimental tools we have been able to unravel the regulation
circuitry of this plasmid. Our current work focuses on the parametrization
of the network as well as the environmental and genetic signals
from the host the plasmid is able to sense.