Functional characterisation and Mutational analysis of a bacterial dynamin-like protein, DynA

Membrane remodeling is a dynamic process that occurs in bacterial
cells to facilitate substrate transport and to provide protection
to bacteria during environmental stress. In eukaryotic cells,
membrane remodeling is carried out by dynamin-like proteins (DLPs).
These proteins are involved in diverse membrane-associated
functions such as cargo transport via vesicles, cytokinesis,
division of cell organelles and resistance to pathogens. DLPs are
also conserved in bacterial species; however, their function is
still not clearly understood. The genome of B. subtilis contains a
gene dynA (ypbR), which encodes a large DLP (136 KDa),DynA, that
can tether membranes and induce membrane fusion in vitro. Deletion
of dynA in B. subtilis strain 168 fails to produce any observable
growth phenotype under standard laboratory conditions. B. subtilis
is a soil bacterium and prey to several environmental stress
factors to which laboratory strains are normally not exposed.
Hence, it was conceivable that DynA might be required when bacteria
are exposed to stress. To address this hypothesis, the behavior of
DynA was examined under conditions causing membrane-stress, such as
exposure to antibiotics and phage infection. A strain lacking dynA
showed impaired growth in the presence of sublethal amounts of
antibiotics that target the cell membrane and was more sensitive to
phage infection compared to wild-type strains. Time-lapse
microscopy and fluorescence loss in photobleaching (FLIP)
experiments showed that ΔdynA cells have compromised membrane
remodeling compared to wild-type strain. In conclusion, all results
propose DynA to play a role in protecting the cell membrane under
stress conditions. Also, for the first time, it is shown that a
bacterial DLP contributes to innate immunity of bacteria. DynA not
only has a unique membrane protection function but also distinctive
structural features. A single DynA polypeptide contains two
dynamin-like subunits, each consisting of a GTPase domain and a
dynamin-like stalk region. Both subunits, D1 and D2, share strong
intra-molecular cooperativity to facilitate GTPase activity. Here,
a combination of mutational analysis and subsequent in vivo and in
vitro investigation was applied to further characterise structural
assembly and biochemical properties of DynA. Size-exclusion
chromatography elucidated that DynA dimerisation requires
C-terminal amino acids 591-620. In addition, in vivo localisation,
in vitro lipid-binding and GTPase analysis revealed arginine at
position 512 of DynA to be a key regulator of GTP hydrolysis as
well as lipid-binding. Furthermore, in vivo localisation and
bacterial two-hybrid experiments were employed to confirm
interaction of DynA with putative interaction partners (YneK, YwpG
and YmdA). YneK was found to interact with D1 and YwpG with D1 and
D2 individually, whereas YmdA required a full-length DynA (D1+D2)
for interaction. Taken together, the results presented here greatly
expand on current knowledge regarding functional, biochemical and
structural properties of a bacterial dynamin-like protein (BDLP).
This thesis not only demonstrates the preserved membrane remodeling
function of DLPs in bacteria but also explain their conservation
from bacteria to higher-organisms.