Genome segregation in bacteria: investigating protein dynamics and mechanisms in the cell at single molecule level

Bacterial multidrug resistance is a global burden on human health worldwide.
Large, low copy number plasmids responsible for antibiotic resistance have
evolved sophisticated strategies to ensure their faithful distribution at cell
division. Multidrug resistance plasmids harbour their own survival system, a
partition cassette, which ensures an accurate segregation of the plasmids from
one generation to the next at cell division. When this system malfunctions, the
plasmid is not stably inherited and is ultimately lost. When the plasmid is not
retained, bacteria become sensitive to antimicrobials. The multidrug resistance
plasmid TP228 replicates at low copy number in Escherichia coli. Its partition
cassette encodes two proteins: ParF is an ATPase capable of assembling into
higher order structures and ParG is a DNA-binding protein that associates to a
specific site on the plasmid. By using super-resolution microscopy on live cells,
we have recently shown that ParG-plasmid complexes are trapped within a
three-dimensional ParF meshwork that assembles through the volume of the
bacterial chromosome. When the ParG protein is defective, the ParG-plasmid
complex is excluded from the chromosome volume and lost at the following cell
division. We have proposed a Venus flytrap model as a potential mechanism for
plasmid segregation (1).
This project will investigate the localization of ParFG-plasmid complexes in the
cell and the dynamics of complex formation at single molecule level to shed light
on the mechanistic details underpinning plasmid segregation. The objectives
are:
• setting up new tools for microscopy visualization of ParF/ParG fused to
recently developed fluorescent monomeric proteins such as mNeon and
mScarlet-I by using molecular biology
• using conventional epifluorescence microscopy to ensure that the setup
is working and the expression level of the genes is appropriate
• using Spatial Light Interference Microscopy, SLIM-field (2, 3), to image
proteins at single molecule level and very rapidly (millisecond time
scale), which is relevant to cytoplasmic processes. This approach will
provide insights into the nature of the ParF matrix and importantly into
the dynamics of ParFG complex assembly. System perturbation will be

studied by investigating informative mutants.
• quantifying individual protein molecules within the cell to achieve a
mechanistic understanding of how this multidrug plasmid segregation
system works. This objective will also provide software development
opportunities.
The study will involve molecular biology and high-resolution fluorescence
microscopy in parallel to biochemical and biophysical approaches to visualize
proteins and plasmid positioning, trafficking and segregation in the cell.
1. McLeod B, Allison-Gamble GE, Barge MT, Tonthat NK, Schumacher MA,
Hayes F, Barillà D (2017) A three-dimensional ParF meshwork assembles
through the nucleoid to mediate plasmid segregation. Nucleic Acids Res 45,
3158-3171
2. Reyes-Lamothe R, Sherratt DJ, Leake MC (2010) Stoichiometry and
architecture of active DNA replication machinery in Escherichia coli. Science
328, 498-501
3. Badrinarayanan A, Reyes-Lamothe R, Uphoff S, Leake MC, Sherratt DJ.
(2012) In vivo architecture and action of bacterial structural maintenance of
chromosome proteins. Science 338, 528-531