2.1 Introduction of LLPS-based compartments into E. coli
Since the establishment of LLPS-based compartment in prokaryotes has not
been recorded in detail in previous studies4, we
introduced several phase modules and recorded condensate nucleation,
growth, and maintenance in E. coli . The major phase module we
tested included FUSLCD (fused in sarcoma low complexity domain) fused
with truncated GCN4. FUSLCD was known for its ability to phase separate
via pi-pi interaction[28, 29], while GCN4 could
further facilitate phase separation via
oligomerization[30] (Figures 2A and 2B). This
fusion protein formed membraneless compartments in the following
experiments. During the dynamic formation process, some condensates were
observed to nucleate around cell poles and move around them (Figure 2C,
Video S1), while others emerged elsewhere in the cytosol, then moved
around along the cellular membrane, and fused with each other,
especially with the ones near cell poles (Figure 2D, Video S2). These
phenomena are much like the behavior of liquid
droplets[31]. Interestingly, nearly all
condensates finally took up the intracellular space in a regular
pattern, much like polar bodies[32], and the
pattern was homogeneous across different cells and different expression
levels of this phase module (Figure 2E, Supplementary Figure 1). Other
phase separating proteins, such as cryptochrome 2 (CRY2), known for its
homo-oligomerization after a period of blue light induction, formed
compartments at only one pole of the cell (Supplementary Figure
2)[33].
To further investigate the mechanism underlying the pole-localized
pattern of LLPS-based compartment, it was attempted to dissect genome
exclusion and pole attraction of protein aggregates by expressing phase
module in elongated E. coli (ΔftsZ ), which had duplicated
genomes but no septum between them. It turned out that the condensates
formed regularly among genomes rather than only targeting the poles
(Supplementary Figure 3). This phenomenon was similar to the results of
Winkler et. al , indicating that the curvature of a cell wall did
not play a role in the pole-localization[34, 35].
Additionally, it was found that the compartments formed only along one
side of the cellular major axis, opposite to the side nucleoids occupied
(Supplementary Figure 3). Combining those results together, it was
reasoned that the unfavorable contact between nucleoid and phase module
and the stochastic movements of the droplet-like condensates could
determine its polar localization.
Then, the fluidity of the compartment formed by the phase module was
investigated, which showed similar recovering capability as that in
eukaryotes (Figure 2F)[36]. Remarkably, the
compartments formed in our experiments were smaller compared to those
formed inside eukaryotic cells or in vitro due to the limited
space inside a bacterial cell[19, 21, 37], which
could be the reason why they showed whole condensate bleaching. The
recovery of fluorescence could be the result of protein exchange from
the other compartments in the cells mediated by diffusing scaffold
proteins in cytosol, instead of the periphery of the bleached site
(Figure 2F). This property indicated that protein (and other molecules)
were allowed to diffuse across the condensate boundary, providing the
basis for POI recruitment[19].