We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.
The bacterial tubulin FtsZ is the central component of the cell division machinery, coordinating an ensemble of proteins involved in septal cell wall synthesis to ensure successful constriction. How cells achieve this coordination is unknown. We found that in Escherichia coli cells, FtsZ exhibits dynamic treadmilling predominantly determined by its guanosine triphosphatase activity. The treadmilling dynamics direct the processive movement of the septal cell wall synthesis machinery but do not limit the rate of septal synthesis. In FtsZ mutants with severely reduced treadmilling, the spatial distribution of septal synthesis and the molecular composition and ultrastructure of the septal cell wall were substantially altered. Thus, FtsZ treadmilling provides a mechanism for achieving uniform septal cell wall synthesis to enable correct polar morphology.
Bacterial cytokinesis is accomplished by the essential ‘divisome’ machinery. The most widely conserved divisome component, FtsZ, is a tubulin homolog that polymerizes into the ‘FtsZ-ring’ (‘Z-ring’). Previous in vitro studies suggest that Z-ring contraction serves as a major constrictive force generator to limit the progression of cytokinesis. Here, we applied quantitative superresolution imaging to examine whether and how Z-ring contraction limits the rate of septum closure during cytokinesis in Escherichia coli cells. Surprisingly, septum closure rate was robust to substantial changes in all Z-ring properties proposed to be coupled to force generation: FtsZ’s GTPase activity, Z-ring density, and the timing of Z-ring assembly and disassembly. Instead, the rate was limited by the activity of an essential cell wall synthesis enzyme and further modulated by a physical divisome–chromosome coupling. These results challenge a Z-ring–centric view of bacterial cytokinesis and identify cell wall synthesis and chromosome segregation as limiting processes of cytokinesis.
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