Abstract
We have investigated the assembly of FtsZ from Mycobacterium tuberculosis (MtbFtsZ). Electron microscopy confirmed the previous observation that MtbFtsZ assembled into long, two-stranded filaments at pH 6.5. However, we found that assembly at pH 7.2 or 7.7 produced predominantly short, one-stranded protofilaments, similar to those of Escherichia coli FtsZ (EcFtsZ). Near pH 7, which is close to the pH of M. tuberculosis cytoplasm, MtbFtsZ formed a mixture of single- and two-stranded filaments. We developed a fluorescence resonance energy transfer assay to measure the kinetics of initial assembly and the dynamic properties at steady state. Assembly of MtbFtsZ reached a plateau after 60-100 s, about 10 times slower than EcFtsZ. The initial assembly kinetics were similar at pH 6.5 and 7.7, despite the striking difference in the polymer structures. Both were fit with a cooperative assembly mechanism involving a weak dimer nucleus, similar to EcFtsZ but with slower kinetics. Subunit turnover and GTPase at steady state were also about 10 times slower for MtbFtsZ than for EcFtsZ. Specifically, the half-time for subunit turnover in vitro at pH 7.7 was 42 s for MtbFtsZ compared with 5.5 s for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ.
Highlights
All measurements were done at room temperature, so the GTPase of EcFtsZ is 2–3 times lower than the values we reported previously at 37 °C [12]
We expected turnover to be independent of the total FtsZ concentration, because once the assembly has reached steady state all protofilaments will reassemble from the pool of subunits at the steady state concentration
We have confirmed most of these observations; assembly of MtbFtsZ is about 10 times slower than EcFtsZ, and produces almost exclusively long, twostranded filaments at pH 6.5
Summary
We developed a fluorescence resonance energy transfer assay to measure the kinetics of initial assembly and the dynamic properties at steady state. Subunit turnover and GTPase at steady state were about 10 times slower for MtbFtsZ than for EcFtsZ. Photobleaching studies in vivo showed a range of turnover half-times with an average of 25 s for MtbFtsZ as compared with 9 s for EcFtsZ. FtsZ of Escherichia coli (EcFtsZ) shows rapid dynamics both in vitro [1] and in vivo [2, 3], with a turnover half-time of about 9 s. The other is a FRET (fluorescence resonance energy transfer)-based assay [1] Both assays showed that assembly of EcFtsZ is a cooperative process with a weak dimer nucleus. Because our FRET assay has several important advantages over light scattering, we used it to study the kinetics and assembly dynamics of MtbFtsZ
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