Assembly dynamics of Mycobacterium tuberculosis FtsZ

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.

FIGURE 1. Electron microscopy of MtbFtsZ polymers

a, at pH 6.5 MtbFtsZ assembled into long, two-stranded filaments. b, at pH 6.9, MtbFtsZ assembled into a mixture of two- and one-stranded filaments. c, at pH 7.7 MtbFtsZ assembled into short, one-stranded filaments. d, when 10 mm calcium was added to the pH 7.7 buffer, MtbFtsZ formed predominantly two-stranded filaments and small bundles but with some one-stranded filaments visible. All experiments contained 10 μm MtbFtsZ and 500 μm GTP. Bar = 100 nm.

FIGURE 2

a−d, light scattering was used to compare the assembly of 10 μm MtbFtsZ before and after labeling with fluorophores. a, unlabeled; b, fluorescein-labeled; c, TMR-labeled; d, a mixture of 5 μm MtbFtsZ-fluorescein and 5 μm MtbFtsZ-TMR. Curve e shows this mixture assayed for assembly by FRET. Light-scattering assays were excited and detected at 350 nm; the FRET assay was excited at 450 nm and detected at 515 nm (assembly is measured by the decrease in donor emission at 515 nm). The FRET signal was normalized to the light-scattering signal. Both light-scattering and fluorescence measurement started immediately after adding GTP.

FIGURE 3

a, MtbFtsZ assembly kinetics measured by FRET with 3.5 μm fluorescein-labeled protein (donor) plus 3.5 μm tetramethylrhodamine-labeled protein (acceptor). Fluorescence at time zero was normalized to the same value, 250. The extent of fluorescence change was greater at lower pH. However, the kinetics of assembly were similar at each pH. b, the homo-FRET signal of fluorescein-labeled MtbFtsZ without acceptor. The protein concentration was 7 μm. c, the equilibrium FRET signal (the absolute change in donor fluorescence from 0 to 120 s) is plotted as a function of FtsZ concentration. The critical concentration is 2 μm in MMK buffer (50 mm MES, pH 6.5, 100 mm KAc, 5 mm MgAc, 1 mm EGTA) and 3.2 μm in HMK buffer (50 mm HEPES, pH 7.7, 100 mm KAc, 5 mm MgAc, 1 mm EGTA). d, MtbFtsZ has different GTPase activity at different pH. The GTPase activity is about 0.08 GTP/min/FtsZ at pH 6.5 and increases to about 0.8 at pH 7.7. The results show an approximately linear increase in GTPase with increasing pH. All measurements were done at room temperature.

FIGURE 4. Assembly kinetics and fitting results at different concentrations of MtbFtsZ in MMK buffer (a) and HMK buffer (b)

The measured donor fluorescence is represented by closely spaced dots. A solid line through each set of data is the computer fit based on the nucleation model. The values of the kinetic parameters giving the best fit are shown in Table 2.

FIGURE 5. GDP-induced disassembly (a) and the rate of subunit turnover measured by FRET techniques (b)

a, 10 μm MtbFtsZ at different pH was assembled to steady state with 100 μm GTP, and disassembly was induced by adding 2 mm GDP. Disassembly caused loss of FRET, indicated here by the increase in donor fluorescence. The solid lines are the single-exponential fit. b, separate preassembled protofilaments of 10 μm MtbFtsZ-fluorescein and MtbFtsZ-TMR were mixed at time zero. The decrease in donor fluorescence is because of the FRET signal that develops when protofilaments disassemble and reassemble into mixed filaments. The solid lines are the single-exponential fit.

FIGURE 6. In vivo FRAP of MsmFtsZ and MtbFtsZ Z-rings in M. smegmatis

a, representative FRAP time series for strain mC²-79 (expressing MsmFtsZ-GFP). The white arrow indicates the half of the ring about to be bleached. b, histogram of FRAP recovery half-times obtained for MsmFtsZ-GFP in M. smegmatis. c, histogram of FRAP recovery times for MtbFtsZ-GFP in M. smegmatis.