MtbFtsZ Assembles into Different Filamentous Structures Depending on pH
The previous study of MtbFtsZ reported assembly into long, two-stranded filaments (8
), in contrast to the much shorter, one-stranded protofilaments of EcFtsZ (1
). The study of MtbFtsZ used an assembly buffer at pH 6.5. We confirmed that MtbFtsZ assembly gave a predominantly long, two-stranded filaments at pH 6.5 (). Even in the early stages, the assembly consisted predominantly of short two-stranded filaments. Single protofilaments were also found, but they were rare. However, we found that at pH 7.7 MtbFtsZ assembled predominantly into short, one-stranded protofilaments, very similar to those of EcFtsZ (). At intermediate pH, between 6.7 and 7.4, MtbFtsZ formed a mixture of two-stranded and one-stranded filaments (). If we added 10 mm
calcium to the pH 7.7 assembly buffer, we obtained long, two-stranded filaments or multistranded bundles. If we increased the KAc from 100 to 350 mm
at pH 7.7, about half of the filaments were two-stranded (not shown).
Electron microscopy of MtbFtsZ polymers
Kinetics of Initial Assembly of MtbFtsZ
We labeled MtbFtsZ with fluorescein as donor and with TMR as acceptor. We first tested whether the fluorophore labeling had any effect on assembly using the light-scattering assay at pH 6.5. shows that there was only a small decrease in the assembly plateau measured by light scattering after labeling with fluorescein or TMR, and there was no significant change in kinetics. We then mixed equal parts donor and acceptor and assayed assembly by FRET using the decrease in donor fluorescence as the measure of FRET. Donor fluorescence showed a rapid 3−4% decrease after mixing and before adding GTP. This may reflect a weak dimer formation. This decrease occurred within 1 s (data not shown). Following the addition of GTP, donor fluorescence slowly dropped by 20%, which we attributed to the assembly of filaments (, curve e). The kinetics measured by FRET are very similar to those measured by light scattering. We also checked the labeled protein by electron microscopy and found similar structures assembled by labeled and unlabeled protein (data not shown).
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 μ (more ...)
shows the FRET assay of assembly kinetics of 7 μm
total MtbFtsZ in three different pH buffers: pH 6.5, pH 6.9, and pH 7.7. The initial donor fluorescence before assembly was higher at pH 7.7 than at pH 6.5, consistent with the well established sensitivity of fluorescein emission to pH (20
). We therefore normalized the fluorescence of each curve to the value before adding GTP. The kinetics of MtbFtsZ are slow at all three values of pH, requiring 60−100 s to reach a plateau. This is 10 times slower than assembly of EcFtsZ. The total change in donor fluorescence was significantly larger at pH 6.5 than at pH 7.7. This may be related to the structures of the polymers. In the two-stranded filaments assembled at pH 6.5, FRET can occur from one strand to the other, as well as along each strand.
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 (more ...)
Because the excitation and emission spectra of fluorescein overlap, fluorescein-labeled MtbFtsZ in the absence of acceptor can produce a homo-FRET or quenching signal. This is shown in . The homo-FRET signal is barely detectable at pH 7.7 but is substantial (9% decrease) at pH 6.5. The Förster distance is 4.0 nm for fluorescein homo-FRET versus 5.5 nm for fluorescein-rhodamine FRET. Because the subunit spacing is 4.3 nm along the filament, homo-FRET may be small for one-stranded filaments and stronger across the two-stranded filament. This signal would be especially strong if the labeled Cys-155 residues were facing each other across the center of the two-stranded filament.
We concluded that some fraction of the total FRET signal in was due to homo-FRET of the fluorescein donor, but this did not affect our interpretation. We used the drop in donor fluorescence as a measure of relative assembly, and it is not important whether the decrease was generated by hetero- or homo-FRET.
shows the total change in donor fluorescence from time zero to the plateau, measured at 120 s. (The curves in show a continuous slow decrease after 120 s. This may be because of an enhanced FRET produced by bundling protofilaments or by bleaching. For the present purpose we have ignored this and defined 120 s as the end point of the primary assembly reaction.) The plots show the characteristic feature of a critical concentration, which is 2 μm at pH 6.5 and 3.2 μm at pH 7.7. The different slopes of the lines reflect the difference in absolute FRET signal due to pH noted above and cannot be interpreted quantitatively.
Steady State GTPase Activity of MtbFtsZ
The GTPase activity of MtbFtsZ was much lower than that of EcFtsZ at all pH values. Because the GTPase is zero below the critical concentration, we calculated the hydrolysis rate (GDP FtsZ−1
, ) relative to the FtsZ concentration above the critical concentration (12
). Note that these rates were determined at room temperature. Because our previous GTPase assays were mostly at 37 °C, we compared the rates at the two temperatures for a single preparation of EcFtsZ. The GTPase of EcFtsZ was 2- or 3-fold lower at 22 °C than at 37 °C. This is similar to the previous results of de Boer et al.
), who reported that the GTPase of EcFtsZ is 2-fold higher at 37 °C than at 30 °C.
Summary of GDP-induced disassembly, subunit turnover, and GTPase at steady state
MtbFtsZ hydrolyzed 0.8 and 0.08 GTP/FtsZ/min at pH 7.7 and 6.5 (, ). In our previous study of EcFtsZ mutants we considered a value of 0.13 to be the noise from contaminating GTPases (12
). However, we believe the 0.08 value measured here is valid because the His tag provided an extra step of purification, eliminating most of the contaminating GTPases. Previous studies of MtbFtsZ at pH 6.5 have reported values of 0.04 ((8
) (a much lower value was quoted in the text, but we calculated this value from the 13 μm
curve in their Fig. 8) and 0.13 (10
), both in good agreement with our measurement. The data in show that MtbFtsZ hydrolyzes GTP 50 and 9 times slower than does EcFtsZ at pH 6.5 and 7.7.
Kinetics Analysis: MtbFtsZ Assembly Is Cooperative with a Weak Dimer Nucleus
We measured assembly kinetics over a range of protein concentration at both pH 6.5 and 7.7 (). Assembly at both pH 6.5 and 7.7 could be fit with the same kinetic model that fit EcFtsZ. This comprised three steps: monomer activation, formation of a weak dimer nucleus, and elongation (see Scheme 1
under “Experimental Procedures”). The model and fitting procedure are described in detail in our previous papers (1
). The main difference between EcFtsZ and MtbFtsZ (at pH 7.7) is that MtbFtsZ has a much slower rate constant for elongation. lists the kinetic parameters derived from the fitting and compares them to the parameters determined previously for EcFtsZ.
Assembly kinetics and fitting results at different concentrations of MtbFtsZ in MMK buffer (a) and HMK buffer (b)
Comparison of the kinetic parameters, critical concentration (Cc), and GTPase activity of MtbFtsZ and EcFtsZ
Protofilament Dynamics at Steady State
A particular advantage of the FRET assay is that it can measure assembly dynamics and subunit turnover at steady state. We did two experiments to measure the assembly dynamics of MtbFtsZ at steady state. First, we induced disassembly by adding a 20-fold excess of GDP, which blocks the reassembly reaction and lets one observe the rate of disassembly. During the depolymerization processes, FRET is lost and donor fluorescence increases. The depolymerization of MtbFtsZ fit a single exponential decay (), F(t) = Feq +ΔF·e−t/τ (where F(t) is the fluorescence at time t, Feq is the final equilibrium fluorescence, ΔF is the change in fluorescence from time zero to equilibrium, and τ is the characteristic decay time; the half-time of the reaction is t½ = τ·ln2). The decay half-time was 28 s at pH 7.7 and increased to 131 s at pH 6.5 (). In a similar experiment EcFtsZ showed a half-time of 4 s (, bottom panel).
GDP-induced disassembly (a) and the rate of subunit turnover measured by FRET techniques (b)
The second experiment directly measured the subunit turnover at steady state by mixing separate pools of preassembled donor and acceptor protofilaments. Initially there was no FRET (except for some homo-FRET by fluorescein) because the donors and acceptors were on separate protofilaments. FRET developed as the protofilaments disassembled and reassembled into mixed polymers. shows the change in the FRET signal over time in three different pH buffers: pH 6.5, 6.9, and 7.7. These curves were all fit well by single-exponentials. The half-time of MtbFtsZ turnover was about 42 s at pH 7.7 and 163 s at pH 6.5. The turnover of EcFtsZ was much faster with a half-time of 5 s at pH 7.7 (, bottom panel). Also, we found that at pH 6.5 only about half of the MtbFtsZ subunits exchanged. Thus, in addition to the slow turnover, half of the subunits remained stably in their original two-stranded filaments and did not exchange.
We repeated these experiments with different total concentrations of protein. 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 tested both the GDP-induced disassembly and the subunit turnover at 7, 10, and 15 μm MtbFtsZ. The rates were essentially identical at the three concentrations (at pH 7.7 and 7, 10, and 15 μm MtbFtsZ, the half-times for GDP-induced disassembly were 28, 28, and 25 s and turnover was 42, 42, and 41 s, respectively).
In Vivo Dynamics of the Z-ring in M. smegmatis
We used FRAP to examine the dynamics of MtbFtsZ polymers in vivo
. As we were not able to culture M. tuberculosis
strains directly for this study, instead we performed FRAP on two strains of M. smegmatis
. Both strains express M. smegmatis
FtsZ (MsmFtsZ) from the genome. The first strain, mC2
-79, also expresses MsmFtsZ fused to green fluorescent protein (GFP) for detection purposes, and the second strain, mC2
-11, expresses MtbFtsZ fused to GFP. When induced by acet-amide, the FtsZ-GFP level was higher than that from genomic expression (17
). In the present study we did not induce with acetamide but relied on the leakiness of the promoter. In this case the FtsZ-GFP is about half the level of the genomic MsmFtsZ (22
One representative FRAP time series for MsmFtsZ is shown in . As in our previous FRAP studies, we attempted to bleach half of a Z-ring in each case, and we observed a range of recovery half-times (see ). The average half-time for strain mC2-79 (MsmFtsZ-GFP) was 34 s (n = 9) and for strain mC2-11 (MsmFtsZ plus MtbFtsZ-GFP) was 25 s (n = 9). In both cases there was a large spread in the half-times, which is shown in the histograms in . These are not experimental errors but represent real variations from one bacterium to another. We should also note that ~20% of the FRAP series obtained showed very little recovery (less than 10%), and these were not included in the averages given above. Thus the average half-times we report here are likely underestimates.
In vivo FRAP of MsmFtsZ and MtbFtsZ Z-rings in M. smegmatis