Method Article
Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.
Bakteriyel hücre bölünmesi esnasında, esas teşkil eden protein FtsZ sözde Z-halka oluşturmak için hücre ortasında toplanır. FtsZ in vitro GTP varlığında, uzun lifler halinde polimerize ve polimerizasyon çeşitli aksesuar proteinler tarafından düzenlenir. FtsZ polimerizasyon yaygın ışık saçılımı, sedimantasyon, GTP hidroliz deneyleri ve elektron mikroskopi dahil olmak üzere temel yöntemler kullanılarak in vitro incelenmiştir. Tampon koşullar etkisi de her iki FtsZ polimerizasyon özellikleri ve düzenleyici proteinlerle etkileşime FtsZ yeteneği. Burada, biz FtsZ polimerizasyon çalışmalar için protokol tanımlamak ve model proteinler olarak Escherichia coli ve Bacillus subtilis FtsZ kullanarak koşulları ve denetimleri doğrulamak. Bir düşük hızlı çökelme tahlili FtsZ polimerleri paket ya tubulate proteinlerle FtsZ etkileşimi çalışma sağlayan sokulur. Geliştirilmiş bir GTPaz deney protokolü test edilmesine olanak tarif edilmektedirfosfat saptama tepki olarak renk gelişimi varyasyonu kaldırılması standart enkübasyon süreleri ile 96 oyuklu bir plaka kurulumu, çeşitli koşullar kullanılarak, zaman içinde GTP hidrolizinin. Işık saçılması çalışmaları ve elektron mikroskopisi için numunelerin hazırlanması tarif edilmektedir. Çeşitli tamponlar FtsZ polimerizasyon çalışmalar için uygun tampon ve pH, tuz konsantrasyonunu belirlemek için kullanılır. KCl yüksek bir konsantrasyonu, deneylerin büyük bir kısmında en iyisidir. Bizim yöntemleri E. değil sadece FtsZ in vitro karakterizasyonu için bir başlangıç noktası sağlar coli ve B. subtilis ama başka bir bakteriden. Bu nedenle, bu buluşun metodları düzenleyici proteinler veya FtsZ polimerizasyonu etkileyen antibakteriyel ilaçların test ile FtsZ etkileşimi çalışmaları için kullanılabilir.
Esansiyel bakteriyel protein FtsZ bakteriyel hücre bölünmesi makine en iyi karakterize edilmiş bir proteindir. FtsZ tubulin prokaryotik homolog olan ve bir GTP bağlı bir şekilde, in vitro olarak polimerize eder. FtsZ bakteri 1,2 onun korunmuş doğası ve teklik nedeniyle yeni antibiyotik için çok cazip bir hedeftir. Hücre bölünmesi başında, FtsZ diğer hücre bölünmesi proteinlerin montajı için bir yapı iskelesi olarak hizmet midcell bir cytokinetic halka oluşturur. Z-halka oluşumu bölme düzlemi doğru lokalizasyonu için çok önemlidir. FtsZ montajı dinamikleri gibi Minc, SepF, Zapa, UgtP, ve Ezra 2 (bakteri türlerine bağlı olarak) gibi çeşitli aksesuar proteinler tarafından düzenlenir. FtsZ polimerizasyon yoğun in vitro ve protofilaments düz, kavisli protofilaments, filament levha, filaman demetleri ve filament tüpler dahil olmak üzere, pek çok farklı yapılarda incelenmiştir des olmuşturmontaj tampon, nükleotid ve deneyde 3 dahil ek protein bağlı cribed. Caulobacter crescentus elektron cryotomography deneyler Z-halkası geniş 4 donatılacak olmadan nispeten kısa, kesintili tek protofilaments monte edilir olduğunu göstermektedir rağmen in vivo FtsZ protofilaments mimarisi henüz tam olarak anlaşılmış değildir.
İn vitro olarak, FtsZ polimerizasyon özellikleri ve düzenleyici proteinleri ile FtsZ etkileşimi reaksiyon tampon bileşimine duyarlıdır. Örneğin, son zamanlarda FtsZ C-terminüsünde SepF için etkileşim sitesi tarif edilen ve bir FtsZ Δ16 arasında C-terminal kesik artık SepF 5 bağlandığını göstermiştir. SepF-FtsZ İşletmeleri etkileşim önceki bir çalışmada, benzer bir FtsZ İşletmeleri Δ16 hala SepF FtsZ üzerinde ikincil bir siteye bağlanan önerdi SepF ile cosedimented kesilmesinden 6. Bu çalışmalar arasındaki fark, pH 6.5 'te cosedimentation yoktu, oysa FtsZ kesik ile SepF hiçbir cosedimentation vardı, tepkime tampon-at pH 7.5 terkibiydi. Gündoğdu et al. SepF fonksiyonel değildir ve pH 6.5 'de görülen cosedimentation FtsZ Δ16 arasında C-terminal kesik ile SepF çökelmesi yerine etkileşimin neden olması muhtemel olduğunu gösteren, pH 6.5 7 çökeltilerini kaydetti. FtsZ polimerizasyonu pH ve KCl konsantrasyonunun etkisi daha önce incelenmiştir. E. polimerleri pH 6.5 coli FtsZ (FtsZ Ec) daha uzun ve daha bol nötr pH 8,9 oluşmuş daha vardır. Tadros et al. K + bağlayıcı FtsZ Ec polimerizasyonu ile bağlantılı ve FtsZ aktivitesi 10 için çok önemli olduğunu belirterek tek değerli katyonların mevcudiyetinde FtsZ Ec polimerizasyonunu inceledik. PH daha critica olduğuSepF önceki örnek ve FtsZ 11 MINC inhibitör etkisinin pH bağımlılığı ile gösterildiği gibi diğer proteinler ile FtsZ etkileşimi, incelenmiştir l. PH ve tuz konsantrasyonu hem de diğer proteinler ile FtsZ etkileşimini etkileyebilir gibi, bu FtsZ polimerizasyon çalışmaları için doğru koşulları ve kontrolleri seçmek önemlidir.
Burada ışık saçılması, elektron mikroskobu, sedimantasyon ve GTPaz deneyleri ile FtsZ polimerizasyonu ve GTPaz aktivitesini incelemek için protokolleri açıklanmıştır. Sağ açılı ışık dağılımı gerçek zamanlı 12 FtsZ polimerizasyon okumak için standart bir yöntemdir. Biz sedimantasyon ve GTPaz tahlil için birkaç iyileştirmeler tanıttı. Biz ışık saçılması ve elektron mikroskobu için numune hazırlamak için nasıl ayrıntılı olarak mevcut. FtsZ polimerizasyonu çalışma literatürde kullanılan çeşitli tamponlar test ve her deney için en iyi şartları tarif edilmiştir. Biz de olmalıdır kontrol eden göstermeken iyi veri elde etmek tanıttı.
Bu yöntemler FtsZ polimerizasyon aktivitesi ve basit bir yöntem ve en laboratuvarlarda mevcuttur ekipman kullanılarak başka proteinlerle etkileşim hızlı bir çalışma sağlar. FtsZ polimerizasyonu incelemek için daha sofistike yöntemler var ama genellikle floresan etiketleri 8,13,14 ile daha özel ekipmana erişim ve / veya FtsZ değiştirilmesini gerektirir. Bu yazıda anlatılan basit yöntemler B. FtsZ kullanılarak gösterilmektedir subtilis ve E. coli, en yaygın Gram + ve Gram-model organizmalar. Protokoller başka FtsZ proteine uyarlanabilir. Bu yeni FtsZs, zaman ile ilgili olarak küçük değişiklikler, tampon veya inkübasyon sıcaklığı ile ön deneylere dayalı olarak bir optimum bir sonuç için gerekli olabilir. Burada açıklanan deneyler bu en uygun koşulları bulmakta yardımcı olmalıdır.
Untagged FtsZ proteins were purified as described earlier11,15, dialyzed against 20 mM Tris/ HCl (pH 7.9), 50 mM KCl, 1 mM EGTA, 2.5 mM MgAc and 10% glycerol and stored at -80 °C. Under those conditions the protein may be stored for over 2 years without significant loss of activity. The protein should be stored in 100 µl aliquots to avoid thawing and freezing the sample. After thawing, the sample can be kept at 4 °C for maximum one week. FtsZ is soluble at high concentrations and may be stored at 7-10 mg/ml. It is important to keep the protein concentration high to avoid unwanted effects of storage buffer components in later experiments. Thus, storage concentration should allow for a dilution of FtsZ of at least 10x to bring glycerol concentration below 1% and to minimize the concentration of the other components of the dialysis buffer in the reaction mix. FtsZ polymerization may also be affected by the presence of high sodium9 or imidazole concentrations thus these components must be removed from the polymerization buffer. SepF was purified as described7 and stored in elution buffer at -80 °C. Alternative published procedures for purification of FtsZ from E. coli and B. subtilis as well as references to the procedures for purification of FtsZ from different sources are summarized in Table 1 (see representative results section).
1. Sample Preparation
1 | 50 mM Hepes/ NaOH, pH 7.5 |
2 | 25 mM PIPES/ NaOH, pH 6.8 |
3 | 50 mM MES/ NaOH, pH 6.5 |
4 | 50 mM Hepes/ NaOH, pH 7.5; 50 mM KCl |
5 | 25 mM PIPES/ NaOH, pH 6.8; 50 mM KCl |
6 | 50 mM MES/ NaOH, pH 6.5; 50 mM KCl |
7 | 50 mM Hepes/ NaOH, pH 7.5; 300 mM KCl |
8 | 25 mM PIPES/ NaOH, pH 6.8; 300 mM KCl |
9 | 50 mM MES/ NaOH, pH 6.5; 300 mM KCl |
2. FtsZ Sedimentation Assay
3. FtsZ Sedimentation Assay at Slow Spin
4. Quantification of the FtsZ Sedimentation Assays
5. 90° Light Scattering
6. Transmission Electron Microscopy
7. GTP Hydrolysis Assay
The setup of the experiment is designed in such a way that GTP hydrolysis of FtsZ is stopped after various reaction times by mixing the reaction with malachite green. In this way, the time of the color development of malachite green is the same for each sample.
I | 24 µM FtsZBs, 20 mM MgCl2, polymerization buffer |
II | 12 µM FtsZEc, 20 mM MgCl2, polymerization buffer |
III (control 1) | 24 µM FtsZBs, 2 mM EDTA, polymerization buffer |
IV (control 2) | 12 µM FtsZEc, 2 mM EDTA, polymerization buffer |
A | B | C | D | E | F | G | H | |
1 | III | III | I | I | IV | IV | II | II |
2 | III | III | I | I | IV | IV | II | II |
3 | III | III | I | I | IV | IV | II | II |
4 | III | III | I | I | IV | IV | II | II |
5 | III | III | I | I | IV | IV | II | II |
6 | III | III | I | I | IV | IV | II | II |
7 | III | III | I | I | IV | IV | II | II |
8 | III | III | I | I | IV | IV | II | II |
9 | PS | PS | PS | PS | PS | PS | PS | PS |
10 | PS | PS | PS | PS | PS | PS | PS | PS |
11 | ||||||||
12 | GTP | GTP | GTP | GTP | GTP | GTP | GTP | GTP |
A | B | C | D | E | F | G | H | |
1' | III | III | I | I | IV | IV | II | II |
2' | III | III | I | I | IV | IV | II | II |
3' | III | III | I | I | IV | IV | II | II |
4' | III | III | I | I | IV | IV | II | II |
5' | III | III | I | I | IV | IV | II | II |
6' | III | III | I | I | IV | IV | II | II |
7' | III | III | I | I | IV | IV | II | II |
8' | III | III | I | I | IV | IV | II | II |
9' | PS | PS | PS | PS | PS | PS | PS | PS |
10' | PS | PS | PS | PS | PS | PS | PS | PS |
Purification of FtsZ from different bacterial sources has been described in the literature and is summarized in Table 1.
Source | Method | Modification | Yield obtained [mg/L of culture]> | References |
B. subtilis | 1) Ammonium sulfate precipitation/ion exchange chromatography | no | 40 | This work, 11,15 |
2) Affinity chromatography | His-tag | ND | 17 | |
E. coli | 1) Ammonium sulfate precipitation/ion exchange chromatography | no | 35 | This work, 11,15 |
2) Calcium precipitation, ion exchange chromatography | no | 40 | 18 | |
Methanococcus jannaschii | 1) Affinity chromatography under denaturing conditions/refolding/ammonium sulfate precipitation/gel filtration | His-tag | 1.3 | 19 |
2) Affinity chromatography/ gel filtration | His-tag | ND | 20 | |
Thermotoga maritima | Ion exchange chromatography/gel filtration | no | 6.7 | 19 |
Pseudomonas aeruginosa | Affinity chromatography/gel filtration | Strep-tag, His-tag | ND | 21 |
Mycobacterium tuberculosis | 1) Affinity/ion exchange chromatography | no | ND | 22 |
2) Affinity chromatography/ gel filtration | no | 30 | 23 | |
Aquifex aeolicus | Affinity chromatography/gel filtration | His-tag, C-terminal truncation (331-367) | ND | 24 |
Caulobacter crescentus | Ion exchange chromatography/ammonium sulfate precipitation/gel filtration | no | ND | 25 |
Table 1. FtsZ purification protocols described. ND: not determined.
Sedimentation of FtsZ polymers
Initially, we used two different velocities to spin down FtsZ polymers. We found that only at a velocity of 350,000 x g single polymers of FtsZEc are spun down (Figure 1) whereas at 190,000 x g only bundles of FtsZBs are present in the pellet fraction (data not shown). Therefore 350,000 x g was used in our further experiments. The percentage of polymerized FtsZEc and FtsZBs is similar at 50 mM KCl even though the light scattering experiments revealed a much higher scattering signal for FtsZBs. This is due to bundles formed by FtsZBs which scatter more light than single polymers of FtsZEc. It was not possible to obtain high amount of FtsZ polymers in the pellet fraction in the experiment with 300 mM KCl for both FtsZEc and FtsZBs (Figure 1). We attribute this to a combination of quick disassembly of the FtsZ structures and decreased bundling of the filaments.
Sedimentation of FtsZ-SepF tubules
To analyze the interaction of FtsZ with certain activators sedimentation assays can be performed at lower centrifugation speeds. At this velocity only large structures of FtsZ may be pelleted, e.g. the large tubules formed by SepF rings and FtsZBs filaments5, or the bundles formed by FtsZ and ZapA. We used lower centrifugation (24,600 x g) to demonstrate the feasibility of this approach for the tubules formed by FtsZ and SepF. FtsZ was recovered in the pellet above background levels only when both SepF and GTP were present in the sample (Figure 2), and the presence of SepF does not influence FtsZ GTPase activity7 showing that FtsZ is fully active in the presence of SepF. Specific sedimentation of SepF and FtsZ is roughly 45% of total SepF and 15% of total FtsZ (compared to material sedimenting when GDP is added). This shows that the SepF-FtsZ tubules contain more SepF than FtsZ. This may be because many SepF rings organize the FtsZ-SepF tubules5,7. The exact stoichiometry of SepF-FtsZ in these tubules is not known but our results suggest that there is more SepF present than FtsZ.
FtsZEc and FtsZBs polymerization and bundling properties
To characterize the polymerization efficiency of FtsZBs and FtsZEc in different buffers we analyzed both proteins by 90° angle light scattering. At 50 mM KCl, FtsZBs gives a 20-40-fold higher light scattering signal than FtsZEc depending on buffer pH (Figures 3A and B) confirming results of Buske et al.26 Increasing the KCl concentration in the buffer did not significantly influence the light scattering signal of FtsZEc (Figure 3D) but the signal of FtsZBs decreased ~80-fold at pH 7.5, ~30-fold at pH 6.8 and ~45-fold at pH 6.5 in 300 mM KCl (Figure 3C) compared to buffers with 50 mM KCl (Figure 3A). Disassembly of FtsZ polymers is faster at higher KCl concentration for both proteins (Figures 3C and D). Studies of Pacheco-Gómez et al. show that E. coli FtsZ polymerization and bundling is pH dependent. These authors found that in a buffer with 50 mM KCl the light scattering signal of FtsZ polymerization was higher, and disassembly of FtsZ took longer at pH 6.0 compared to pH 7.0 8. These results are not in agreement with our data from polymerization of FtsZEc at 50 mM KCl (Figure 3B), but it has to be noted that we have used three different buffers (HEPES, MES and PIPES) where Pacheco-Gómez et al. only used MES. Thus, not only the pH, but also buffer composition (ionic strength) affects the kinetics of FtsZ polymers at 50 mM KCl. However, at 300 mM KCl neither buffer composition nor pH influenced FtsZEc assembly in a detectable manner.
A light scattering experiment of FtsZBs in buffers without KCl was not possible due to precipitation of the protein under these conditions. When the concentration of FtsZBs was lowered to 3 µM, precipitation did not occur. However, 3 µM is not the physiological concentration of FtsZ in the cell. In plastic cuvettes, FtsZBs did not precipitate at 12 µM at pH 7.5, but at pH 6.8 and 6.5 FtsZ still precipitated in the absence of KCl.
Morphologies of FtsZ structures from E. coli and B. subtilis
The structures formed by FtsZ were inspected by TEM. FtsZBs assembled into closely compacted polymers that covered the whole grid in all buffers at low salt (Figures 4A-C). FtsZEc formed long filaments, cables and bundles in all buffers at low salt (Figures 4D-F). However, the observable amount of polymers formed by FtsZEc was lower than the amount formed by FtsZBs. In high salt buffers FtsZBs formed longer single-stranded protofilaments which did not associate into bundles (Figures 4G-I). While FtsZBs protofilaments changed structure at higher salt concentration, FtsZEc formed structures indistinguishable from those of low salt buffer (Figures 4J-L). There was no observable pH influence on polymerization of FtsZEc but FtsZBs forms more bundles at pH 6.5 which are visible as closely compacted polymers and sheets (Figure 4B). These results are in accord with our light scattering experiments and previously published TEM work8,11,26.
The GTPase activity of FtsZ at high and low KCl concentrations
The GTP hydrolysis activity of FtsZ was measured under different conditions using a colorimetric assay for free phosphate. As reported previously15 the GTPase activity of FtsZ increased with increasing KCl concentration: depending on the buffer used FtsZBs. showed a 3-7 fold increase, and FtsZEc showed a 1.5-2.5 fold increase in GTPase activity at 300 mM KCl compared to 50 mM KCl. The reduced GTPase activity at 50 mM KCl is due to bundling of FtsZBs filaments. At 50 mM KCl FtsZEc had a 3-6 fold higher GTPase activity than FtsZBs due to quicker disassembly of the FtsZEc polymers. The difference in GTP hydrolysis activity between FtsZBs and FtsZEc was reduced at 300 mM KCl, possibly because of reduced bundling of FtsZBs filaments (Figure 5).
Figure 1. Quantification of FtsZ polymerization by sedimentation. (A) 12 µM FtsZ was polymerized in the presence of 2 mM GTP or GDP at pH 7.5 (black bars), 6.8 (grey bars) or 6.5 (white bars). The amount of protein pelleted was determined by densitometric analysis of Coomassie stained gels. GDP served as a control for a specific sedimentation and the percentage of FtsZ sedimented with GDP was subtracted from the percentage of FtsZ sedimented with GTP to obtain the values plotted in the graph. On the left: FtsZ sedimented at 50 mM KCl, on the right: FtsZ sedimented at 300 mM KCl. (B) Representative results from Coomassie stained gels. Polymerization of FtsZBs (upper gel) and FtsZEc (lower gel) at 50 mM KCl. (S) supernatant, (P) pellet fractions from the experiment. Click here to view larger image.
Figure 2. Sedimentation of SepF/FtsZ tubules at low speed. (A) 12 µM FtsZBs was polymerized with 2 mM GDP (white bars) or GTP (black bars). The amount of protein pelleted was determined by densitometric analysis of Coomassie stained gels. The + and - signs under the x-axis indicate the presence or absence of FtsZ and SepF in the reaction. (B) Representative results from a Coomassie stained gel. Polymerization of FtsZBs in the presence and absence of SepF. As a control SepF without FtsZ was used. Polymerization was carried out with GTP and GDP. (S) supernatant, (P) pellet fractions from the experiment. Click here to view larger image.
Figure 3. Light scattering of 12 μM FtsZEc and 12 μM FtsZBs. FtsZs were assembled in the presence of 2 mM GTP and polymerization was monitored by 90° angle light scattering. Polymerization of FtsZBs (A) and FtsZEc (B) at 50 mM KCl at pH 7.5, pH 6.8, pH 6.5. Polymerization of FtsZBs (C) and FtsZEc (D) at 300 mM KCl at pH 7.5, pH 6.8 and pH 6.5. Click here to view larger image.
Figure 4. Structures of FtsZBs and FtsZEc polymers visualized by electron microscopy. (A-L) Images of 12 µM FtsZBs (A-C and G-I) and FtsZEc (D-F) and (J-L) polymerized with 2 mM GTP. (A-C) FtsZBs in buffer with 50 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (D-F) FtsZEc in buffer with 50 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (G-I) FtsZBs in buffer with 300 mM KCl and pH 7.5, 6.8, and 6.5 respectively. (J-L) FtsZEc in buffer with 300 mM KCl and pH 7.5, 6.8, and 6.5 respectively. Scale bar: 100 nm. Click here to view larger image.
Figure 5. GTP hydrolysis during FtsZ polymerization in 6 different buffers. In all experiments 2 mM GTP was used. As a control sample with no MgCl2 was used. Activity of FtsZ without MgCl2 was subtracted from the activity of FtsZ in the presence of MgCl2. On the left: GTPase activity of FtsZ at 50 mM KCl, on the right: GTPase activity of FtsZ at 300 mM KCl. Click here to view larger image.
We describe a set of methods that allows a quick analysis of FtsZ activity and its interaction with other proteins. Light scattering, sedimentation and GTPase assays as well as electron microscopy have been widely used to study FtsZ polymerization. We have made some improvements to existing protocols, we showed the influence of different conditions on FtsZ assembly, and we propose controls that should be included in FtsZ studies.
We introduce low speed centrifugation to distinguish large structures formed by the association between FtsZ and its interacting proteins from FtsZ polymers. This method shows two advantages over the standard sedimentation assay. First, no background is formed by the FtsZ polymers in the pellet fraction as they are not spun down at 24,600 x g. Second, the amount of FtsZ present in the structure formed with an interacting protein may be calculated from the gel. Two critical steps in this method are the incubation time and the GTP concentration. It is important to centrifuge the large protein structure when it is complete but before it disassembles when all GTP is hydrolyzed. The best control for this study is polymerization of FtsZ with GDP. There is one potential limitation of the assay. FtsZ forms a stable complex with SepF, which can easily be spun down at 24,600 x g. If the sedimentation with another activator or a drug that bundles FtsZ polymers is performed, it may be necessary to adapt the assay. It may be done by changing the incubation time, or increasing the speed of centrifugation.
Proper preparation of the sample is the most important for light scattering experiments. Proteins must be precleared by spinning and all the buffers should be filtered prior to use. If any aggregates are present in the sample, they will disrupt a stable signal obtained from FtsZ polymers. For the analysis of the FtsZ structures by electron microscopy, preparation of a grid is the main step. The time of sample incubation on the grid will have the effect of producing more or less compacted polymers. For bundles of FtsZBs, the time of incubation must be shorter than for FtsZEc and FtsZBs at high KCl concentration. We used a concentration of 12 µM for every sample to be able to compare the results. However, for FtsZBs at 50 mM KCl a lower FtsZ concentration should be used, as 12 µM resulted in a full saturation of the grid. This makes the polymers highly compacted and difficult to detect. Less compacted polymers are better to detect on EM.
The GTPase assay is the only experiment used to study the activity rather than the structures of FtsZ. Mg2+ is necessary for GTP turnover in FtsZ polymers. Thus, in the absence of Mg2+, FtsZ does not hydrolyze GTP. Therefore, a sample with no Mg2+ is the right control in this assay but cations of Mg are present in the FtsZ storage buffer. They may be removed by addition of 1 mM EDTA to the control sample. The critical step in this assay is the incubation time. It is important to stop FtsZ activity after a given time. This is achieved by transferring the FtsZ sample to a malachite green solution in a 96-well plate. However, development of the malachite green color is a continuous process. Thus the measurements must be taken at the same time for every sample. Using a well-planned GTP addition protocol with measurements taken each 30 sec apart in an established order, it is possible to obtain the same incubation and sample handling time for every time point. Another critical step is choosing the concentration of the protein for the experiment. In the experiment we used two different concentrations for FtsZEc and FtsZBs. GTP hydrolysis is much quicker for FtsZEc compared to FtsZBs. The GTPase activity of FtsZEc under chosen conditions and at 12 µM is linear only for maximum 5 min and after that time the hydrolysis rate plateaus. Thus, it is difficult to interpret data from the experiment when performed under these conditions. In this case FtsZEc must be used at lower concentration than FtsZBs to be able to compare activities of both proteins. The GTPase activity of FtsZs from different sources may vary. Thus, the right concentration must be chosen. The concentration for FtsZ polymerization should be well above the critical concentration (in general from 2.5-10 µM). The dynamics of FtsZ assembly and disassembly is also important. Some proteins show a significant lag in polymerization after addition of GTP, as shown for FtsZBs at 50 mM KCl. It is useful to perform the light scattering assay before the GTPase assay to approximate the time of assembly and disassembly of FtsZ polymers. After that, the time of incubation and concentration of protein may be chosen. Since the conditions chosen for FtsZ polymerization are crucial, it is important to use the right pH and KCl concentration in each method. In this work we studied 9 different buffers with pH ranging from 6.5-7.5 and KCl concentrations from 0 M to 300 mM. We noticed that the best condition to analyze FtsZs from B. subtilis and E.coli and their biological activity is at pH that is close to physiological level (7.5) together with a high KCl concentration. At a high KCl concentration, FtsZ has a higher GTPase activity and produces polymers that are better detectable by electron microscopy. We also confirmed that the physiological pH and a high KCl concentration are better for the study of the interaction between FtsZ and regulatory proteins than any other buffers mostly used to study FtsZ assembly. FtsZBs shows a similar activity to FtsZEc when studied at high KCl concentration. In addition, at low salt concentration the influence of pH is more visible than in the buffers with high salt concentration. FtsZ sometimes precipitates when using buffers without KCl, as a result, buffers without salt should be avoided. Sedimentation of FtsZ polymers is low when using buffers with high KCl concentrations. This may be an advantage when studying interactions between FtsZ and proteins that assemble FtsZ filaments such as SepF and ZapA as these higher order structures are easy to detect with centrifugation. In all our experiments we used MgCl2 at a 10 mM concentration. It was shown that a relatively high Mg2+ concentration stabilizes FtsZ polymers and reduces the GTPase activity of FtsZ. In Table 2 results from various studies are summarized describing FtsZ polymerization and GTPase activity at different Mg2+ concentrations using otherwise identical buffer conditions 27. The measured concentration of free cytoplasmic Mg2+ is 0.9 mM3. It should be noticed that GTP will chelate an equivalent amount of Mg2+. Thus, the optimal Mg2+ concentration for GTPase experiments is around 2-2.5 mM, which is close to physiological level3. However, in our experiments we used MgCl2 at a 10 mM concentration to obtain an easily detectable light scattering signal and to stabilize FtsZ polymers during the sedimentation assay.
Although we applied our protocols to FtsZ from the model organisms E. coli and B. subtilis, they can be adapted to FtsZ from any other organism. It has to be noted that the physiological pH, and concentrations of monovalent, and divalent cations differ among organisms. Thus, the optimal conditions for FtsZ polymerization may vary. Differences in doubling time and growth conditions of different bacteria may result in different assembly kinetics of FtsZ and optimal conditions of the experiments. However, our protocol provides a good starting point for the experiments with FtsZs from other organisms. The protocols should be useful for the study of FtsZ with regulatory proteins or the study of effects of small compounds and drugs on FtsZ.
Source | Polymerization [% of FtsZ sedimented] | GTPase [Pi/FtsZ/min] | Mg2+ concentration [mM] | FtsZ concentration [µM] | References |
FtsZEc | ~ 28% | ~ 2.1 | 10 | 12 | This work |
~ 50% | ~ 2.4 | 10 | 12.5 | 27 | |
~ 43% | ~ 3.5 | 5 | 12.5 | 27 | |
~ 27% | ~ 4.6 | 2.5 | 12.5 | 27 | |
ND | ~ 5.4 | 2.5 | 5 | 26 | |
FtsZBs | ~ 30% | ~ 0.8 | 12 | This work | |
~ 52% (with DEAE dextran) | ~ 0.5 | 10 | 10 | 11 | |
ND | ~ 2.25 | 2.5 | 5 | 26 |
Table 2. Effect on Mg2+ on FtsZ polymerization and GTPase. Results from this work compared to published data. All experiments were carried out in 50 mM MES/ NaOH, pH=6.5, 50 mM KCl.
No conflicts of interest declared.
Work in our laboratory is funded by a VIDI grant from the Netherlands Organisation for Scientific research (to DJS). We thank Marc Stuart and the Department of Electron Microscopy at our university, for assistance with and providing access to the transmission electron microscope.
Name | Company | Catalog Number | Comments |
GTP | Roche | 10106399001 | Part 1, 2, 3, 4, 5, 6, 7 |
Thickwall Polycarbonate Tubes | Beckman Coulter | 343776 | Part 2 |
Optima MAX-XP Ultracentrifuge | Beckman Coulter | 393315 | Part 2, 3 |
Polyallomer Tube with Snap-on Cap | Beckman Coulter | 357448 | Part 3 |
AIDA Bio-package, 1D, 2D, FL | Raytest Isotopenmessgeräte GmbH | 15000001 | Part 4 |
Luminescence Image Analyzer LAS-4000 | Fujifilm | Part 4 | |
Thermo Spectronic AMINCO-Bowman Luminescence Spectrometer | Spectronic Instruments | Part 5 | |
Fluorescence Cell | Hellma Analytics | 105-250-15-40 | Part 5 |
Square 400 Mesh, Copper, 100/vial | Electron Microscopy Sciences | G400-Cu | Part 6 |
CM120 Electron Microscope Operating at 120 kV | Philips | Part 6 | |
96 ml x 0.2 ml Plate | BIOplastics | B70501 | Part 7 |
Malachite Green Phosphate Assay Kit | BioAssay System | POMG-25H | Part 7 |
PowerWave HT Microplate Spectrophotometer | BioTek | Part 7 |
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