Design of FtsZ(R85Q) and FtsZ(E83Q) mutants
We choose as candidates for mutation two conserved residues located in the lateral face of FtsZ, specifically in the bend of H3 helix. This was based on the similarities in the crystal structures between FtsZ and tubulin and the degree of conservation of amino acids among FtsZ sequences. The three dimensional structure of an E. coli FtsZ modela (Figure 1A) was compared with the structure of the αβ-tubulin heterodimer. The helix H3 of tubulin, located on the lateral face and known for the role in the lateral interactions, is also present in FtsZ. The helix H3 of FtsZ is located in the left lateral face (left, right, top and bottom face are the nomenclature defined by analogy between FtsZ protofilaments and tubulin microtubules) [27]. Helix H3 is present in most of the FtsZ sequences from gram positive and negative bacteria, mitochondria and chloroplasts. Within the H3 helix, glutamate at position 83 is 100% conserved and the positive charge of arginine at 85 is highly conserved (80% using non-redundant sequences) among FtsZ sequences from 11 different organisms (Figure 1B). Interestingly, the two residues are located in the bend of the helix H3 and are exposed to the surface of the left face. Hence, we hypothesized that these residues, E83 and R85, participate in the interactions of FtsZ to form filaments, tubules or sheets. To this end, the bend in H3 could be necessary to maintain both the topography of the monomer for the interactions and the GTPase activity in the polymer. In microtubules, the lateral interactions are principally mediated between the helix H3 from a protofilament and the M loop of the adjacent protofilament [29]. The M loop is even present in bacterial tubulin A/B, though smaller than its counterpart tubulin, and is also believed to participate in the lateral interactions to form a bacterial microtubules (bMTs) composed by five protofilaments [30]. However, FtsZ lacks the M loop hence, the lateral interactions are expected to be weaker between FtsZ protofilaments [13], hence an effect on longitudinal interactions should affect both the length and the GTPase activity of the polymers.
Viability of E. coliVIP2(DE3) cells in the presence of FtsZ(E83Q) and FtsZ(R85Q)
To evaluate the functionality of the FtsZ mutants in vivo, the E83Q, and R85Q proteins were expressed independently and the cell viability was determined in a strain containing ftsZ thermonull mutant, VIP2(DE3)/pLAR9. The parental strain, VIP2/pLAR9 has been successfully employed to demonstrate that FtsZ is essential for cell division and survival in E. coli[31]. For the purpose of our study we modified the parental strain to create a DE3 lysogen of VIP2, that contains a copy of T7 RNA polymerase gene under the control of PlacUV5, so the expression of a target protein is inducible by IPTG addition. Thus, if VIP2(DE3)/pLAR9 is transformed with a second plasmid that contains the FtsZ mutants (pMFV-derivatives: pMFV-wt, pMFV-rq and pMFV-eq, see Methods), the transformed cells will contain a ftsZ mutant allele from pMFV-derived plasmid under the control of a T7 promoter, a wild-type allele encoded on plasmid pLAR9 (a temperature sensitive replication plasmid) and a genomic null allele. In summary, at the permissive temperature (30°C), VIP2(DE3)/pLAR9/pMFV- will express both ftsZ alleles (from pLAR9 and pMFV-derivatives), whereas at the restrictive temperature (42°C) pLAR9 cannot replicate, so the ftsZ expression originates from the pMFV-derivatives. The production of FtsZ ceases completely after 2–3 h of shifting the temperature, once pLAR9 is segregated by lack of replication. In this regard, this system is different from those utilizing a FtsZ mutant nonfunctional at 42°C, because in this case after shifting to the non-permissive temperature FtsZ will be nonfunctional almost instantly.
To simplify the nomenclature we will refer henceforth to the strain VIP2(DE3), containing pLAR9, as VIP5.
VIP5/pMFV-wt cell grown at 42°C (positive control) and at 30°C show similar cfu/ml in the absence of IPTG, indicating that the basal non-induced expression level from pMFV-wt (a high copy number plasmid) was enough to complement the lack of FtsZ from pLAR9 at 42°C (Figure 2). Hence, in order to avoid an excessive expression of FtsZ, IPTG was not added in all our experiments. As expected, the survival of VIP5 at 42°C (negative control) decreases about four orders of magnitude. The small survival fraction observed in VIP5 is explained as due to the reversion frequency of the chromosomal mutation [31].
Figure 2 shows that the viability of cells expressing EcFtsZ mutants E83Q and R85Q at the restrictive temperature diminished about 1.5 and 3 orders of magnitude, respectively, compared to the non-restrictive temperature. These values indicate that the cells could not support normal cell division but at the same time the EcFtsZ mutants did not completely lose functionality in vivo. Hence, the survival assay was useful to characterize the functionality of EcFtsZ mutants in vivo and the quantification of the viability of the cells expressing EcFtsZ mutants was used as an indication of the efficiency in the cell division. Consequently, the efficiency of the different forms of EcFtsZ in terms of cell viability can be interpreted as: wild type > E83Q > R85Q.
Cell morphology and in situlocalization of FtsZ mutants using immunofluorescence microscopy
The FtsZ ring is formed in the middle of the cell and is attached to the cytoplasmic membrane through ZipA [2]. In order to evaluate the effect of these mutations on cell morphology and FtsZ localization, VIP5 cells in exponential phase of growth carrying plasmids with FtsZ(WT), FtsZ(E83Q), and FtsZ(R85Q) mutants were incubated in the absence of IPTG at 30°C (control) and at 42°C. Figure 3 shows that, in the negative control at 42°C (pLAR9 is lost at this temperature) the cell division is blocked, and FtsZ localization is dispersed along the very long filamentous cells (a’), while at 30°C the cells are normal. Cells carrying the plasmid FtsZ(WT) at 42°C (b’) reverts the cell filamentation, and at 30°C (b) as expected the morphology and FtsZ localization is normal. The co-expression at 30°C of FtsZ(WT) from pLAR9 with the mutants FtsZ(E83Q) (c) and FtsZ(R85Q) (d) also display a normal phenotype. However, the expression of the FtsZ mutants at 42°C (in the absence of FtsZ(WT)) have an effect on cell division, producing filamentous cells (c’ and d’). Interestingly, FtsZ(E83Q) still localizes as discrete bands along the filamentous cells, probably at the potential cell division sites (c’). In contrast, the expression of FtsZ(R85Q) produced shorter filamentous cells with a disperse localization of the protein along the filaments, and no clear bands were observed (d’). The same experiments were performed with IPTG 0.2 mM and similar results were obtained (not shown). In order to keep the cells in exponential phase of growth for a longer time, the morphology experiments were repeated starting the culture for the temperature shift at an OD600 of 0.06. After 3 h of incubation no difference in the cell morphology of the mutants respect to the experiments presented in Figure 3 was observed.
Localization of the FtsZ mutants in the single cell population (not filaments) that appear at early stationary phase (Additional file 1: Figure S1) shows that FtsZ(E83Q) is located with greater intensity at the middle of the cell but presents some degree of dispersion, while FtsZ(R85Q) is rather distributed in a gradient shape from the mid-cell toward the cell poles. The ZipA distribution was similar to that observed for FtsZ mutants (Additional file 1: Figure S2), which suggests that the mutations in FtsZ do not affect its interaction with ZipA. Binding experiments between FtsZ (WT and mutants) and ZipA inserted into proteoliposomes confirmed this hypothesis (not shown).
In summary, the residues glutamate 83 and arginine 85 located in bend of helix H3 on the left face of FtsZ plays a role in the formation of the Z-ring (Figure 3) and consequently ZipA presents an altered localization in cells expressing the FtsZ mutants (Additional file 1: Figure S2).
Structural characterization of R85Q and E83Q
In order to evaluate the impact of the point mutations E83Q and R85Q on the protein structure, we performed circular dichroism on purified FtsZ wt, R85Q and E83Q. All three purified proteins showed the expected molecular mass and had a very similar secondary structure content (not shown); therefore, presumably the mutations did not cause significant changes in the tertiary structure of FtsZ.
GTPase activity of E83Q and R85Q
In order to understand the possible effects of the FtsZ mutations on the formation of the Z ring, we purified the mutant proteins and measured their abilities to hydrolyze GTP. The wild-type FtsZ and E83Q exhibited a constant initial velocity of GTP hydrolysis for at least 10 minutes at a protein concentration of 12.5 μM (Figure 4A). By contrast, R85Q showed a lag time (τ) of 86.3 minutes and a reduced GTPase activity. We next compared the GTP hydrolysis efficiency by calculating the apparent catalytic constants (i.e., the ratio between the initial velocity and the protein concentration). Wild-type FtsZ, E83Q and R85Q hydrolyzed GTP with apparent catalytic constants of 2.90 ± 0.16 min-1, 1.13 ± 0.06 min-1 and 0.13 ± 0.02 min-1 respectively. R85Q was further characterized by measuring the dependence of GTP hydrolysis on protein concentration (Figure 4B). The progress curves show a sigmoidal behaviour, i.e., the lag time was dependent on the protein concentration (inset Figure 4B). This lag time should disappear at the extrapolated concentration of 130 μM. The similarity of the progress curves at 36.8 and 49.0 μM of protein is probably due to GTP becoming limiting. These results suggest that the R85Q mutation does not directly alter the rate of GTP hydrolysis, but rather affects FtsZ polymerization, which is known to induce GTPase activity. Therefore, the effect is reversed when the concentration of R85Q is increased to stimulate polymerization.
The GTPase activity of FtsZ wt and E83Q is not affected by ZipA, which is in agreement with previous study [2]. Interestingly, ZipA increased around twofold the GTPase activity of R85Q (Figure 4C and Additional file 1: Figure S3b), suggesting that ZipA stimulates the longitudinal interactions of R85Q protofilaments during polymerization, similar to the effect of MAPs on microtubules [32].
Polymerization of FtsZ mutants with and without ZipA
In vitro, FtsZ polymerizes into protofilaments in the presence of GTP [33], and these protofilaments associate to form bundles in the presence of ZipA [15]. Single protofilaments are monomers that interact longitudinally whereas double protofilaments (thick filaments) consist of two parallel protofilaments interacting laterally [24]. Antiparallel lateral interactions of these double filaments induce the formation of sheets or bundles [34]. In order to evaluate the effect of the mutations on the polymerization capability, we followed the polymerization of FtsZ mutants by 90° light scattering at 350 nm and the polymers morphology by electron microscopy.
The polymerization reaction was started with the addition of the protein, and the reaction was followed for one hour for wild type and E83Q, while R85Q was followed for almost three hours due to the long lag period observed for the GTPase activity (Figure 4B). The mutant R85Q apparently did not form long filaments at the protein concentration used for FtsZ wild type and E83Q (Figure 5C), producing a small increment in turbidity that was stimulated around two-fold by ZipA Additional file 1: Figure S3a), whereas FtsZ wild type and E83Q showed a fast polymerization kinetics that could not be measured. E83Q formed polymers that remained in solution around three times longer than those formed by FtsZ wild type, with a rate of depolymerization 3.9 times slower. This increase in the time in which the filaments remain polymerized can be explained by the small apparent catalytic constant for the GTPase activity. The time traces of light scattering (Additional file 1: Figure S4) indicate that the polymerization and the subsequent depolymerization of FtsZ are altered by the mutations on the arginine 85 and glutamate 83. Additional file 1: Figure S5 shows that calcium increased the ligth scattering, and for FtsZ wild type and E83Q this was due to the formation of bundles, as shown in Additional file 1: Figure S6. On the other hand, R85Q presented a smaller increase in light scattering and no formation of bundles was observed.
Figure 5A shows the maximum light scattering value reached by FtsZ in 1 mM GTP in the presence and absence of ZipA. The mutants presented a decrease in the polymerization levels of ~20% and ~75% for E83Q and R85Q, respectively. In the presence of ZipA, the maximum light scattering value of FtsZ wild type increased slightly, (~20%, and bundles were observed by electron microscopy, Additional file 1: Figure S6), while for both mutants the increment was significantly larger: 90% and 150% for E83Q and R85Q, respectively (Figure 5A). In the absence of ZipA, FtsZ wild type polymerized into long and mostly straight filaments of 8 to 24 nm width (media around 14 nm) suggesting that the main population of these filaments consists of 4 assembled protofilaments of 4 nm width for each protofilament (Figure 5Ba and Ca'). E83Q polymerized into curved filaments with a width of 8 to 12 nm (Figure 5Bb and Cb'). The greatest effect was caused by the mutation R85Q, in which the polymers were mainly short and straight, and occasionally, spirals probably composed of single or double protofilaments were found on the grids (Figure 5Bc and Cc'). ZipA induced bundling of wt and E83Q, which is in agreement with previous studies [34]; however, we did not observe a clear bundling effect in the R85Q mutant, even given the increase in light scattering. The light scattering contribution of ZipA was subtracted, therefore we can rule out the possibility that the increase in the light scattering of R85Q is due to ZipA. Further characterization of R85Q will be done in a future study.
All the results together indicate that arginine 85 and glutamate 83 participate in the longitudinal and in the lateral interactions, shown by their reduced GTPase activity and the effect on polymerization, respectively. Interestingly, the addition of a stabilizer of the lateral interactions, such as ZipA to R85Q, did not induce bundles (Additional file 1: Figure S6) but stimulated the GTPase activity (Figure 4B and Additional file 1: Figure S3b), indicating that protofilaments are induced. On the other hand, in the case of E83Q, even though the protofilament lateral interactions were diminished (Figure 5B) and ZipA induced bundles that were thinner than those with FtsZ wild type, the GTPase activity was not greatly compromised. Therefore, we believe that the ability of FtsZ to form bundles, presumably through lateral interactions, is important to form the Z-ring in the cell, and consequently for cell division, a conclusion consistent with our immunofluorescence experiments (Figure 3) and cell viability results (Figure 2).