YeeV is an Escherichia coli toxin that inhibits cell division by targeting the cytoskeleton proteins, FtsZ and MreB

Authors


Summary

Toxin–antitoxin (TA) systems of free-living bacteria have recently demonstrated that these toxins inhibit cell growth by targeting essential functions of cellular metabolism. Here we show that YeeV toxin inhibits cell division, leads to a change in morphology and lysis of Escherichia coli cells. YeeV interacts with two essential cytoskeleton proteins, FtsZ and MreB. Purified YeeV inhibits both the GTPase activity and the GTP-dependent polymerization of FtsZ. YeeV also inhibits ATP-dependent polymerization of MreB. Truncated C-terminal deletions of YeeV result in elongation of cells, and a deletion of the first 15 amino acids from the N-terminus of YeeV caused lemon-shaped cell formation. The YeeV toxin is distinct from other well-studied toxins: it directs the binding of two cytoskeletal proteins and inhibits FtsZ and MreB simultaneously.

Introduction

All free-living bacteria studied to date contain a number of toxins that are co-transcribed with cognate antitoxins from single operons and termed toxin–antitoxin or TA operons (Pandey and Gerdes, 2005; Yamaguchi et al., 2009). These TA systems are typically encoded by a small antitoxin (65–85 amino acid residues) and a slightly larger adjacent toxin (95–135 residues) (Brown and Shaw, 2003). TA pairs form a stabilized protein complex in the cell preventing toxicity under normal growth conditions (Engelberg-Kulka et al., 2004; Buts et al., 2005). However, antitoxin stability is typically substantially lower than that of the cognate toxin due to an inherent sensitivity to proteases that are expressed from cellular damage or stress. Because of this characteristic, damage or growth inhibition causing the induction of proteases will alter the balance between the toxin and antitoxin, leading to the toxin being released from the TA complex. The unbound toxins are thus freed to exert damaging effects on the cell.

To date, 37 TA systems have been found in the Escherichia coli genome (Y. Yamaguchi and M. Inouye, unpubl. results). Some of these TA systems have been well characterized and their cellular targets have been identified, including relB-relE (Pedersen et al., 2003), chpBI-chpBK (Zhang et al., 2005), mazE-mazF (Kamada et al., 2003; Zhang et al., 2005), yefM-yoeB (Kamada and Hanaoka, 2005; Zhang and Inouye, 2009), dinJ-yafQ (Motiejunaite et al., 2007; Prysak et al., 2009), hipB-hipA (Wang et al., 2003; Korch and Hill, 2006), hicA-hicB (Makarova et al., 2006; Jorgensen et al., 2009), yafN-yafO (Zhang et al., 2009), mqsR-YgiT (Yamaguchi et al., 2009) and higB-higA (Hurley and Woychik, 2009). The cellular targets of some of these toxins have been identified. For example, RelE, which has no endoribonuclease activity alone, acts as a 70S ribosome-associating factor that promotes mRNA cleavage at the ribosome A site (Neubauer et al., 2009). ChpBK (Zhang et al., 2005), MazF (Zhang et al., 2003) and MqsR (Yamaguchi et al., 2009) function as sequence-specific endoribonucleases or mRNA interferases, cleaving mRNAs and effectively inhibiting protein synthesis and cell growth. Interestingly, all described TA operons appear to use a similar mode of regulation: namely the formation of TA complexes to neutralize toxin activity, and the autoregulation of their operon expression by a protein complex binding to the promoter region.

Here we report a new E. coli TA system, YeeUV, that was initially identified as a putative TA pair based on the characteristic operon arrangement (Brown and Shaw, 2003). These authors demonstrated that YeeV is indeed a toxin that inhibits cell growth. This TA system is intriguing, as its cellular target appears to be very different from that of other TA systems studied to date, as described below. We found that YeeV inhibits cell division, which alters the cell morphology from rod- to lemon-shaped cells. Interestingly, YeeV interacts with FtsZ, a tubulin-like protein, and MreB, a prokaryotic actin-like protein. FtsZ is a highly conserved GTPase (de Boer et al., 1992; RayChaudhuri and Park, 1992), which forms a ring structure at septa and is essential for cell division (Bi et al., 1991). Purified YeeV inhibits the GTPase activity and GTP-dependent polymerization of FtsZ in vitro. YeeV also interacts with MreB and inhibits its polymerization in vitro. MreB is required for maintenance of the typical rod shape of cells, and assembles into a helical structure that coils along length of cell (van den Ent et al., 2001; Jones et al., 2001). MreB is also involved in cell division (Wachi and Matsuhashi, 1989; Kruse and Gerdes, 2005), chromosome segregation (Kruse et al., 2003; Kruse and Gerdes, 2005) and cell polarity (Gitai et al., 2004). We also demonstrate that both the N- and C-terminal ends of YeeV have different effects on cell morphology. A C-terminal truncation of 52 residues inhibits only septum formation, forming filamentous cells; an N-terminal deletion of 15 residues results in the formation of lemon-shaped cells. This is the first report to date of a member of TA systems in which the toxin inhibits cell growth by binding and inhibiting the cytoskeletal proteins, FtsZ and MreB. On the basis of these findings, we propose to rename YeeV to CbtA for Cytoskeleton Binding Toxin.

Results

YeeV induction causes spherical cell formation

Deletion of the yeeV gene from E. coli BW25113 showed no overt effect on cell growth and morphology (data not shown), indicating that the yeeV gene is not essential for cell growth. However, when YeeV expression was induced using the arabinose-inducible vector pBAD33, cell growth was completely inhibited after 6 h (Fig. 1A). Interestingly, cell morphology was significantly affected by this method of YeeV expression. At 2 h post induction, the central part of every cell became inflated; and at 4 and 6 h post induction, the cells became lemon-shaped with the two polar ends still intact. At 24 h post induction, most of the cells were spherical with polar regions highly diminished. Some cells lysed as can be seen from the cell debris surrounding the spherical cells (Fig. 1B). This phenotype is highly reminiscent of the morphology of rod-shaped E. coli carrying a mutation of mreB, which also gradually changed into spherical cells (van den Ent et al., 2001; Jones et al., 2001; Gitai et al., 2004). To test whether the change in morphology following YeeV overexpression may be due to inhibition of MreB, we treated BW25113 cells with the compound S-(3,4-Dichlorobenzyl) isothiourea (A22) to inhibit MreB function (Gottesman et al., 1981). A22 has been shown to directly disrupt MreB assembly causing formation of spherical cells (Noguchi et al., 2008; Bean et al., 2009). A22 is the well-known inhibitor of MreB. The cells treated with A22 grew as smooth round cells (Fig. 1C1), while cells with overexpression of YeeV became lemon-shaped with two extensions at the two pole sites (see Fig. 1B). This is consistent with previous reports that induction of a lemon-shaped cell morphology occurred when FtsZ and MreB or FtsZ and PBP2 were inhibited simultaneously (Varma et al., 2007). When FtsZ alone was inhibited by expression of the SOS gene SulA from pET28c plasmid, every cell becomes filamentous (Figs 1 and 2). When these filamentous cells were treated with A22, the central regions of the cells inflated (Fig. 1C3). A similar effect was seen when YeeV was induced by arabinose using pBAD33-yeeV in filamentous cells (Fig. 1C4). Thus, we speculated that YeeV may inhibit the functions of both FtsZ and MreB or other proteins involved in maintaining the typical rod shape of E. coli.

Figure 1.

Ectopic expression of yeeV affects cell growth and cell shape.
A. Growth curve shows cell growth inhibition after overexpression of YeeV. E. coli BW25113 cells harbouring plasmid pBAD33-yeeV were grown at 37°C in glycerol M9 medium in the presence of 0.2% arabinose.
B. Phase-contrast images of E. coli BW25113 cells carrying pBAD33-yeeV shows altered cell morphology after overexpression of YeeV. Cells were grown and induced as in (A).
C. Morphological manifestation of inhibition of FtsZ and MreB. 1. E. coli BL21 (DE3) carrying both pET28c-sulA and pBAD33-yeeV were grown as described in (A). 1. MreB was inhibited by A22 (5 µg ml−1). 2. FtsZ was inhibited by SulA induced from pET28c by adding 1 mM IPTG. 3. After 1 h induction of SulA, cells were treated with A22 (5 µg ml−1) to inhibit MreB. 4. After 1 h induction of SulA, YeeV was induced from pBAD33 by adding 0.2% arabinose. All the bars represent 2 µm.

Figure 2.

Isolation and identification of proteins interacting with YeeV. Proteins interacting with YeeV were isolated by a His-tag pull-down assay as described in Experimental procedures. Lane 1, protein markers; lane 2, cell lysates of the yeeUVW deletion strain passed through Ni-NTA resin; lane 3, cell lysates of the yeeUVW deletion strain passed through the Ni-NTA column to which YeeV is bound. The bands indicated by arrows in lane 3 were subjected to mass spectrometer.

It has been long established that low-molecular-weight penicillin-binding proteins (PBPs) are also involved in cell morphology. The overproduction of PBP5 is known to cause round-shaped morphology (Markiewicz et al., 1982). Indeed, we observed increased production of PBP5 upon YeeV induction (data not shown) by a penicillin binding assay. To rule out the possibility that the YeeV-induced morphological change was due to increased expression of PBP5, we expressed YeeV in a pbp5-deletion strain. These cells still showed the same lemon-shaped cell morphology as the parent pbp5+ strain when YeeV was induced (data not shown), indicating that PBP5 is not a target for YeeV.

YeeV directly interacts with FtsZ and MreB in vitro and in vivo

To identify the cellular target(s) of YeeV, we carried out a pull-down experiment with purified His-tagged YeeV. Overexpressed His-tagged YeeV protein was mixed with a cell lysate of a yeeUVW-deletion strain. Elution fractions were analysed by SDS-PAGE and silver-stained (Fig. 2). Three new bands were observed compared with the control of ∼70 kDa and an apparent doublet of ∼40 kDa (indicated by arrows in Fig. 2). These bands were then subjected to trypsin digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. The band at approximately 70 kDa was found to be DnaK (69 kDa), a molecular chaperon known to co-purify with proteins bound to a Ni-NTA column. The band at approximately 39 kDa was composed of two proteins, FtsZ and MreB. Despite their small but significant differences in molecular masses (40 kDa for FtsZ and 37 kDa for MreB), they were not easily resolved by SDS-PAGE. To further verify the interaction between FtsZ and YeeV or the interaction between MreB and YeeV, we coexpressed FtsZ and His-YeeV, and MreB and His-YeeV in BL21(DE3) cells and purified co-immunoprecipitated the resulting complexes. Both FtsZ and MreB and proteins were recovered after coexpression with YeeV (data not shown).

To further verify the direct interaction between FtsZ and YeeV or between MreB and YeeV, we performed yeast two-hybrid analyses. We fused ftsZ to the activation domain (AD) of GAL4 in pGAD-C1 (ftsZ fused with the binding domain showed self-activation and could not be used) and yeeV or mreB to the binding domain in pGBD-C1. Interaction between protein pairs were assessed by colony formation on selected plates and quantitatively measured by β-galactosidase activity (see Experimental procedures). In these assays, the direct interactions between YeeV and FtsZ, and between YeeV and MreB were detected (Table 1). The yeast two-hybrid validates the MreB pull down by YeeV and strongly suggests that the physical interaction between MreB and YeeV was not through indirect interaction between YeeV and MreB by way of FtsZ. In our yeast two-hybrid assay, MreB also directly interacted with FtsZ. This is the first demonstration to support the hypothesis previously proposed by a number of researchers that MreB may directly interact with FtsZ for the assembly of the MreB-associated cytoskeletal ring structure (Vats et al., 2009).

Table 1. Analysis of the interaction between FtsZ and YeeV, MreB and YeeV, FtsZ and MreB using yeast two-hybrid systems.
DNA-binding domain (BD)Activation domain (AD)β-Galactosidase activity (Miller units)aGrowth on –His and –Ade platesb
  • a. 

    β-Galactosidase activity of yeast cells carrying combinations of proteins was quantified using an ONPG (O-nitrophenyl-β-d-galactopyranoside) liquid assay. Each number represents average from at least three independent experiments.

  • b. 

    The interaction was also selected on –His and –Ade plates: + represents growth and − represents no growth.

BDAD4.7 (± 0.61)
BDYeeV5.3 (± 0.13)
YeeVAD10.8 (± 0.51)
BDFtsZ4.9 (± 0.18)
MreBAD9.8 (± 0.21)
YeeVFtsZ419 (± 81)+
YeeVMreB98 (± 2.7)+
MreBFtsZ345 (± 29)+

YeeV and MreB interact with both N-terminal and C-terminal end of FtsZ

We also demonstrated that both the N-terminal and C-terminal ends of FtsZ are required for interaction with both YeeV and MreB. It has been shown that the C-terminal end (FtsZ321–383 in E. coli) is responsible for both self-interaction between FtsZ molecules and interaction with other proteins (i.e. ZipA, an essential membrane-associated division protein) (Hale et al., 2000).

We observed that either a deletion of the first 32 N-terminal residues [FtsZΔN(-32)] or a deletion of 66 residues from the C-terminal end [FtsZΔC(-66)] of FtsZ resulted in loss of interaction between FtsZ and YeeV, and also between FtsZ and MreB (Table 2), indicating that both the N-terminal and C-terminal regions of FtsZ are indispensible for this interaction. The interaction domain of N-terminal end of FtsZ for interacting with YeeV overlaps with the domain for interacting with SulA. The previous results has been shown that deletions from the end of N-terminus of FtsZ (which deleted first 32 amino acids) eliminated interaction between FtsZ and SulA (Huang et al., 1996).

Table 2. Map the interaction domains of FtsZ with YeeV and MreB using yeast two-hybrid system.
DNA-binding domain (BD)Activation domain (AD)β-Galactosidase activity (Miller units)aGrowth on –His and –Ade platesb
  • a. 

    β-Galactosidase activity of yeast cells carrying combinations of proteins was quantified using an ONPG (O-nitrophenyl-β-d-galactopyranoside) liquid assay. Each number represents average from at least three independent experiments.

  • b. 

    The interaction was also selected on –His and –Ade plates: + represents growth and − represents no growth.

BDFtsZΔC(-66)4.2 (± 0.31)
BDFtsZΔN(-32)10.9 (± 1.70)
YeeVxFtsZΔN(-32)4.8 (± 0.27)
YeeVFtsZΔN(-32)4.7 (± 0.34)
MreBFtsZΔC(-66)10.42 (± 0.35)
MreBFtsZΔN(-32)9.2 (± 0.91)

YeeV inhibits GTP-dependent polymerization and GTPase activity of FtsZ

On the basis of the above results, we asked if the direct interaction between YeeV and FtsZ, YeeV and MreB affects on the polymerization FtsZ and MreB, and GTPase activity of FtsZ. Previous work has shown that a well-characterized inhibitor of cell division, SulA, directly interacts with FtsZ, and that the induction of SulA inhibits the GTPase activity in vitro via blocking the polymerization of FtsZ (Mukherjee et al., 1998). The effect of YeeV on the polymerization of FtsZ was tested by sedimentation assay as described in Experimental procedures. Briefly, FtsZ monomers were incubated with different concentration of purified His-YeeV in the presence or absence of GTP. The FtsZ monomer and polymers were separated and visualized by SDS-PAGE and band intensity was quantified. With GTP, 80% of the total FtsZ was polymerized in the pellet in the conditions described in Experimental procedures. This is consistent with the previous results of others (Bramhill and Thompson, 1994). When FtsZ (final concentration is 160 µg ml−1 in the reaction) was mixed with increasing amounts of YeeV in the presence of GTP, the amounts of FtsZ polymers in the pellet fraction decreased in a dose-dependent manner. With the addition of His-YeeV at 100 and 200 µg ml−1, the amount of FtsZ polymers in pellet was 24% and 12% of total FtsZ respectively. These results clearly demonstrated that YeeV inhibits FtsZ polymerization.

The effect of YeeV on the GTPase activity of FtsZ was also examined (Fig. 3B). The GTPase activity of FtsZ was reduced as increasing amounts of YeeV were added to the reaction mixture, demonstrating that YeeV inhibits the GTPase activity of FtsZ. A low level of GTPase activity observed only with added YeeV (column 6 in Fig. 3B) is possibly due to small amount of FtsZ co-eluted with His-YeeV during its purification.

Figure 3.

YeeV inhibits the GTP activity and polymerization of FtsZ.
A. Effects of YeeV on the polymerization of FtsZ. The effect of YeeV on FtsZ polymerization was determined by sedimentation assay. FtsZ (final concentration 160 µg ml−1) was polymerized for 20 min at 20°C in the buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 10 mM MgCl) with addition of 2 mM GTP in the presence or absence of His-YeeV (from 100 µg ml−1 to 200 µg ml−1). Reaction was centrifuged at 50 000 r.p.m. at 20°C for 20 min. Equivalent volumes of total (T) and supernatants (S) and pellet (P) were separated on a 17% SDS-PAGE and Coomassie Blue-stained bands were quantified.
B. YeeV inhibits FtsZ's GTPase activity. The GTPase activity of FtsZ was measured in polymerization buffer as described in Experimental procedures in the presence of increasing amounts of His-YeeV. From lane 2 to 6, 100 mM GTP was added and from lane 1 to 5, His-FtsZ (final concentration is 160 µg ml−1 in the reaction) was added. Increasing amount of His-YeeV was added from lane 3 to 6 (50 µg ml−1 in lane 3, 100 µg ml−1 in lane 4, and 200 µg ml−1 in lanes 5 and 6).

YeeV inhibits ATP-dependent polymerization of MreB

In a similar manner to the experiments with FtsZ described above, we tested the effect of YeeV on the ATP-dependent polymerization of MreB. The polymerization properties of MreB from Thermotoga matitima have been characterized and it was shown that polymerization of MreB is temperature and concentration dependent (van den Ent et al., 2001; Bean and Amann, 2008). Here, MreB polymerization assay was performed as described previously (Bean et al., 2009). Approximately 4 µM MreB (see Experimental procedures) was incubated with 5 mM ATP at 20°C for 20 min. In the absence of YeeV, more than 90% of MreB was recovered in the pellet fraction, which contains the polymerized MreB (Fig. 4). This is consistent with previous results (Bean and Amann, 2008). In the presence of YeeV at 100 and 200 µg ml−1, the amount of MreB polymers in the pellet fraction was approximately 60% and 40% respectively (Fig. 4). In the similar manner, MreB polymerization was shown to be inhibited by A22 (Bean et al., 2009).

Figure 4.

Effect of YeeV on the polymerization of MreB. Approximately 4 µM MreB was polymerized in the buffer same as described in Fig. 3A with addition of 200 µM ATP in the presence or absence of His-YeeV (from 100 µg ml−1 to 200 µg ml−1). Proteins were separated on a 17% SDS-PAGE. Equivalent volumes of total (T) and supernatants (S) and pellet (P) were quantified.

These results clearly indicated that YeeV also inhibits MreB ATP-dependent polymerization. Interestingly, however, YeeV was not able to inhibit the ATPase activity of MreB, in contrast to the effect that YeeV has on the GTPase activity of FtsZ (Fig. 3B). This result suggests that YeeV may not inhibit MreB polymerization via interfering with ATP hydrolysis. Rather, YeeV may act by blocking those conformational changes in MreB molecules normally induced by ATP hydrolysis, which is a required step for polymerization (van den Ent et al., 2001).

Dissection of YeeV function

In order to dissect the function of YeeV, we truncated YeeV at both the N- and C-terminal ends as shown in Fig. 5A. We also examined the effect of truncated YeeV on toxicity and morphology. The 52-residues at the C-terminal of yeeV was deleted, the remaining N-terminal 73-residue fragment [YeeVΔC(-52)] still retained toxicity (Fig. 5B). Interestingly, the overexpression of YeeVΔC(-52) only inhibited cell division leading to the formation of filamentous cells (Fig. 5C). However, the cells did not inflate or become lemon-shaped, as seen in cells following overexpression of intact YeeV, indicating that the N-terminal fragment of YeeV lost the ability to inhibit MreB but still retained the ability to interact with FtsZ and inhibit its function. Further deletion of the C-terminal end with additional 11 residues [YeeVΔC(-63)] resulted in the complete loss of the YeeV toxicity (Fig. 5B), probably because this region is required for stability and/or functional integrity of the protein. The deletion analysis of the N-terminal domain demonstrated that the deletion of the N-terminal 15 residues [YeeVΔN(-15)] affect the toxicity (Fig. 5B), and even caused the faster formation of lemon-shaped cells (Fig. 5C). The interaction between truncated YeeV and FtsZ or MreB was determined by coexpression of truncated His-YeeV and FtsZ or MreB. YeeVΔN(-15) showed interaction with FtsZ and MreB (data not shown). However the interaction between YeeVΔC(-52) and FtsZ or MreB was not detected. A more likely possibility was that truncated C-terminal end of YeeV reduced stability of the protein. Examination of protein expression of His-YeeVΔC(-52) and His-YeeVΔN(-15) showed that in the presence of IPTG, His-YeeVΔN(-15) accumulated in the cell, and His-YeeVΔC(-52) does not (data not shown).

Figure 5.

Effect of truncated YeeV on cell growth and morphology.
A. Schematic representation of truncated YeeV used for (B) and (C).
B. E. coli BW25113 carrying either a wild-type or truncated yeeV in pBAD33 was grown at 37°C in glycerol M9 plates in the presence or absence of 0.2% arabinose overnight. The numbers on the plate represents: 1. the empty vector pBAD33, 2. YeeV (WT), 3. YeeVΔC(-52), 4. YeeVΔC(-63), 5. YeeVΔN(-15), 6. YeeVΔN(-28).
C. Phase-contrast images of same cells in (B) carrying truncated yeeV in pBAD33 show altered cell morphology after overexpression of truncated yeeV in the presence of 0.2% arabinose.

Discussion

Based on the observation that severity of growth inhibition is increased as the cellular concentration of YeeV toxin increased, it has been suggested that YeeV may inhibit cell growth by binding to and titrating out some essential protein (Brown and Shaw, 2003). The major finding in this study is that we identified YeeV inhibits the functions of two essential cytoskeletal proteins, FtsZ and MreB, through physical interaction. Several protein inhibitors of cell division targeting FtsZ have been previously identified in E. coli. One of them is SulA, which is expressed when SOS response is induced and inhibits cell division. Its overexpression has been shown to result in the formation of filamentous cells but not lemon-shaped cells (Gottesman et al., 1981). Similar to YeeV, SulA is known to inhibit FtsZ polymerization and GTPase activity in vitro (Mukherjee et al., 1998), by interacting with the N-terminal region of FtsZ (Dajkovic et al., 2008). Our results show that purified His-YeeV markedly inhibits the GTPase activity and polymerization of FtsZ in vitro. How dose YeeV inhibit the GTPase activity and polymerization of FtsZ? Our yeast two-hybrid results have demonstrated here that YeeV interacts with both the N- and C-terminal ends of FtsZ. The interaction domain of N-terminal end of FtsZ with YeeV and SulA is overlapped. Unlike YeeV, SulA does not interact with C-terminal end of FtsZ (FtsZ321–383 in E. coli). It has been shown that the C-terminal end (FtsZ321–383 in E. coli) is accountable for both self-interaction between FtsZ molecules and interaction with other proteins (Hale et al., 2000). It is likely that YeeV binds to FtsZ, blocking self-interaction of FtsZ, that is essential for polymerization and GTP activity.

In addition to interacting with FtsZ, YeeV also interacts with MreB and disrupts its polymerization. A22, a specific inhibitor of MreB, has been shown to directly target the ATP-binding pocket in MreB and decrease the affinity between the MreB monomers and polymers (Bean et al., 2009). Like A22, purified His-YeeV could significantly reduce formation of MreB polymers at certain concentration and at 20°C, but not inhibits completely. We did not find evidence that YeeV inhibits the ATPase activity of MreB. Because of this, it is likely that YeeV disrupts the interaction between MreB and MreB-associated proteins by directly binding to MreB.

Interestingly, we examined the effects of both N- and C-terminal ends of YeeV on cell morphology and growth. The N-terminal 73-residue fragment only causes cell elongation, similar to that observed with SulA overexpression. Clearly, this fragment failed to interact with MreB, but still retained the ability to interact with FtsZ to inhibit cell division. In contrast to the C-terminal deletion, the truncation of the N-terminal 15-residue segment resulted in formation of lemon-shaped cells, the same morphology observed when both FtsZ and MreB were inactivated (Fig. 5).

It has been hypothesized that FtsZ might induce the MreB-associated cytoskeletal ring assembly by directly interacting with cytoskeletal proteins (Vats et al., 2009). However, no evidence has been previously shown to support this hypothesis. In this study, we demonstrated the direct interaction between FtsZ and MreB using the pull-down and yeast two-hybrid system, providing a compelling link between FtsZ and the cytoskleletal ring. It appears that YeeV simultaneously inhibits both FtsZ and MreB, as YeeV is able to directly interact with both cytoskeleton proteins. However, it is possible that YeeV may first inhibit the FtsZ ring formation which results in blocking the interaction between FtsZ and MreB. Further experiments have to be carried out to elucidate these possibilities.

The physiological function of chromosomal TA systems remains unclear so far. The environmental or internal cellular condition which triggers the toxicity of the YeeV seems to be a novel mechanism which will be elucidated in further studies. On the basis of our findings on the toxic functions of YeeV, we propose to rename YeeV of previously unknown function to CbtA, for Cytoskeleton Binding Toxin. Furthermore, the structural studies of YeeV and the YeeV–FtsZ complex, as well as the YeeV–MreB complex likely reveal how YeeV interacts with FtsZ and MreB to inhibits their functions, which may provide important insights into designing new antibiotics.

Experimental procedures

Strains, plasmids and growth conditions

Escherichia coli BW25113 (ΔaraBD) (Datsenko and Wanner, 2000) and BL21 (DE3) were grown in M9 medium at 37°C. Plasmids pET-28a-yeeV, pET-28a-ftsZ, pET-28a-mreB and pET-28a-sulA were constructed in pET-28a (Novagen) with an N-terminal His6 tag. Plasmids pBAD33-yeeV and pBAD33-sulA were constructed from pBAD33 (Guzman et al., 1995) to tightly regulate gene expression by the addition of arabinose. To generate the N-terminal GFP-fusion plasmids, a DNA fragment of gfp from plasmid pAcGFP1 (Clontech laboratories) was cloned into pBAD33 to generate pBAD33-gfp. ftsZ, mreB and yeeV genes were then cloned into the pBAD33-gfp vector.

Protein expression, purification and pull-down assay

YeeV, FtsZ and MreB were expressed with the N-terminal (His)6-tag using the pET28a plasmid in E. coli BL21(DE3), and purified using Ni-NTA agarose according to the manufacturer's instructions (Qiagen). Expression was induced for 3 h by adding 1 mM IPTG when the OD600 reached 0.5 (expression of FtsZ was induced when OD600 reached 1.0). His-tag pull-down assays were carried out by incubating purified His-tagged YeeV (10 mg ml−1) with 0.5 ml of Ni-NTA agarose overnight at 4°C. Twenty millilitres of cell lysate (lysis buffer: 50 mM HEPES-KOH, pH 7.5; 10 mM MgCl2; 200 mM KCl; 0.1 mM EDTA; 10% glycerol; 1% Triton X-100) was applied to the beads bound with YeeV. The beads were washed three times with 20 ml of the same lysis buffer plus 20 mM imidazol. Protein complexes were then analysed on 12% SDS-PAGE and visualized by silver staining and identified by LC-MS/MS using a LTQ ion trap mass spectrometer (Thermo Scientific).

Yeast two-hybrid assay

The GAL4 DNA-binding (pGBD-c1, from Clontech) or DNA-activation domain (pGAD-c1, from Clontech) was fused to indicated genes and expressed in the Saccharomyces cerevisiae strain PJ69-4A (James et al., 1996). Positive interactions were determined by X-gal assay and colony formation on high-stringency Minimal Synthetic Dropout medium (SD) lacking leucine, tryptophan, histidine and adenine. β-Galactosidase activity was quantified using an O-nitrophenyl-β-d-galactopryanoside (ONPG) liquid assay as described previously (Moriyoshi, 2009).

Polymerization assays

The effect of YeeV on FtsZ and MreB polymerization was determined by a sedimentation method previously described (Mukherjee et al., 1998; Anand et al., 2004; Bean et al., 2009) with a few modifications. Purified His-FtsZ or MreB was mixed with different amounts of purified His-YeeV in 50 µl of polymerization buffer P (5 mM MgCl2; 100 mM Tris-HCl; pH 7.0, 100 mM NaCl) and pre-incubated at room temperature for 20 min before GTP or ATP was added to initiate polymerization. The polymerization reaction was carried out at room temperature for 20 min. FtsZ or MreB polymers were precipitated by centrifugation at 50 000 r.p.m. in a Beckman TLA100 rotor for 20 min at 20°C, pellets were suspended in buffer P, using the same volume as for supernatant. Both the supernatant and pellet fractions were visualized by 17% SDS-PAGE gel and Coomassie Blue staining and quantified with AlphaEaseFCTM software.

GTPase assay of FtsZ

The GTPase assay of FtsZ was carried out by mixing purified His-FtsZ with different amounts of purified His-YeeV in 50 µl of buffer P (described in the polymerization assay). After pre-incubation at room temperature for 30 min, a GTP mixture containing [α-32P]-GTP was added to a final concentration of 10 mM to initiate the GTPase reaction at room temperature for 10 min. The reaction was stopped by adding 200 µl of 6% active charcoal, and then centrifuged at 14 000 r.p.m. for 2 min. One hundred microlitres of the supernatant was removed to quantify the radioactivity of phosphate released from GTP.

Acknowledgements

The authors are grateful to Dr S. Phadtare, Dr Meredith H. Prysak, Dr Hisako Masuda, Dr Y. Yamaguchi and Dr Michael Parisi for their critical reading of the manuscript. This work was supported by grants from the National Institute of Health (RO1GM081567) and Takara Bio.

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