Direct Interaction between the Two Z Ring Membrane Anchors FtsA and ZipA

Engineering cross-linkable derivatives of FtsA.To investigate potentially weak in vivo interactions between FtsA and ZipA and identify interacting residues, we chose to use BpA as a genetically encoded cross-linker. To generate sites for BpA in FtsA, we initially engineered a series of single TAG (amber) codons to replace charged residues in FtsA that are predicted to lie at the surface and be involved in protein-protein interactions. Five of these, R122, E124, R126, R153, and R177, lie in domain 1C and are candidates for coordinating FtsA-FtsA interactions, whereas R202, R260, R286, and R300 are in regions of the protein implicated in FtsA-FtsA or FtsA-FtsZ interactions. In each of these constructs, these codon replacements represent the sole amber codon in ftsA, as the stop codon at the end of native ftsA is TAA. Cells expressing FLAG-FtsA with these engineered TAG codons in a plasmid require pEVOL, a compatible plasmid that expresses the orthogonal tRNA and tRNA synthetase, inducible with l-arabinose, which can incorporate BpA at the residue changed to TAG (39). By growing cells in the presence of BpA, a proportion of FtsA protein incorporates BpA at the targeted residue. The exposure of these cells to long-wave UV light triggers the formation of a single covalent linkage between the BpA residue on FtsA and a target amino acid, if it is within ∼3 to 4 Å.

To confirm that the mutant FtsA proteins properly incorporated BpA at their respective sites, we cotransformed cells carrying a chromosomal thermosensitive ftsA allele (ftsA12, in strain WM1115) with pEVOL and pDSW210F expressing FLAG-tagged FtsA amber mutants to determine the capacity of FtsA-BpA derivatives to suppress the thermosensitivity (Ts) of WM1115. While optimizing the experimental conditions, we found that high concentrations of l-arabinose or BpA caused cell filamentation, indicating that cell division was inhibited. As we wanted division to proceed normally in cells being probed for interactions between cell division proteins, we ended up using lower inducer concentrations to minimize division defects.

To test the ability of the BpA-substituted amber mutants to complement, we spotted various dilutions of cells onto LB agar plates at permissive (30°C) or nonpermissive (42°C) temperatures supplemented with ampicillin (Ap), chloramphenicol (Cm), 100 µM l-arabinose, 100 µM BpA, and different isopropyl-β-d-thiogalactopyranoside (IPTG) concentrations (see Fig. S1A in the supplemental material). As expected, wild-type FtsA produced from pDSW210F was toxic when induced at >100 µM IPTG and completely inviable at 1 mM IPTG at 30°C (40). In contrast, none of the mutant proteins were toxic even at 1 mM IPTG (Fig. S1A). At the nonpermissive temperature of 42°C, three mutants (R122X, R153X, and R300X) failed to suppress ftsA12, indicating that they were inactive. However, all the other mutants complemented ftsA12 at some level of IPTG, indicating that they were functional but were not toxic like wild-type FtsA (Fig. S1A). Interestingly, the substitution at R286, which is the same residue altered in FtsA*, fully complemented with no added IPTG, whereas the other substitutions all required at least 10 µM IPTG for optimal complementation.

To determine how efficiently the various mutants incorporated BpA, we grew the same WM1115 (ftsA12) strains containing the various derivatives used for the viability assays in the presence or absence of BpA, followed by UV cross-linking. We then probed cell extracts for the expressed FLAG-FtsA on Western blots by using anti-FLAG antibody. All mutants except R122 displayed a band corresponding to full-length FLAG-FtsA-BpA (∼45 kDa) only when grown in medium containing BpA (Fig. S1B, “+” lanes), indicating that BpA was incorporated into the FtsA amber derivatives. The band intensities for R153X and R300X were lower than the others, suggesting that their inability to complement (Fig. S1A) might be caused by protein instability. In addition to the full-length BpA-containing proteins, we also detected protein bands from the various derivatives that migrated at smaller sizes than expected if their translation were terminated at the engineered premature TAG codon. In the samples grown in the presence of BpA, the bands of truncated protein that were large enough to be retained by SDS-PAGE (R153X, R177X, R202X, R260X, R286X, and R300X) were less abundant than the respective full-length protein (Fig. S1B), with R202X, R260X, and R286X incorporating 64%, 78%, and 87%, respectively, of the total detectable FtsA into the full-length protein, suggesting that BpA was incorporated efficiently into these proteins under these conditions.

BpA at residue 260 of FtsA can cross-link with ZipA or additional FtsA subunits.In the initial screening, we used anti-FLAG to detect whether the FLAG-tagged BpA derivatives of FtsA shifted into higher-molecular-weight protein bands that might form cross-linked species (CLS) under these conditions, but we observed only very faint potential CLS in the “+ UV” lanes. However, when we probed a blot of the same samples with anti-ZipA, we observed a strikingly strong UV-dependent CLS for the R260X protein, suggesting that the BpA at residue 260 might be able to cross-link with native ZipA (Fig. S1C, asterisk). No UV-dependent CLS were observed for any of the BpA derivatives in this initial screen by using antibodies against FtsZ or FtsN (Fig. S1D and E). Using anti-ZipA, we further confirmed that FtsAR260-BpA generated CLS containing ZipA only in the presence of BpA and only when irradiated by UV (Fig. 1B, lane 8, compare with lanes 5 to 7). When we used anti-FLAG on a blot with the same samples to detect FLAG-FtsA, this time we detected a faint higher-molecular-weight CLS in the sample with BpA and UV (Fig. 1A, lane 8), probably because there was less nonspecific staining. In addition, the R260 amber derivatives of FtsA that failed to incorporate BpA were detectable as prematurely truncated proteins (Fig. 1A, lanes 5 to 8); the amount of truncated protein was reduced, as expected, in the presence of BpA (Fig. 1A, lanes 7 and 8, compare with lanes 5 and 6).

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FIG 1

FtsA-R260-BpA specifically cross-links with ZipA. (A and B) Strains expressing either FLAG-FtsA or FLAG-FtsA with an amber codon replacing R260 (R260X) were grown with the antibiotics specified, with or without BpA, and either subjected to UV cross-linking or not. Protein extracts were separated by SDS-PAGE, followed by Western blotting using anti-FLAG to detect the FLAG-FtsA (A) or anti-ZipA (B). Full-length FtsA or ZipA bands are indicated by arrowheads, as are the most prominent cross-linked species (CLS). The band representing FtsA protein terminated at codon 260 is indicated by an unlabeled arrowhead. (C to E) Strains expressing FtsA or FtsAR260X, carrying ftsA12 (WM1115), ΔzipA ftsA* (WM1657) or WM1657 with ZipA-Strep-Tag produced from plasmid pPK9 (selection with spectinomycin), were grown with BpA and either UV cross-linked or not, and their proteins were separated by SDS-PAGE. Western blots were probed with anti-Strep-Tag to detect ZipA-Strep-Tag (C), anti-ZipA (D), or anti-FtsA (E). Bands representing CLS are indicated by arrowheads. Molecular weight markers in kilodaltons are shown to the left of all blots.

Although the predicted molecular weights for FtsA and ZipA are 45.3 and 36.5 kDa, respectively, ZipA migrates aberrantly at ∼50 kDa in SDS-PAGE (19), probably because of its internally disordered P/Q-rich domain. Even accounting for the slow migration of ZipA, the size of the CLS (∼135 kDa) we observed after SDS-PAGE was not the expected ∼95-kDa size of one FtsA molecule cross-linked to one ZipA. To resolve this issue, we first wanted to confirm that the CLS corresponded to FtsA cross-linked to ZipA and not a larger ∼90-kDa protein that potentially cross-reacted with anti-ZipA.

We addressed this in several ways. To determine whether the CLS detected with anti-ZipA actually represented ZipA, we repeated the cross-linking by expressing FtsAR260-BpA not only in WM1115 but also in WM1657, a mutant lacking zipA that harbors the ftsA* allele (which bypasses zipA), and WM1657 transformed with a zipA-expressing plasmid (pPK9-ZipA-StrepTag, compatible with pEVOL and pDSW210F-FtsA) as an isogenic zipA⁺ control. As expected, in WM1115 (ftsA12 zipA⁺), CLS were observed by probing Western blots with anti-ZipA (Fig. 1D, lane 4) or anti-FtsA (Fig. 1E, lane 4). The CLS, as mentioned above, were only detectable after incubating with BpA and after UV irradiation. Importantly, in the absence of ZipA (WM1657 ftsA* ΔzipA), CLS were detected only by anti-FtsA (Fig. 1E, lane 8) and not by anti-ZipA or anti-Strep-Tag (Fig. 1C and D, lane 8). When ZipA was restored back to the ΔzipA strain by expressing it from a plasmid (WM1657/pPK9-ZipA-Strep-Tag), CLS were observed under all conditions (Fig. 1C to E, lane 12). These results strongly suggested that the CLS correspond to FtsA-ZipA, but we also noticed that ∼135-kDa CLS were generated even in the absence of ZipA (Fig. 1E, lane 8). This raised the possibility that three FtsAR260-BpA monomers could cross-link as a trimer (45 × 3 = 135 kDa) or that FtsA could, in the absence of ZipA, cross-link to another protein of ∼90 kDa.

A search for ∼90-kDa proteins involved in E. coli cell division revealed that PBP1A and/or PBP1B, both 89-kDa proteins that could theoretically interact with FtsA, might be targets of FtsA cross-linking. To test this idea genetically, we constructed mutants lacking either the mrcA or mrcB genes encoding PBP1A or PBP1B, respectively (deleting both is lethal) and transformed them with pEVOL and the plasmid carrying FLAG-FtsAR260X. Both ΔmrcA and ΔmrcB strains still generated the same CLS at intensities at least as high as the mrc⁺ parent strain (see Fig. S2), suggesting that PBP1A or PBP1B is not crucial for forming the CLS under these conditions.

Finally, to confirm this and to explore whether other potential targets of FtsA-R260-BpA cross-linking existed, we purified CLS from the WM1657 ΔzipA strain expressing ZipA-Strep-Tag from pPK9-ZipA-Strep-Tag (WM5952), only after exposure to BpA and UV light, with anti-Strep-Tag affinity chromatography. We identified three bands in the 135-kDa area after SDS-PAGE and silver staining and analyzed the contents of the excised bands by mass spectrometry. The contents of the three bands were similar, with band 2 being the most prominent. The analysis identified FtsA and ZipA as top hits, but no other known cell division-related protein was identified, including PBP1A or PBP1B (see Fig. S3). These results suggest that the CLS contains only FtsA-R260-BpA cross-linked to FtsA and/or ZipA. It remains possible that other protein partners interact with FtsA in the absence of ZipA.

Residues in FtsA helix 7 are part of an FtsA-ZipA-interacting domain.According to the atomic structure of Thermotoga maritima FtsA, R260 lies within helix 7 (41) (see Fig. S4). Although R260 is not well conserved in FtsA proteins from diverse species, several nearby residues in helix 7, such as E255 and K258, are highly conserved (Fig. 2D; see also Fig. S5) and interact with the adenosine moiety of ATP (41). To determine whether residues immediately adjacent to R260 might be involved in the FtsA-ZipA interaction, we constructed a new set of FtsA amber codons to replace K258, V259, H261, and G262. As for previous mutants, we tested the ability of these amber mutant FtsA proteins to complement ftsA12 in WM1115 at 30°C and 42°C in the presence of BpA (Fig. 2A). The results at 30°C were similar to those shown in Fig. S1A and B: wild-type (WT) FtsA was toxic at high induction levels, but none of the BpA derivatives were toxic (Fig. 2A, mutants row and data not shown). As shown at the bottom of Fig. 2A, FtsAR260X and H261X complemented but needed higher IPTG levels than WT FtsA. On the other hand, substitutions at V259 and G262 complemented poorly, with only a slight increase in viability at higher IPTG levels. The K258 amber mutant complemented slightly better but still was unable to complement without the addition of IPTG.

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FIG 2

Residues on the outer face of helix 7 of FtsA preferentially cross-link with ZipA. (A) WM1115 strains expressing FtsA amber substitutions at all the residues within helix 7 were grown in the presence of BpA and tested for their ability to complement ftsA12 at 42°C. After growth at 30°C, all strains shown were serially diluted 10-fold, spotted on plates containing different concentrations of IPTG to induce expression of the ftsA derivatives at different levels plus arabinose to induce pEVOL and BpA, and incubated either at 30°C or 42°C for 24 h. To save space, the “Mutants” row shows one of the mutants that grew at all IPTG levels at 30°C, because all of the other mutants behaved similarly. (B and C) The same strains in panel A were UV cross-linked, and protein extracts were separated by SDS-PAGE. Western blots were probed with either anti-FtsA (B) or anti-ZipA (C), and arrowheads indicate the general position of CLS. Molecular weight markers in kilodaltons are shown to the left. (D) Shown at the left is the position of each residue in FtsA helix 7 (including P250, which is outside the helix) and the relative ability of BpA at each position to cross-link with ZipA. The protein sequence of ZipA from E. coli was used to generate a model PDB file with the Phyre2 server and subsequently analyzed in UCSF Chimera. Summarized in the table at the right is the ability of each amber substitution under the conditions of the experiments in panels A and C to cross-link with ZipA and to complement at 42°C, along with the relative conservation of each substituted residue. The scoring system for CLS reflects the intensity of CLS bands, from ++++ (strongest) to − (undetectable). Scoring for complementation reflects the ability to grow at no IPTG like WT FtsA (++++), weak at no IPTG but strong growth with 10 µM IPTG (+++), weak with 10 µM IPTG but stronger growth at higher IPTG levels (++), or little growth at any induction level (+). Scoring for conservation depends on the degree of conservation across the species aligned in Fig. S5 in the supplemental material: ++++, identity across all species; +++, identity across most species; ++, identity in at least 4 other species in the list; +, some identity or similar characteristics in other species; −, no obvious conservation.

We then tested cells expressing these amber substitution derivatives under permissive growth conditions in the presence of BpA for the ability to cross-link ZipA. As before, CLS specific for FtsA expression were observed only in the presence of BpA and UV cross-linking. Consistent with our previous data, FtsAR260-BpA showed a strong ∼135-kDa CLS via anti-ZipA (Fig. 2C, lane 12) and a prominent CLS in the same part of the gel via anti-FtsA (Fig. 2B, lane 12), visible here as a doublet. As a doublet, CLS was also visible with anti-FtsA in other cases (e.g., Fig. 1E); we suspect that a portion of the protein sometimes undergoes cleavage near the FLAG-FtsA junction and these two populations then form cross-links, resulting in variations in CLS molecular weights. An additional weak higher-molecular-weight CLS at ∼200 kDa was also detectable by anti-FLAG or anti-ZipA and was reproducible under different conditions (see below). However, although a weak CLS was visible for H261X probed with anti-ZipA (Fig. 2C, lane 13), analogous BpA-specific CLS bands above background were not detectable with anti-ZipA for K258X, V259X, or G262X despite detectable CLS bands with anti-FtsA (Fig. 2A and B, lanes 10, 11, and 14).

One possible reason for the sharp drop in cross-linking efficiency on either side of R260 as detected by anti-ZipA is that residue 260 is an exclusive FtsA-ZipA contact point. However, this is unlikely given that strong cross-linking is achieved when BpA replaces the arginine at this position. A more likely scenario is that the interaction with ZipA only occurs on the exposed surface of the helix, oriented away from the FtsZ binding face of FtsA (Fig. 2D). If true, we considered that other exposed residues in helix 7 distal from R260 might exhibit a similar capacity to generate ZipA-containing CLS as R260.

To test this hypothesis, we selected two putative exposed residues, D253 and A256, on the same side of the helix as R260 (Fig. 2D). When we tested amber codon replacements of these two residues in the cross-linking assay, we reproducibly detected prominent CLS with anti-ZipA, and they were specific for the presence of BpA and UV irradiation, although the CLS bands were weaker than those for R260X (Fig. 2C, lanes 5 and 8). These data support the idea that multiple residues in FtsA helix 7 can cross-link with ZipA, provided they are on the exposed face of the helix pointing away from the FtsZ binding face of FtsA. Notably, BpA substitutions at both D253 and A256 complemented the ftsA12 mutant nearly as well as WT FtsA (Fig. 2A and D).

To further test our hypothesis and more fully characterize the role of helix 7 in protein-protein interactions, we generated single amber mutants for the remaining residues in the helix (Fig. 2D). BpA substitutions at P251, A254, and E255, all predicted to face inward, showed no detectable CLS with anti-ZipA (Fig. 2B and C, lanes 3, 6, and 7), although all three substitutions showed CLS of various intensities with anti-FtsA (Fig. 2B and C, lanes 3, 6, and 7), suggesting that they can interact with another molecule, probably FtsA, more readily than with ZipA. The BpA substitution at S252, which is at the top of the helix and is predicted to face outward, behaved similarly to substitutions at D253, A256, and R260 in that it showed robust CLS bands at ∼135 kDa with both anti-FtsA and anti-ZipA and complemented ftsA12 efficiently (Fig. 2A to C, lane 4). Altogether, our data indicate that only residues on the outward-facing surface of FtsA helix 7 are capable of generating strong FtsA-ZipA cross-links (Fig. 2D).

Disruptions in helix 7 differentially inhibit FtsA-FtsA and FtsA-ZipA interactions.Given the key involvement of helix 7 in generating CLS, we wanted to determine the role of some of the residues that cross-link with ZipA (D253, A256, and R260) in FtsA function in vivo. Using pDSW210F-FtsA as the template for site-directed mutagenesis, we generated D253R, D253W, A256R, A256W, R260E, and R260W substitutions to test the effects of charge changes and the introduction of bulky hydrophobic residues at these positions. These mutants in pDSW210F were transformed into wild-type (MG1655) and ftsA12 (WM1115) strains to assess their function under permissive (30°C) and nonpermissive (42°C) conditions.

Notably, the D253R and R260E opposite-charge mutants mainly behaved like wild-type FtsA, complementing ftsA12 at 42°C and exhibiting toxic effects with IPTG concentrations higher than 100 µM (Fig. 3A and B). D253R consistently conferred slightly more toxicity than WT FtsA. In contrast, A256R was unable to complement and was not toxic (Fig. 3A and B), indicating that it is nonfunctional. As A256 is not well conserved in other FtsA proteins, it is possible that replacing a small neutral residue with a bulkier charged residue at the center of the helix (A256R) causes more dramatic effects on structure and function. Indeed, the A256R protein was undetectable by immunoblotting even with high levels of IPTG induction (see Fig. S6A). Interestingly, replacing D253 or A256 with W showed no toxicity and was able to suppress ftsA12, similar to the phenotype of ftsA*-like alleles (Fig. 3A and B). In contrast, R260W complemented ftsA12 weakly at low levels but was extremely toxic under all conditions. Given their FtsA*-like nontoxic phenotype, we asked whether the D253W and A256W replacements could bypass ZipA, but they did not (see Fig. S7).

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FIG 3

Disruption of FtsA helix 7 reduces FtsA-ZipA interaction. (A and B) Various FtsA constructs with residue changes in helix 7 were expressed in ftsA12(Ts) cells (WM1115) and tested for viability at different concentrations of inducer under either permissive (A) or nonpermissive (B) conditions. (C) A subset of these residue changes were converted into BpA-containing derivatives and tested for their ability to cross-link with ZipA. Protein extracts from cultures supplemented with BpA and cross-linked with UV were separated by SDS-PAGE and probed on Western blots with anti-FtsA or anti-ZipA. FtsA, ZipA, and cross-linked species (CLS) are highlighted. Blots contain spliced lanes from the same gel. Molecular weight markers in kilodaltons are shown at the left.

As all BpA substitutions are by definition already missense changes like those mentioned above, we chose to generate a set of small deletions (ΔD253, ΔA256, ΔR260, ΔA256-R260, and ΔD253-R260) to investigate further the role of these residues in cross-linking with ZipA. We first tested their capacity to suppress ftsA12. At 42°C, the single residue deletions at the ends of helix 7, ΔD253 and ΔR260, suppressed ftsA12 without toxicity (Fig. 3A and B). In contrast, FtsA derivatives harboring ΔA256, ΔA256-R260, or ΔD253-R260 complemented ftsA12 poorly, either not at all (Δ256-260) or required 500 µM IPTG to achieve weak growth (ΔA256 and ΔA253-260) (Fig. 3B). All three of these deletion proteins were expressed and inducible by IPTG but at approximately half the levels of the intact FtsA, which correlate with the phenotypes observed (Fig. S7). Combined with the amino acid substitution data described above, these data suggest that A256 itself is not required for proper FtsA function, but even a single residue deletion in the middle of helix H7 can potentially disrupt the helical structure and inactivate FtsA function in vivo.

We next determined the effects of the deletions in helix 7 on the ability of FtsA to form cross-links with ZipA. To use R260-BpA as the donor residue for cross-linking, we combined the D253R, D253W, or ΔD253 mutant with R260X. Conversely, we combined ΔR260 with D253X so that D253-BpA would serve as the cross-linking residue. When we probed Western blots with anti-FtsA, CLS were detected for R260-BpA as expected (Fig. 3C, lane 2), as well as for R260-BpA plus ΔD253 (Fig. 3C, lane 5), but band intensities were significantly reduced for R260-BpA plus D253R (Fig. 3C, lane 3) and further reduced for R260-BpA plus D253W (Fig. 3C, lane 4). By analogy with R260-BpA plus ΔD253, significant CLS were observed for D253-BpA plus ΔR260 (Fig. 3C, lane 6). In contrast, by using the anti-ZipA antibody, strong CLS were detected for R260-BpA plus D253R as well as the control R260-BpA (Fig. 3C, lanes 8 and 9), but only weak CLS were detected for R260-BpA plus D253W (Fig. 3C, lane 10) and almost undetectable for R260-BpA plus ΔD253 and D253-BpA plus ΔR260 (Fig. 3C, lanes 11 and 12). Altogether, the data suggest that alterations in helix 7 can differentially affect FtsA-FtsA and FtsA-ZipA interactions, especially as the levels of endogenous ZipA and plasmid-expressed FtsA are relatively unchanged in the different strains used for cross-linking (Fig. 3C). In particular, deletion of D253 when BpA replaces R260, or deletion of R260 when BpA replaces D253, can drastically inhibit the ability of FtsA to interact with ZipA, possibly because two residues at the interface are affected simultaneously.

FtsA*-like mutants cross-link similarly to wild-type FtsA.The overproduction of ZipA inhibits cell division, but FtsA* confers resistance to these toxic effects (31). Although the mechanism of resistance is not known, it is possible that the lower oligomerization capacity or higher membrane binding capacity of FtsA* (11, 42) plays a role. We reasoned that if this were the case, FtsA*-R260-BpA might exhibit reduced cross-linking to ZipA compared with that of FtsA-R260-BpA. We engineered a double replacement carrying R260X and R286W (FtsA* allele) to test this idea. However, when this double replacement was produced from pDSW210F, we saw no significant differences in the generation of CLS by FtsA-R260-BpA versus that by FtsA*-R260-BpA (Fig. 4B), either in the ftsA12 strain background (WM1115, at the permissive temperature) or a strain with ftsA* replacing ftsA in the chromosome (WM1657). As shown in Fig. 1, WM1657 also lacks ZipA, as seen by the lack of CLS detectable by anti-ZipA (Fig. 4B, lane 6), but the ZipA-containing CLS is restored upon ectopic expression of ZipA from a plasmid (Fig. 4B, lane 8).

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FIG 4

FtsA* forms CLS in the absence of ZipA and cross-links as well as FtsA. Cross-linking experiments with FtsAR260-Bpa or FtsA containing both R286W (FtsA* allele) and R260-BpA tested whether the presence of the R286W allele (ftsA*), which decreases FtsA oligomerization, influences the relative levels of cross-linking to ZipA or another FtsA. The zipA⁺ strain WM1115 or the ftsA* ΔzipA strain WM1657, with or without pPK9-ZipA to overproduce ZipA, was used to express either FtsAR260-BpA (lanes 1 and 2) or FtsA*R260-BpA (lanes 3 to 8) in the presence of BpA, UV cross-linked or not, followed by separation of proteins by SDS-PAGE and probing Western blots with anti-FtsA (A) or anti-ZipA (B). Un-cross-linked FtsA and ZipA, as well as major and minor CLS bands, are denoted with arrowheads. Molecular weight markers are shown at the left. Lanes 1 and 2 and lanes 3 to 8 were from separate gels.

These results suggest that the ZipA-resistant phenotype of FtsA* is not a consequence of poor interactions with ZipA. Moreover, the strong CLS observed from the ΔzipA strain detected by anti-FtsA (Fig. 4A) indicates that our cross-linking conditions may not be able to distinguish between the strong oligomerization activity of FtsA and the weaker oligomerization predicted for FtsA*. This may be because FtsA’s oligomerization activity is regulated over the course of cell division, and our cross-linking experiments, which use relatively low levels of protein that do not significantly perturb cell division, may capture its time-averaged oligomerization activity instead of one specific oligomerization state. Finally, these results clearly demonstrate that FtsA*-R260-BpA (Fig. 4A) or FtsA-R260-BpA (Fig. 1D and E, lane 8) is capable of cross-linking to other proteins, probably other FtsA subunits, independently of ZipA.

FtsA interacts with ZipA in bacterial two-hybrid assays.To provide independent support for the FtsA-ZipA interaction observed by cross-linking, we investigated whether FtsA and ZipA could give a positive signal in a bacterial two-hybrid assay. This assay relies on the ability of the T18 and T25 domains of adenylate cyclase to generate beta-galactosidase in cells and thus a Lac⁺ phenotype that can be scored colorimetrically on agar plates, only when proteins fused to these two domains interact sufficiently closely to bring the two domains together. When we combined pUT18c-FtsA with pKNT25-ZipA, the colonies were strongly positive, consistent with the ability of FtsA and ZipA to interact in cells (Fig. 5, A5).

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FIG 5

Bacterial two-hybrid assay for ZipA-FtsA interactions. BTH101 cells containing pUT18c derivatives carrying full-length FtsA (WT) or small deletions within helix 7 plus pKNT25 or pKT25 derivatives fused to FtsA, FtsN, FtsZ, or ZipA (or empty vector) were spotted onto LB X-Gal plates with antibiotics and 50 µM IPTG, incubated for 24 h at 30°C, and photographed. All six replicates for each pair had identical phenotypes, and so only one example of each pair is shown.

We then asked whether disruptions of residues in helix 7 of FtsA might disrupt the two-hybrid signal in the same way they inhibited FtsA-ZipA cross-linking. When pUT18c-FtsAΔ260 was combined with pKNT25-ZipA, the Lac⁺ phenotype was at least as strong as that with WT FtsA, indicating that deleting only R260 had no effect on the interaction (Fig. 5, B5). This is different from the result for FtsA A253-BpA R260 in cross-linking experiments, which showed a significant diminution of CLS; however, this could easily be explained by the simultaneous loss of both A253 and R260 in the cross-linking experiment (A253 is changed to BpA), as opposed to only the loss of R260 in the bacterial two-hybrid assay.

When we tested a deletion of 5 residues (Δ256-260) in helix 7, which we showed earlier cannot complement ftsA12, it no longer interacted with ZipA in the two-hybrid assay (Fig. 5, C5). To rule out the possibility that this pUT18c-FtsA Δ256-260 construct is inactive, we combined it with T25 versions of FtsN, FtsA, and FtsZ and found that it gave positive two-hybrid interactions with FtsA and FtsN but not with FtsZ (Fig. 5, C2 to 4). The ability of the deleted FtsA to interact with FtsN or another FtsA indicated that the loss of interaction with ZipA was fairly specific, although the loss of interaction with FtsZ also suggested that the deletion resulted in allosteric conformational changes. A larger deletion of residues 253 to 260, essentially removing the entire helix 7, abolished all interactions, including those with FtsA, FtsN, and FtsZ (Fig. 5, D2 to 5), perhaps because the removal of helix 7 destabilizes the entire protein structure when expressed from pUT18c. Yet, FtsA Δ253-260 expressed from pWM2785 retains some ability to complement ftsA12, suggesting in that context, it is not completely inactive.

Role of the Z ring in FtsA-ZipA interaction.Although the FtsA-ZipA cross-linking data indicate that the in vivo interaction is direct, we asked whether this interaction is dependent on an intact Z ring. We took two approaches. In the first approach, we tested FtsAR260-BpA for the ability to cross-link to ZipA after a relatively brief treatment with nalidixic acid (NA), an inhibitor of DNA gyrase that rapidly induces the SOS response and disassembly of Z rings (43). By introducing the FtsAR260X and pEVOL plasmids into a functional ftsZ-mCerulean chromosomal reporter strain (44) and adding BpA to the medium, we confirmed that many midcell FtsZ-mCerulean rings remained after growth in BpA, resuspension of the culture in buffer, and UV cross-linking (see Fig. S8C). ZipA-green fluorescent protein (GFP) rings also persisted at midcell after UV cross-linking in experiments expressing that fusion protein (Fig. S8D). In contrast, nearly all cells grown in parallel but in the presence of NA for 1 h lacked Z rings (Fig. S8C). When we compared anti-FtsA or anti-ZipA immunoblots after UV irradiation, we observed that the ∼135-kDa CLS containing ZipA and FtsA from the NA-treated cells were present at levels equivalent to the CLS from untreated cells (Fig. S8A and B, compare lanes 6 and 8). We also repeated the cross-linking experiment in our WT strain background containing native FtsZ and again observed equivalent CLS bands with or without NA treatment (Fig. S8A and B, compare lanes 2 and 4).

In a more targeted approach, we tested FtsAR260-BpA for the ability to cross-link to ZipA in a ftsZ84(Ts) strain (WM1125), which forms Z rings and divides fairly well at 30°C but mostly lacks Z rings when shifted to the nonpermissive temperature of 42°C. Comparing the formation of CLS in a WT strain with that in the ftsZ84 strain, we found that CLS formed readily at both 30°C and 42°C, with more intense bands at 42°C for both the WT and ftsZ84 backgrounds (Fig. S8E), with either anti-FtsA (lanes 1 to 4) or anti-ZipA (lanes 5 to 8). Even with the expression of FtsAR260-BpA, cells of this mutant divided fairly well at 30°C, although not at 42°C, as expected (Fig. S8F). Taken together, these results suggest that the FtsA-ZipA interaction through FtsA’s helix 7 can occur independently of the Z ring.