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Journal of Bacteriology, March 2004, p. 1546-1555, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1546-1555.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Switch I and II Regions of MinD Are Required for Binding and Activating MinC

Huaijin Zhou and Joe Lutkenhaus*

Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160

Received 1 October 2003/ Accepted 19 November 2003


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ABSTRACT
 
MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.


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INTRODUCTION
 
In Escherichia coli, the min system prevents Z-ring assembly away from the midcell (1, 6). This spatial regulation of Z-ring assembly requires all three products of the min locus, MinC, MinD, and MinE. MinC and MinD cooperate to form a division inhibitor that is topologically regulated by MinE (6). MinC is an antagonist of FtsZ assembly (15) that is recruited to the membrane by MinD and, together, induced to oscillate between the poles of the cell by MinE (11, 28). Through this oscillation, the time-averaged concentration of MinC and MinD at the membrane is highest at the poles and lowest at the midcell (24). Although it appears that FtsZ has the capacity to assemble into a Z ring at many points on the cell membrane, the Min system helps to limit the position to the midcell (37).

Overexpression of MinC, but not MinD, in a {Delta}min strain prevents Z-ring formation, establishing MinC as an inhibitor of division (1, 7). In vitro, MinC antagonizes FtsZ assembly without affecting GTP hydrolysis, indicating that it does not block assembly but instead promotes disassembly (15). MinC can be divided into two domains, an N-terminal domain responsible for antagonism of FtsZ assembly and a C-terminal domain responsible for dimerization and interaction with MinD (12, 34). The crystal structure of MinC confirmed that it is a dimer with two structurally independent domains and that it dimerizes through its C-terminal domain (3).

Although MinC is the inhibitor and can antagonize FtsZ assembly in vitro, MinD is required for it to be effective at its physiological concentration (7). MinD recruits MinC to the membrane (11, 16, 21, 28) and confers a high affinity on the complex for some septal component (19). This affinity was revealed by examining the localization of a green fluorescent protein (GFP)-tagged MinC mutant that is unable to antagonize FtsZ assembly. The MinD-dependent localization of such GFP-tagged MinC mutants to Z rings is readily observed, since the complex localizes to rings but is unable to cause their destruction. DicB, encoded by the defective Kim prophage, can also confer high affinity to MinC for a septal component (19).

MinD is at the center of the min system and has been shown to interact with itself, the membrane, MinC, and MinE. MinD self-interaction and interaction with MinC and MinE have been demonstrated with the yeast two-hybrid system (17, 32). Mutations that affect the nucleotide binding site of MinD reduce the interaction of MinD with MinC and MinE as well as self-interaction and binding to the membrane, suggesting that ATP is required for all MinD interactions (9, 10, 14, 27).

In vitro studies have shown that MinD binds cooperatively to vesicles in an ATP-dependent manner and is released by MinE stimulation of the MinD ATPase (10, 21, 25). MinE stimulation of the MinD ATPase requires the presence of phospholipid vesicles, indicating that only the membrane-bound form of MinD is susceptible to MinE stimulation (13). MinC is recruited to the MinD-vesicle complex and can be displaced by MinE, even in the absence of ATP hydrolysis (16, 21). However, MinC cannot displace MinE bound to the MinD-vesicle complex (16). Furthermore, once MinD is bound to the vesicle, it is able to undergo assembly into polymers that are able to tubulate the vesicles (10). Assembly of MinD into polymers on vesicles could help to recruit MinD to the site of assembly and restrict MinD diffusion until it is released by MinE. Recently, GFP-MinD was observed in spiral-like patterns in vivo, indicating that the oscillation of MinD involved the formation of MinD polymers on the membrane (30).

Binding of MinD to the membrane is ATP dependent and requires a short, conserved C-terminal region that encodes an amphipathic helix (14, 35). Hydrophobic residues within this helix mediate binding by inserting into the bilayer (38). Deletion of this helix or mutations that substitute charged residues for hydrophobic residues prevent MinD from binding to the membrane (35, 38). Importantly, such mutations do not prevent MinD binding to MinC in the yeast two-hybrid system but decrease the targeting of MinC to the septal ring (14). This result indicated that MinD's interaction with MinC may not require the membrane but that membrane-bound MinD is required for efficient targeting of MinCD complexes to the septum. Together, these results have led to a model in which the MinCD complex assembles on the membrane at the poles of the cell and acquires a high affinity for a septal component. In this model, the polar targeting and subsequent polymerization of MinD restrict the MinCD complex from diffusing in the membrane so that it does not come into contact with the cell center (14). However, a Z ring attempting to form in the polar MinCD zone would come into contact with MinCD and be destabilized.

MinD-like proteins have been crystallized as monomers from several Archaea species with ADP or no nucleotide bound (4, 9, 29). However, MinD has been reported to be a dimer. In one study, dimerization was ATP dependent (14), whereas in the other, it was not (32). The structure of MinD is similar to that of NifH, which has been compared to small G proteins (4, 8, 23). These proteins have regions designated switch I and II that undergo nucleotide-dependent conformational changes that are responsible for interactions with their partners. In this study, we have isolated MinD mutants that are unable to cooperate with MinC to block division to further explore the MinD-MinC interaction. Our results demonstrate the importance of the switch I and II regions in the interaction between MinD and MinC.


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MATERIALS AND METHODS
 
Strains and plasmids. The E. coli K-12 strain JS964 (MC1061 malP::lacIq {Delta}min::kan) was used in this study. The plasmids used in this study are listed in Table 1. To construct the plasmid pGB2CD2, the minCD genes, including the promoter region and ribosome binding site upstream of minC, were cloned into the low-copy-number plasmid pGB2 (2). The primers used to amplify the minCD fragment were 5'MTNCD2, 5'-TAGCATGAATTCACGACGGCAATGGGTTGATTG-3', and 3'MTNCD2, 5'-TAGCATAAGCTTAACTTATCCTCCGAAGAAGCG-3', with the locations of restriction sites indicated by underlining. To construct the plasmid pHJZ109, a fragment containing minC116-231 and minD was amplified from pJPB210 (minCDE) by PCR with the primers 5'SalIminC116-231, 5'-TAGCATGTCGACGCGCAAAATACAACGCCGGTC-3',and 3'HindIII minD, 5'-TAGCATAAGCTTCTTATCTCTCGAACAAGCGTTTGA-3'. The fragment was cloned such that the C-terminal fragment of MinC was in frame downstream of gfp (mut2) in pSEB181. pSEB181 was constructed by S. Pichoff and contains the EcoRI-SspI fragment from pJC106 (11) cloned into pAM238 (11) between the FspI and EcoRI sites. pHJZ109-D9 (minD-E126A) was constructed similarly to pHJZ109, except that pSEB12-D9 (minD-E126A) (as described below) was used as the template for PCR.


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  		TABLE 1. Plasmids used in this study

Isolation of minD mutations. A PCR-based, random mutagenesis approach (22) was used to introduce mutations into minD. The mutagenic PCR contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.25 mM MnCl2, 20 µM concentrations of each deoxynucleoside triphosphate, 1 mM concentrations of each primer, 50 ng of template DNA (pGB2CD2), and 1.25 U of Taq DNA polymerase in a 100-µl reaction mixture. The initial denaturing step was carried out at 94°C for 5 min followed by 40 cycles of temperature cycling (94°C for 30 s, 55°C for 30 s, and 72°C for 90 s) and a final incubation at 72°C for 10 min. Primers used in the reaction were 3'MTNCD2 and 5'MluC, 5'-TATTAATCGCGTTTTTGTGACCGGCGT-3', which amplified a fragment of 1.16 kb containing minC116-231 minD. The pool of mutated fragments was digested with BstXI and HindIII, generating a 0.7-kb fragment containing codons 26 to 270 of minD. By doing this, we excluded the first 25 codons to avoid mutations in the deviant Walker A motif already known to affect the MinD-MinC interaction (5, 9). The pool of mutated fragments was used to replace the wild-type fragment on pGB2CD2. The resultant plasmids were electroporated into JS964 ({Delta}min), and transformants were selected on plates containing spectinomycin. The transformants were inoculated into liquid culture, and cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting to assess the level of MinD. Forty-nine of the 111 clones examined expressed MinD at a level comparable to the wild-type MinD expressed from pGB2CD2. Of these 49 clones, 30 were further analyzed by sequencing and 20 of them were found to contain a single mutation. DNA fragments containing these minD mutations were subcloned into different vectors by PCR to generate various fusions, pZH106 (gfp-minD), pJC41(AD-minD), pJC42-1 (BD-minD), and pZH115 (pJF118EH-minD), with the same primers that were used for cloning the wild-type minD into these vectors. In addition, several minD mutations were made by site-directed mutagenesis. The switch II mutations minD-IE125,126AA, minD-I125E, and minD-E126A were introduced into minD contained on pZH106 by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The minD-I126E mutation was also introduced into minD on pSEB12 (minCDE) by site-directed mutagenesis, giving rise to pSEB12-D9 (minD-I126E).

Yeast two-hybrid assay. Interaction between MinD and itself and MinC can be detected in the yeast two-hybrid system (17, 19, 32). The effect of various minD mutations on the interaction between MinD and MinC and the interaction of MinD with itself were assessed by using the yeast two-hybrid assay as described previously (17). The corresponding yeast two-hybrid plasmids described above were transformed in various combinations into the yeast reporter strain SFY526 as described in the Clontech manual (BD Bioscience, Palo Alto, Calif.). Double transformants were selected on media without tryptophan and leucine. Transformants were examined for ß-galactosidase activity both qualitatively by the colony lift assay and quantitatively as described in the Clontech manual.

Fluorescence microscopy. The effect of minD mutations on the membrane localization of GFP-MinD fusions were examined as described previously (13). To test the ability of minD9 to target GFP-MinC116-231 to the septum, plasmid pHJZ109-D9 (minD-E126A) was transformed into JS964 ({Delta}min) by selecting for Spcr. pHJZ109 was used as a control. Cultures of the transformants were grown at 37°C until the optical density at 600 nm reached 0.05. At this point, 40 µM of isopropyl-ß-D-thiogalactopyranoside (IPTG) was added, and 1 h later, samples were removed and fixed with 2% glutaraldehyde. Aliquots were placed on microscope slides, and cells were photographed with a Nikon fluorescence microscope with a MagnaFire charge-coupled device camera (Optronics). Images were imported into Adobe PhotoShop for assembly.

Phenotypic analysis of minD mutants. To test the effect of minD mutations on the ability of MinD to activate MinC to block cell division, derivatives of pZH106 (gfp-minD) were cotransformed with pZH110 (minC) into JS964 ({Delta}min) and selected on Luria-Bertani plates containing ampicillin, spectinomycin, and 0.2% glucose. Colonies were restreaked onto plates with ampicillin and spectinomycin with arabinose added at 0.0001% and 0.001% as indicated. The effect of minD mutations on cell morphology was determined by phase-contrast microscopy after overnight incubation.

Protein purification. MinD-S121T, MinD-R44G, MinD-G42A, MinD-G42D, MinD-I141N, and MinD-E126A were overexpressed and purified with plasmids pJF118EH-minD-S121T, -minD-R44G, -minD-G42A, -minD-G42D, -minD-I141N, and -minD-E126A, respectively. The purification procedure was the same as that for the wild-type MinD protein. All of the mutants behaved the same as the wild-type protein during the purification except MinD-G42D, which proved to be unstable. Protein concentrations were determined by protein assay (Bio-Rad Laboratories, Hercules, Calif.), and the purity was judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MinE and MalE-MinC116-231 were purified as described before (16).

MinD ATPase and membrane binding assay. The MinD ATPase assay was as described previously (13). MinD, MinD mutants (9 µM), and phospholipid vesicles (400 µg/ml) were mixed with 1 mM [{gamma}-32P]ATP in an ATPase buffer (25 mM Tris · HCl [pH 7.5], 50 mM KCl, 5 mM MgCl2). MinE (9 µM) was added to reaction mixtures as indicated. Reaction mixtures were incubated at 30°C, the amount of released Pi was determined, and the specific activities were calculated. In the sedimentation assay, MinD mutants (4 µM), phospholipid vesicles (400 µg/ml), and nucleotide (1 mM ADP or ATP) were mixed at room temperature in 50 µl of ATPase buffer. MalE-MinC116-231 and MinE were added at 4 µM as indicated in the figure legends. The reaction mixtures were incubated at room temperature for 20 min. For reaction mixtures containing MinE, the samples were incubated at 30°C. Samples were centrifuged at 10,000 x g at room temperature in a tabletop centrifuge for 2 min. The supernatants were carefully removed, and the pellets were resuspended in 50 µl of SDS sample buffer. Twenty-microliter aliquots of the samples were electrophoresed on SDS-12.5% PAGE and stained with Coomassie brilliant blue.


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RESULTS
 
Isolation of MinD mutants unable to activate MinC. A low-copy-number plasmid containing minCD cannot be introduced into a {Delta}min strain in the absence of MinE. This offers a simple approach to isolate mutations that inactivate minC or minD. We used a plasmid containing the minC and minD genes cloned into the low-copy-number plasmid pGB2 (Spcr). This plasmid, pGB2CD2, could not transform JS964 ({Delta}min) unless this strain also contained pJPB216, which expresses minE under lac promoter control, and was grown in the presence of IPTG. To generate mutations, the minD gene was amplified from pJPB210 (minCDE) by a mutagenic PCR procedure as described in Materials and Methods. The approach we used excluded the first 25 codons to avoid mutations in the deviant Walker A motif already known to be required for MinD function. The pool of PCR fragments was used to replace minD in pGB2CD2, and transformants of JS964 ({Delta}min) were selected on plates containing spectinomycin. Transformants were screened by immunoblot analysis for the level of expression of MinD to try to eliminate mutations that lead to instability. Of 111 transformants, 49 were found to express MinD at a level comparable to a control containing the wild-type minD (pGB2CD2). DNA sequence analysis of 30 of these 49 revealed that 20 contained a single mutation. These 20 were chosen for further study. The locations of the mutations are summarized in Fig. 1. Despite screening for protein stability, many of these mutations contain proline substitutions which are likely to disrupt the protein structure. However, other mutations affect charged residues that probably do not affect folding and are likely to have other defects.



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  		FIG. 1. Location of mutations in minD. This diagram indicates the conserved motifs that have been identified within MinD. The location of the mutations and the amino acid substitutions that were analyzed in this study are indicated. The mutations above the diagram were obtained by random mutagenesis and screening for loss of MinD function. The mutations below the diagram were constructed by site-directed mutagenesis.

Effect of minD mutations on self-interaction and interaction with MinC. One possible explanation for the inability of the MinD mutants to inhibit division and prevent colony formation is that they no longer interact with MinC. To test this possibility, we utilized the yeast two-hybrid test in which a strong interaction is observed between MinD and MinC. We found that MinD-I141N interacted strongly with MinC, although somewhat less than the wild type. The other 19 mutants, however, showed no interaction with MinC (Table 2). Possible explanations for the failure of these mutants to bind MinC include that they don't fold properly, they don't interact with ATP, which is required for MinD to bind MinC, or they have an alteration in the surface of MinD that interacts with MinC.


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  		TABLE 2. Summary of the effects of minD mutations isolated in this study

MinD self-interaction can also be assayed by the yeast two-hybrid system (19, 32). This interaction may reflect the interaction to form dimers. We observed that the MinD self-interaction is weaker than the interaction observed between MinC and MinD in this test system. Of the 20 mutants, only 4, MinD- R44G, MinD-G42A, MinD-G42D, and MinD-I141N, displayed self-interaction. In fact, two of these, MinD-R44G and MinD-G42A, displayed stronger interaction than the wild type. If we assume that this test reflects dimerization, then these four mutants are able to dimerize, whereas the other 16 are not (Table 2, three are listed under mutations affecting ATP binding or hydrolysis motifs, and the other 13 are grouped separately). Since three of the four mutants that self-interact do not interact with MinC, the corresponding mutations may define residues specifically required for MinC interaction. The fourth mutant, MinD-I141N, binds MinC and must be defective in another step.

Effect of MinD mutations on MinD localization. MinD is a peripheral membrane protein that binds to the membrane through a C-terminal amphipathic helix (14, 35). In vitro it has been shown that this binding requires ATP, which also promotes MinD dimerization (16). To determine the effect of the mutations isolated in this study on MinD localization, each of the mutations was introduced into a gfp-minD fusion downstream of the arabinose promoter. Each of the plasmids was introduced into JS964 ({Delta}min) and examined by fluorescence microscopy.

The 16 mutants, such as MinD-S121T (Fig. 2), that failed to display self-interaction in the yeast two-hybrid test also failed to localize to the membrane. In contrast, three of the mutants which displayed self interaction, MinD-R44G, MinD-G42A, and MinD-G42D, were clearly on the membrane, as they produced a halo-like appearance comparable to that seen with the wild-type MinD (Fig. 2). However, MinD-I141N, the other mutant that displayed self interaction and the only one that bound MinC, was present throughout the cytoplasm. The failure of MinD-I141N to bind to the membrane would explain its reduced ability to activate MinC. The behavior of MinD-I141N is similar to mutants reported previously (14) that fail to bind to the membrane due either to deletion of the C-terminal helix or to substitution of charged for hydrophobic amino acid residues within this helix. Such mutants are still able to bind MinC in the yeast two-hybrid system but are unable to activate it as effectively as the wild-type MinD due to a deficiency in recruiting it to the membrane and targeting it to the septum (14). Interestingly, the minD-I141N mutation does not lie in the C-terminal helix but lies near the middle of the protein (Fig. 1).



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  		FIG. 2. Effect of minD mutations on membrane localization. N-terminal GFP fusions to the various MinD mutant proteins were examined in JS964 ({Delta}min). Cells containing the fusions were fixed with 2% glutaraldehyde 1 h after induction of the fusion with 0.001% arabinose. Cells were analyzed by fluorescence microscopy. (A) Wild type; (B) MinD-R44G; (C) MinD-G42A; (D) MinD-S121T; (E) MinD-I141N.

Biochemical analysis of MinD mutants. To determine the effects of minD mutations on the biochemical activities of MinD, selected minD alleles were cloned into an expression vector for purification of the mutant proteins. The four mutants that displayed self-interaction (MinD-R44G, MinD- G42A, MinD-G42D, and MinD-I141N) and one that did not (MinD-S121T) were chosen. MinD-G42D proved to be unstable during purification and was not studied further. The other mutant proteins were stable and behaved similarly to the wild-type protein during purification.

Wild-type MinD displays a basal ATPase activity that is stimulated about 10-fold by MinE in the presence of phospholipid vesicles (13, 21, 31). MinD-R44G and MinD-G42A expressed a basal ATPase that was three to five times the level of the wild-type protein (Fig. 3). Nonetheless, this basal level was stimulated by MinE such that the stimulated level was higher than the wild-type stimulated level. In contrast, the basal ATPase activity of MinD-I141N was slightly lower than the wild-type level and was not stimulated by MinE. Also, the basal ATPase activity of MinD-S121T was comparable to the wild-type activity but was only stimulated twofold by MinE (Fig. 3).



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  		FIG. 3. ATPase activity of MinD mutants. MinD (D) proteins (9 µM) were mixed with phospholipid (PL) vesicles (400 µg/ml) and analyzed for ATPase activity in the presence or absence of MinE (E) (9 µM) as indicated.

Of the four mutant proteins examined for ATPase activity, the two that showed a level of stimulation by MinE similar to that of the wild type localized to the membrane in vivo. In contrast, the two that showed little or no stimulation failed to localize to the membrane. This result is consistent with the previous finding that ATPase stimulation by MinE requires that MinD binds to the membrane (13, 14). Further support for this interpretation was provided by examining binding to phospholipid vesicles in vitro. Both MinD-R44G and MinD-G42A showed ATP-dependent binding to phospholipid vesicles, whereas MinD-I141N and MinD-S121T did not display significant binding (Fig. 4A). Since MinD-R44G and MinD-G42A bound to vesicles, we could also test whether they were released by MinE and whether they were able to recruit MinC. Both MinD-R44G and MinD-G42A displayed an approximately 50% reduction in the amount of bound protein when MinE was added (Fig. 4B). Importantly, both mutants failed to recruit MinC (Fig. 4C) to the vesicles. This latter result is consistent with their failure to interact with MinC in the yeast two-hybrid system and explains their failure to inhibit division in the presence of MinC.



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  		FIG. 4. Interaction of MinD mutants with phospholipid vesicles. (A) MinD switch I mutants bind to phospholipid vesicles. MinD proteins (4 µM) were mixed with 400 µg of phospholipid vesicles/ml in the presence of 1 mM ATP. After adding the ATP, the samples were centrifuged and the pellets were analyzed by SDS-PAGE. (B) MinE stimulates release of MinD switch I mutants from vesicles. MinD proteins were incubated with phospholipid vesicles as described for panel A in the presence (+) or absence (-) of MinE (4 µM). (C) Analysis of the ability of switch I and switch II mutants to recruit MinC to vesicles. MinD (4 µM) was incubated with phospholipid vesicles in the presence of 1 mM ADP or ATP. MalE-MinC116-231 (4 µM) was added, the samples were centrifuged, and the pellets were analyzed by SDS-PAGE.

Mutations in the switch II region. Proteins in the extended ParA family, NifH, ArsA, and MinD, have been compared to G proteins, and regions have been identified that correspond to the switch I and II regions (4, 8). These regions correspond to signal transduction pathways and communicate information about the state of the bound nucleotide to other regions of the protein. Each region has an aspartate at one end of the switch that contacts the bound nucleotide indirectly through H2O molecules bound to an Mg2+ ion. Two of the mutations that we isolated in the first part of this work contain mutations that alter these aspartic acid residues. MinD-D38A at the base of switch I and MinD-D120G at the base of switch II produce proteins that do not bind MinC or the membrane (Table 2). These mutants also fail to display self-interaction, suggesting that they fail to dimerize. Presumably, such mutants do not bind ATP or are unable to differentiate ATP from ADP. This is in contrast to mutations that alter residues in the switch I region adjacent to D38A and produce proteins (MinD-R44G and MinD-G42A) that failed to bind MinC but retain other functions such as self-interaction, membrane binding, and MinE stimulation. We, however, obtained no mutations in the switch II region. We therefore constructed three mutations in the switch II region by site-directed mutagenesis (MinD- I125E, MinD-E126A, and MinD-IE125,126AA) to try to determine what role, if any, the switch II region has in MinD function.

The MinD mutants MinD-I125E, MinD-E126A, and MinD-IE125,126AA were constructed in the GFP-MinD expression vector under arabinose promoter control. All three mutant proteins localized to the membrane, suggesting that the switch II region, like the switch I region, did not have a significant role in membrane binding (Fig. 5). Next, the three mutants were examined for the ability to activate MinC to block cell division (Table 2). Plasmids containing the corresponding mutations were transformed into JS964 ({Delta}min) containing pZH110 (minC) and selected on plates containing glucose, which causes maximum repression of the arabinose promoter. Under these conditions, a plasmid containing the wild-type GFP-MinD fusion only yields a few transformants and the cells are extremely filamentous. Thus, the glucose plates probably most closely represent the physiological situation, since minC and minD cannot be introduced together in a single copy into a {Delta}min strain. In contrast, the switch II mutants yielded ~1,000 transformants with a minicell phenotype similar to that of a plasmid control containing GFP-MinD-K16Q, a mutant that appears to be completely inactive (5, 10).



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  		FIG. 5. Switch II mutants localize to the membrane in vivo. GFP fusions to the various MinD switch II mutants were analyzed for membrane localization as described in the legend to Fig. 2. (A) MinD- IE125,126AA; (B) MinD-E126A; (C) MinD-I125E.

Restreaking the transformants on plates lacking glucose or containing various amounts of arabinose gives some indication of the degree of MinD attenuation. For example, transformants with the MinD-K16Q mutation gave a minicell phenotype at all arabinose concentrations, which is consistent with MinD-K16Q being completely inactive. In contrast, transformants with MinD-I125E started to filament at high arabinose concentrations, whereas transformants with MinD-E126A or MinD-IE125,126AA started to filament in the absence of both glucose and arabinose. The degree of attenuation of these latter mutants is similar to that observed with MinD{Delta}3, which is missing the three carboxyl amino acids and binds poorly to the membrane (14). Thus, all three switch II mutants are attenuated, which suggests that they are either deficient in binding MinC, similar to switch I mutants, or deficient in a subsequent step such as targeting the MinCD complex to the septum.

The yeast two-hybrid test revealed that one of the three switch II mutants, MinD-I125E, failed to bind MinC while the other two still bound MinC (Table 2). This result indicated that the switch II region has some role in interaction with MinC. However, the fact that two of the switch II mutants still bound MinC and the membrane indicated that the switch II region is also required at a step after the binding of MinD to MinC and the membrane.

To further explore the ability of these switch II mutants to activate minC, we examined the ability of MinD-E126A to target the C terminus of MinC to the septal ring. Plasmids were constructed with minD (pHJZ109) or minD-E126A (pHJZ109-D9) downstream of a gfp-minC116-231 fusion and transformed into JS964 ({Delta}min). Cells from the colonies were examined by fluorescence microscopy to determine whether the GFP fusion was localized in bands indicative of localization to septal rings. Control cells containing MinD contained fluorescent bands as described previously (19), indicating that the GFP-MinC116-231-MinD complexes were being targeted to septal rings (Fig. 6). Cells containing MinD-E126A also contained fluorescent bands, indicating that it could still target GFP-MinC116-231 to the septal ring.



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  		FIG. 6. The switch II mutant MinD-E126A targets MinC116-231 to septal rings. The ability of the MinD-E126A mutant to target MinC to the septal machinery was assessed by fluorescence microscopy. JS964 ({Delta}min) containing the control plasmid pHJZ109 (gfp-minC116-231-minD) (A) or pHJZ109-D9 (gfp-minC116-231-minD-E126A) was grown in Luria-Bertani broth at 37°C until the optical density at 600 nm reached 0.05. IPTG was added at 40 µM, and the cells were fixed with 2% glutaraldehyde 1 h later. Cells were analyzed by fluorescence microscopy.

Since MinD-E126A could still target GFP-MinC116-231 to septal rings, we checked the Min phenotype of this mutation following introduction on a single-copy plasmid carrying the intact min operon. Introduction of a plasmid carrying the wild-type min operon conferred a wild-type morphology on JS964 ({Delta}min) (Fig. 7A). In contrast, JS964 ({Delta}min) carrying the plasmid pSEB12-D9 (minC minD-E126A minE) displayed a typical Min phenotype with minicells and nucleated cells of heterogeneous length (Fig. 7B). This result confirms that the minD-E126A mutation confers a typical Min phenotype and is unable to spatially regulate cell division.



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  		FIG. 7. MinD-E126A is unable to provide MinD function to spatially regulate division. To verify that minD-E126A lacked the ability to spatially regulate division, it was placed in the context of the min operon on a single-copy plasmid and introduced into JS964 ({Delta}min). Phase-contrast microscopy of exponentially growing cells is shown. (A) JS964 ({Delta}min) pSEB12 (minCDE); (B) JS964 ({Delta}min) pSEB12-D9 (minC minD-E126A minE).

To support the in vivo results, MinD-E126A was purified and examined for MinD functions. MinD-E126A expressed a basal ATPase activity that was stimulated by MinE similar to wild-type MinD (data not shown). As expected from this result and the in vivo localization, MinD-E126A bound to phospholipid vesicles in an ATP-dependent fashion (Fig. 4C). Furthermore, MinD-E126A was able to recruit MinC to the vesicles, consistent with its ability to interact with MinC in the yeast two-hybrid system (Fig. 4C; Table 2). Thus, this mutant is able to bind to the membrane, recruit MinC, and undergo MinE stimulation. It is also able to target the MinCD complex to the septal ring, leading to the conclusion that the MinC-MinD-E126A complex is unable to carry out destruction of the Z ring.


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DISCUSSION
 
MinC and MinD associate to form an inhibitor of division that prevents Z rings from forming away from the midcell in the presence of MinE. The formation of an efficient MinCD inhibitor involves several steps, including (i) the interaction of MinD with ATP, (ii) the binding of MinD to itself, to MinC, and to the membrane, (iii) the acquisition by the MinCD inhibitor of high affinity for some septal component following membrane binding, and (iv) destabilization of Z rings upon MinCD binding to a septal component. Also, MinD must interact with MinE for MinCD to be spatially regulated.

The purpose of this study was to investigate the requirements for MinD activation of MinC. We isolated loss-of-function mutations in minD coupled with site-directed mutagenesis to gain information about these steps and to identify the regions of MinD that are required. Several loss-of-function mutations affected residues important for interaction with ATP. As a result, they failed to self-interact, bind to the membrane, or interact with MinC, indicating that ATP is required for all MinD interactions. Several mutations were isolated that affected residues that correspond to the switch I and II regions of MinD. Analysis of these mutations revealed that the switch I and II regions are primarily involved in the binding and activation of MinC, as the other activities of MinD were retained. Another mutation (minD-I141N) affected membrane binding without significantly affecting MinC binding.

Mutations leading to loss of MinD interaction with itself, the membrane, and MinC. Most of the loss-of-function mutations we isolated are defective in self-interaction, localization to the membrane, and binding to MinC. Many of these mutations result in either proline substitutions that probably alter protein structure or substitutions that alter residues located in motifs required for ATP binding and/or hydrolysis, including S121T, D38A, and D120G. The two aspartic acid residues are at the bases of the switch I and switch II regions (Fig. 8) and are involved in hydrogen bonding to H2O molecules that in turn are bound to the Mg2+ ion required for ATP binding and hydrolysis (9). Loss of either of these aspartic acid residues would likely disrupt ATP binding. Serine 121 is at the base of the pocket near the {gamma}-phosphate of the bound ATP. It is likely that replacing it with the larger threonine residue occludes the space that would be occupied by the {gamma}-phosphate and would therefore interfere with ATP binding. These results suggest that ATP binding is required for dimerization, binding to the membrane, and binding to MinC.



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  		FIG. 8. Model of MinD indicating the locations of the residues investigated in this study. The MinD protein sequence of E. coli was modeled on the structure of the MinD-like protein of P. furiosus (PDB accession no. 1G3R). The C-terminal 25 residues are not in the structure, since the corresponding region is not present in the MinD-like protein of P. furiosus. The positions of the residues altered in this study are indicated. The switch I residues are colored red, and the switch II residues are colored cyan.

Other mutations that affect residues in the deviant Walker A motif involved in ATP binding and hydrolysis have been analyzed and found to be deficient in interaction with MinC (5, 9). In this study, we have analyzed one of these, MinD-K16Q, and found that it is also deficient in self-interaction in the yeast two-hybrid test. Previous results have shown that this mutant is also unable to bind to the membrane (10). Thus, the behavior of this mutant is typical of MinD mutants that are defective in interaction with ATP; they don't interact with themselves, MinC, or the membrane.

The effects of mutations in minD genes from other bacteria have also been reported. Mutations in the minD gene from Bacillus subtilis that alter the same conserved aspartic residues at the bases of the switch I and switch II regions investigated here have been studied previously (20). The mutations were found to prevent the polar targeting of MinD (which is dependent upon DivIVA), but they did not appear to prevent MinD from binding to the membrane. We are not sure of the basis for this apparent difference, since MinD from B. subtilis also binds to the membrane through a C-terminal amphipathic helix (35). A K16Q substitution in the Neisseria gonorrhoeae minD largely eliminated the self-interaction in the yeast two-hybrid test (27), similar to what we observed here.

MinD binding to the membrane. Analysis of MinD has revealed that a C-terminal motif, which has the potential to form an amphipathic helix, is required for MinD to bind to the membrane (14, 33, 35). More recently, evidence for this model was provided by demonstration that tryptophan residues, substituted for the hydrophobic residues within this helix, are embedded in the bilayer (38). This has led to a model in which ATP binding by MinD activates this helix so that it can interact with the membrane. One possibility is that ATP binding promotes dimerization, resulting in a tighter bivalent association with the membrane (33, 38).

Although most of the loss-of-function mutations we isolated failed to bind to the membrane, we isolated one mutation, minD-I141N, which primarily affected membrane binding, since it displayed self-interaction and interaction with MinC in the yeast two-hybrid system. In vitro, this mutant displayed poor binding to vesicles and expressed a basal ATPase that was poorly stimulated by MinE. These results are very similar to a mutant, MinD{Delta}10, with a deletion of the C-terminal amphipathic helix (14). Interestingly, however, the minD-I141N mutation does not affect a residue in the C-terminal amphipathic helix but one that maps near the middle of the primary sequence (Fig. 1). Since this mutant appears to dimerize, it is likely that it is unable to regulate the C-terminal amphipathic helix in response to ATP binding.

Interaction of MinD with MinC and MinE. The mutations we isolated that mapped to the switch I and II regions of MinD are primarily defective in the interaction with MinC. Three of these mutations (G42A, R44G, and I125E) lead to a loss of interaction between MinD and MinC in the yeast two-hybrid system; however, none of the mutations affected MinD self-interaction or localization to the membrane. Detailed study of the effect of two of the switch I mutations on MinD function in vitro revealed that the mutant proteins had an elevated basal ATPase activity but were still stimulated by MinE. Consistent with this, the proteins underwent ATP-dependent vesicle binding and were released by MinE. The mutants, however, were unable to recruit MinC to the membrane. These results are consistent with these mutants being primarily deficient in the binding of MinC. The switch I region has been designated the MinD box, as it is highly conserved among MinD proteins but not among other members of the ParA family, of which MinD is a member (36).

Interestingly, MinE can displace MinC from a MinC-MinD-vesicle complex made with a nonhydrolyzable ATP analogue, suggesting that it may compete with MinC for binding to MinD (16, 21). However, the binding of the two proteins to MinD must be distinct, since MinE stimulates the MinD ATPase, whereas MinC has no stimulatory effect and does not interfere with the MinE stimulation (13). Furthermore, MinC cannot displace MinE from a MinE-MinD-vesicle complex. This distinction between the binding of MinC and MinE is further documented here, as the switch I mutations eliminate MinC binding without having an observable effect on the interaction between MinD and MinE. These results are consistent with the previous suggestion that MinE induces a conformation change in MinD, resulting in release of MinC (16). Furthermore, we have also observed that the switch I mutants can assemble on vesicles to cause tubulation, indicating that the switch I region is not required for MinD polymerization that occurs on vesicles (data not shown).

In addition to the two switch I mutations, one of the mutations in the switch II region (I125E) prevented MinC binding. This mutation affects a residue, based upon homology modeling with the MinD-like protein from Pyrococcus furiosus, which lies on the surface of the MinD molecule (Fig. 8). Although similar in sequence, it should be noted that the P. furiosus protein is unlikely to carry out the same function as MinD, since it lacks the C-terminal amphipathic helix and therefore is unlikely to bind to the membrane. It has not been tested in vivo but was shown to bind very poorly to phospholipid vesicles in vitro (31). Although the residues in the switch I region are on the surface in a monomer, they are not readily accessible to the surface in the postulated dimer model (23). Thus, these residues are unlikely to interact with MinC directly but may function as a link to surface residues. This would be similar to what is postulated for the switch I region of NifH (18). On the other hand, residue I125 in the switch II region is on the surface in both the monomer and dimer models and therefore might interact directly with MinC.

MinD activation of MinC. The MinCD complex acquires a high affinity for some septal component upon binding to the membrane (19). This affinity for a septal component is easy to visualize experimentally provided that a mutant MinC is used that is unable to cause disassembly of the Z ring. In contrast, MinCD complexes formed between MinC and MinD mutants unable to bind to the membrane fail to acquire this high affinity and to decorate the septum (14). One of the switch II mutants (E126A) was able to target the GFP-MinC116-231 fusion; however, it was unable to inhibit division efficiently, since placing the minD-E126A mutation in the context of the min operon resulted in a Min phenotype. There appears to be two possible explanations for this mutant. Either MinD is required for an additional step beyond targeting MinC to the septum or the MinD-E126A-MinC complex has a lower affinity for the septum, but this is overcome by the expression of the GFP fusion protein, which is visualized at a level higher than the physiological level.

Although MinC or the N-terminal half of MinC is able to antagonize FtsZ assembly in vitro (15), this must represent a basal activity of this inhibitor. In vivo, MinC requires MinD to be an efficient inhibitor. This activation involves MinD bringing MinC to the membrane and targeting it to the septum, steps that require the C-terminal domain of MinC (19). However, the behavior of MinD-E126A raises the possibility that MinD may have an additional role. It may be required at a step subsequent to targeting, possibly activating the N-terminal domain of MinC following binding to the C-terminal domain.

Order of MinD interactions. The binding of MinD to itself, the membrane, and MinC are all dependent upon ATP; however, the order of these interactions is not clear. Earlier experiments demonstrated that MinD recruited MinC to vesicles (16, 21) but did not address whether MinD bound to MinC in the absence of vesicles. ATP-dependent but vesicle-independent dimerization of MinD and binding to MinC have been observed by size-exclusion chromatography; however, that analysis was done at relatively high protein concentrations (~30 µM) (16). Interestingly, we have found that MinD mutants that do not bind to the membrane, such as MinD-I141N (this study) and MinD{Delta}10 (14), are able to self-interact and bind MinC quite well in the yeast two-hybrid system. These results indicate that these MinD mutants dimerize and bind MinC quite well independent of the membrane.

MinD binding to vesicles and its ATPase both show cooperative behavior suggesting oligomerization (13, 21, 25). MinD is a monomer in the presence of ADP but undergoes ATP-dependent dimerization at high concentrations (~30 µM) in the absence of phospholipid vesicles (14). It is quite possible, however, that at lower concentrations the membrane promotes MinD dimerization. Evidence for membrane-dependent dimerization has been obtained from analysis of MinD proteins tagged with different fluorophores. Fluorescence resonance energy transfer between the differentially tagged MinD proteins was only observed after the addition of phospholipid vesicles to MinD at a concentration of 1 µM (25).

How can we reconcile the observations that MinD may require a membrane for dimerization and MinC interaction, whereas MinD-I141N and MinD{Delta}10 do not? One possible explanation may lie in the recent observation that removal of the amphipathic helix from MinD facilitates dimerization. It is possible that removing the helix, or possibly affecting its availability (as with MinD-I141N), bypasses the membrane requirement (C. Saez and J. Lutkenhaus, unpublished data). This result suggests that the C-terminal amphipathic helix masks the dimerization domain in the wild-type monomer and that the membrane promotes MinD dimerization upon the addition of ATP, and therefore MinC binding, by sequestering the helix away from the dimer interface.


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ACKNOWLEDGMENTS
 
This work was supported by grant GM2974 from the National Institutes of Health.

We thank Gerry Lushington for modeling the E. coli MinD structure.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160. Phone: (913) 588-7054. Fax: (913) 588-7295. E-mail: jlutkenh{at}kumc.edu. Back


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Journal of Bacteriology, March 2004, p. 1546-1555, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1546-1555.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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