Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors

Abstract

Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinC_Ng) and E. coli MinC (MinC_Ec) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinC_Ec interacts with itself and MinD_Ec; however, no interactions involving MinC_Ng were detected. Therefore, a recombinant MinC protein, with the N terminus of MinC_Ec and the C terminus of MinC_Ng, was designed to test for a MinC_Ng-MinD_Ng interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinC_Ec G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinC_Ec G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the α-7 helix of MinD.

The MinC protein plays a pivotal role in determining where cell division occurs in several bacterial species. In gram-negative bacteria, such as Neisseria gonorrhoeae and Escherichia coli, this protein acts as a cell division inhibitor in concert with MinD and MinE (5, 23). In E. coli, MinC has been shown to block cell division by destabilizing FtsZ rings (10), a protein involved in septal ring formation (20). Overexpression of MinC in E. coli produces a filamentous phenotype indicative of cell division inhibition (5, 23). In contrast, inactivation of this protein results in minicell morphology, which is mediated by irregular cell division at the cell poles (5). In the diplococcus N. gonorrhoeae, which, unlike E. coli, divides in alternating perpendicular planes (32), inactivation of minC results in enlarged cells with disrupted placement of the septum (23). Interestingly, gonococcal MinC complements an E. coli minC mutant, and its overexpression causes E. coli cells to filament, indicating the common functionality of MinC in the genera Neisseria and Escherichia (23).

Size exclusion chromatography, as well as limited proteolytic analyses, indicated that E. coli MinC (MinC_Ec) exists as a dimer (30). The MinC_Ec monomer consists of two separate domains joined by a flexible linker; the N-terminal domain comprises amino acid residues 1 to 99, and the C-terminal domain includes residues 125 to 231 (11). The N terminus of MinC_Ec interacts with FtsZ_Ec (10), while the C terminus has been implicated in self-interaction and interaction with MinD_Ec (11). It has been proposed that the interaction of MinC_Ec with MinD_Ec brings the N terminus of MinC_Ec close to its target, FtsZ_Ec (15).

The crystal structure of Thermotoga maritima MinC (MinC_Tm) indicated that it comprises four molecules arranged as two dimers (4). The N- and C-terminal domains of MinC_Tm are bound by a flexible linker, which is hypervariable between species (4). The N-terminal domain of each monomer contains two α-helices and five β-strands, and the C-terminal domain consists of a right-handed β-helix, which resembles a triangular barrel with three surfaces, A, B, and, C (4). The dimerization interface for MinC_Tm was found to be located on the hydrophobic A surface, although no specific amino acid residues were implicated in this function (4). The MinC residues implicated in the MinC-MinD interaction have not been identified.

In a current E. coli model explaining how the Min proteins act to determine the septation site, it is proposed that MinD dimerizes in the cytoplasm in the presence of ATP and then binds to a MinC dimer (12, 13). This MinCD_Ec complex then interacts with the cell membrane (12, 21, 31). The subsequent binding of MinE_Ec to the MinCD_Ec complex causes the displacement of MinC_Ec and stimulates the ATPase activity of MinD_Ec (13, 19, 21). As a result of ATP hydrolysis, MinD_Ec and MinE_Ec are released from the membrane. Through membrane-associated coiled structures, MinD_Ec oscillates from one end of the cell to the other and at the same time recruits MinC_Ec away from the cell center, allowing accumulation of FtsZ in this region (19, 21, 28). Gonococcal Min proteins also exhibit dynamic behavior in both rod-shaped and round E. coli cells, which divide in alternating perpendicular planes as N. gonorrhoeae does (24). Since the MinC_Ec and N. gonorrhoeae MinC (MinC_Ng) proteins play a critical role in the functioning of the min system in their hosts and since the C-terminal domains of these proteins are highly conserved, we were interested in establishing what role could be attributed to specific completely conserved residues in the function of MinC_Ng or MinC_Ec.

Alignment of MinC sequences from 36 different bacterial species indicated that while the MinC C terminus is highly conserved, only five residues (four glycines and one arginine) are identical. Glycine is unique among the amino acids due to its small size, the enhanced backbone flexibility that it confers (1), and its involvement in protein stability (16) or in protein-protein interactions (2). For these reasons, we investigated the functional roles of the conserved glycines in the C terminus of MinC_Ng and MinC_Ec by amino acid substitution using site-directed mutagenesis.

In this study, we found that the completely conserved C-terminal glycines are essential for maintaining MinC functionality as a cell division inhibitor and for the interaction of MinC with other Min proteins. On the basis of existing crystal structures of the MinC and MinD proteins and taking into account which MinC glycine residues should be surface exposed, we propose a specific interaction between residues on the α-7 helix of MinD and the B-C surface junction of MinC.

MATERIALS AND METHODS

Strains and growth conditions.

Strains and plasmids used in this study are shown in Table 1. E. coli DH5α was used for cloning purposes. E. coli PB103 (wild type) and E. coli DR105 (minC) were used for expression and complementation studies, respectively. E. coli C41(DE3) was used as an expression strain for purification of six-His-tagged MinC_Ng. E. coli cells were grown at 37°C for 6 to 8 h in Luria-Bertani broth (Difco) supplemented with 100 μg of ampicillin per ml and 40 μM isopropyl-β-d-thiogalactopyranoside (IPTG), when required. Saccharomyces cerevisiae strain SFY526 (Clontech) was used in yeast two-hybrid assays to examine protein-protein interactions. Yeast was grown at 30°C on yeast extract-peptone-adenine-dextrose medium or on the appropriate synthetic dropout media to select for transformants, as described in the Clontech yeast two-hybrid manual.

TABLE 1.

Strains and plasmids used in this study

Strains or plasmid	Relevant genotype	Source or reference
Strains
E. coli DH5α	supE44ΔlacU169 (φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	Gibco-BRL
E. coli PB103	dadR1 trpE61 trpA62 tna-5 purB+	23a
E. coli DR105	zcf-117::Tn10/dadR1 trpE61 trpA62 tna-5 minC3 Tetr	23a
E. coli C41 (DE3)	F−ompT hsdSB(rB− mB−) gal dcm Δ(srl-recA)306::Tn10 (Tetr) (DE3)	23b
S. cerevisiae SFY526	MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 Canrgal4-542 gal80-538 URA3::GAL1UAS− GAL1TATA-lacZ	Clontech
Plasmids
pUC18	Ampr Plac	Amersham Pharmacia
pEGFP	Ampr Plac::gfp	Clontech
pSR2	Ampr Plac::minCNg	23
pSR23	Ampr Plac::minC(MinCEc1-99)	This study
pSR24	Ampr Plac::minC(MinCCh:MinCEc1-99-MinCNg103-237)	This study
pSR32	Ampr Plac::minCEc	This study
pSR32G135	Ampr Plac::minCEc(G135D)	This study
pSR32G154	Ampr Plac::minCEc(G154D)	This study
pSR32G161	Ampr Plac::minCEc(G161S)	This study
pSR32G171	Ampr Plac::minCEc(G171E)	This study
pSR32P141	Ampr Plac::minCEc(P141A)	This study
pVG4	Ampr Plac::minCNg(G138D)	This study
pVG6	Ampr Plac::minCNg(G157D)	This study
pVG8	Ampr Plac::minCNg(G164S)	This study
pVG10	Ampr Plac::minCNg(G174E)	This study
pPF1	Ampr Plac::minCNg(E144I)	This study
pGAD424	Ampr PADH1::gal4 (AD)c,d	Clontech
pSRAD-C	Ampr PADH1::gal4 (AD)-minCEc	This study
pSRAD-D	Ampr PADH1::gal4 (AD)-minDEc	29
pGADminC	Ampr PADH1::gal4 (AD)-minCNg	This study
pGADminD	Ampr PADH1::gal4 (AD)-minDNg	29
pSRAD-G135	Ampr PADH1::gal4 (AD)-minCEc (G135D)	This study
pSRAD-G154	Ampr PADH1::gal4 (AD)-minCEc (G154D)	This study
pSRAD-G161	Ampr PADH1::gal4 (AD)-minCEc (G161S)	This study
pHDAD-G171	Ampr PADH1::gal4 (AD)-minCEc (G171E)	This study
pSRAD-P141	Ampr PADH1::gal4 (AD)-minCEc (P141A)	This study
pSRAD-Ch	Ampr PADH1::gal4 (AD)-minC(MinCEc1-99-MinCNg103-237)	This study
pGBT9	Ampr PADH1::gal4 (BD)e	Clontech
pSRBD-C	Ampr PADH1::gal4 (BD)-minCEc	This study
pSRBD-D	Ampr PADH1::gal4 (BD)-minDEc	29
pGBT9minC	Ampr PADH1::gal4 (BD)-minCNg	This study
pGBT9minD	Ampr PADH1::gal4 (BD)-minDNg	29
pSRBD-G135	Ampr PADH1::gal4 (BD)-minCEc (G135D)	This study
pSRBD-G154	Ampr PADH1::gal4 (BD)-minCEc (G154D)	This study
pSRBD-G161	Ampr PADH1::gal4 (BD)-minCEc (G161S)	This study
pHDBD-G171	Ampr PADH1::gal4 (BD)-minCEc (G171E)	This study
pSRBD-P141	Ampr PADH1::gal4 (BD)-minCEc (P141A)	This study
pSRBD-Ch	Ampr PADH1::gal4 (BD)-minC(MinCEc1-99-MinCNg103-237)	This study
pSRBD-ChG135	Ampr PADH1::gal4 (BD)-minCCh(G135D)	This study
pSRBD-ChG154	Ampr PADH1::gal4 (BD)-minCCh(G54D)	This study
pSRBD-ChG161	Ampr PADH1::gal4 (BD)-minCCh(G161S)	This study
pSRBD-ChG171	Ampr PADH1::gal4 (BD)-minCCh(G171E)	This study
pPF2	Ampr PADH1::gal4 (BD)-minCCh(E141I)	This study
pET30a	Kanr PT7::6-His	Novagen
pHLCC	Kanr PT7:: minCNg-6-His	This study
pETminCG138	Kanr PT7:: minCNg(G138D)-6-His	This study
pETminCG157	Kanr PT7:: minCNg(G157D)-6-His	This study
pETminCG164	Kanr PT7:: minCNg(G164S)-6-His	This study
pETminCG174	Kanr PT7:: minCNg(G174E)-6-His	This study

^aGift from P. de Boer (Case Western Reserve University, Cleveland, Ohio).

^bGift from J. E. Walker (Medical Research Council, Cambridge, England).

^cP_ADH1 is yeast promoter which regulates expression of gal4.

^dEncodes the AD of GAL4.

^eEncodes the DNA BD of GAL4.

PCR and IPCR.

Oligonucleotide primers used for PCR and inverse PCR (IPCR) in this study are listed in Table 2. Primers were designed by using Primer Designer (Scientific and Education Software) and were synthesized by the University of Ottawa Biotechnology Research Institute. All PCRs and IPCRs were carried out with a Perkin-Elmer GenAmp PCR System 9600 thermocycler (Perkin-Elmer Corp.) as follows: 3 min at 94°C; 30 cycles of denaturation for 15 s at 94°C, annealing for 15 s at temperatures varying from 48 to 51°C (depending on the primer pair used), and extension for 1 to 7 min (depending on the expected product size) at 72°C; 5 min at 72°C; and hold at 4°C. The PCRs were carried out in 100-μl (final volume) mixtures containing 75.5 μl of double-distilled H₂O (ddH₂O), 10 μl of 10× PCR buffer containing 1.5 mM MgCl₂ (Roche Diagnostics Corp.), 2 μl of deoxynucleoside triphosphates (Boehringer Mannheim Corp.), 1 μl of each primer (0.2 μg/ml), 0.5 μl of Taq DNA polymerase for PCR amplification (Roche) or 0.1 μl of Vent DNA polymerase (New England Biolabs) for IPCR amplification, and 10 μl of template DNA. N. gonorrhoeae CH811 and E. coli PB103 cell suspensions were prepared by diluting cells from overnight cultures in ddH₂O. Cell concentrations were adjusted by using McFarland equivalence turbidity standard 0.5 (Remel) to provide chromosomal DNA templates for PCRs. The concentrations of plasmid DNA templates were adjusted to 0.01 μg/ml with ddH₂O for IPCRs.

TABLE 2.

Oligonucleotide primers used for PCRs in this study

Primer	Sequence	Product
min10	5′ GCG CCT GCA GAT GAT GGT TTA TAT AAT 3′	minCNg
min29	5′ GCG CGG ATC CCA AAC AAT TAC TCT GAG CC 3′c
NEc-up	5′ GCG CCT GCA GAT GTC AAA CAC GCC AAT C 3′	N terminus of minCEc
NEc-down	5′ GCG CAC TAG TCA GGA TAG GCA GCC C 3′a,c
CNg-up	5′ GCG CAC TAG TCA TTC GGA AAA TGT TAA AG 3′a	C terminus of minCNg
CNg-down	5′ GCG CGG GCC CTT ACT CTG AGC CAA TTG C 3′c
EcminC-up	5′ GCG CGA ATT CAT GTC AAA CAC G 3′a	minCEc
EcminC-down	5′ GCG CGG ATC CTC AAT TTA ACG GTT GAA CGG 3′a,c
EC G135-1	5′ TCC GAT CAG CGT ATT TAT GCT 3′b	minCEc G135D
EC G135-2	5′ ACG CAC CGG GGT ATC TAT TAA 3′c
EC G154-1	5′ GCT GAC GCC GAA TTG ATT GC 3′b	minCEc G154D
EC G154-2	5′ GCT AAC GTG GCT TGT AAC AA 3′c
EC G161-1	5′ GAT TCG AAC ATT CAT GTC TAT GG 3′b	minCEc G161S
EC G161-2	5′ GGC AAT CAA TTC GGC CCC AGC GC 3′c
EC G171-1	5′ GCG GAA CGT GCG CGT GCA GGG G 3′b	minCEc G171E
EC G171-2	5′ CAT CAT GCC ATA GAC ATG AAT G 3′c
EC P141-1	5′ GCT GCT CAA TGT GAT CTG ATT GTT AC 3′b	minCEc P141A
EC P141-2	5′ ATA AAT ACG CTG ACC GGA ACG CAG CG 3′c
minCG138-1	5′ ACC GAT CAG CAG GTT TAT GCC 3′b	minCNg G138D
minCG138-2	5′ ACG GAC AGG GGA TGT AAT CAA 3′c
minCG157-1	5′ CAG GAC GCG GAA TTG ATT GCA 3′b	minCNg G157D
minCG157-2	5′ GCT GAC CGC CCC CGT AAC AAT 3′c
minCG164-1	5′ GAT TCG AAT ATG CAT ATT TAT 3′b	minCNg G164S
minCG164-2	5′ TGC AAT CAA TTC CGC CCC CTG 3′c
minCG174-1	5′ AGA GAT CGT GCT TTG GCC GGC 3′b	minCNg G174E
minCG174-2	5′ CAT CGG CGC ATA AAT ATG CAT 3′c
minCE144-1	5′ GCC ATC GAT GGC GAT TTG ATT GTT ACG 3′b	minCNg E144I
minCE144-2	5′ ATA AAC CTG ACC GGT ACG GAC AGG 3′c
HLC2	5′ GCG CCA TAT GAT GGT TTA TAT AAT TGA ATG 3′a	minCNg
HLC3	5′ GCG CAA GCT TCT CTG AGC CAA TTG CAC TG 3′a,c

^aRestriction sites for the following enzymes are underlined: SpeI (CNg-up, NEc-down), EcoRI (EcminC-up), BamHI (EcminC-down, min29), PstI (min10, NEc-up), ApaI (CNg-down), ClaI (minCE144-1), TseI (EC P141-1), NcoI (HLC2), and HindIII (HLC3).

^bNovel restriction sites introduced for screening PCR products are underlined. Recognition sites for the following enzymes were used: Sau3AI (EC G135-1); BsaHI (EC G154-1), HinfI (EC G161-1), loss of AciI (EC G171-1), Sau3AI (minCG138-1), HgaI (minCG157-1), TaqI (minCG164-1), and Sau3AI (minCG174-1).

^cAnneals to complementary strand.

Site-directed mutagenesis of minC_Ec and minC_Ng.

Wild-type minC_Ec was PCR amplified by using primers EcminC-up and EcminC-down containing EcoRI and BamHI restriction sites, respectively (Table 2). This gene was ligated with pUC18, which had been previously digested with the same restriction enzymes, to generate pSR32 (Table 1). Wild-type minC in pSR32 was mutagenized by IPCR by using primers containing point mutations that extended in opposite directions around pSR32, as described by Sambrook and Russell (25). Five mutations in minC_Ec (G135D, G154D, G161S, G171E, and P141A) were introduced into pSR32 by using primer pairs EC G135-1 plus EC G135-2, EC G154-1 plus EC G154-2, EC G161-1 plus EC G161-2, EC G171-1 plus EC G171-2, and EC P141-1 plus EC P141-2 (Table 2), generating plasmids pSR32G135, pSR32G154, pSR32G161, pSR32G171, and pSR32P141, respectively (Table 1).

Similarly, wild-type minC_Ng was mutagenized in pSR2 (Table 1) by IPCR. Mutated minC_Ng genes with G138D, G157D, G164S, G174E, and E144I substitutions were created by using primer pairs minCG138-1 plus minCG138-2, minCG157-1 plus minCG157-2, minCG164-1 plus minCG164-2, minCG174-1 plus minCG174-2, and minCE144-1 plus minCE144-2 (Table 2), generating plasmids pVG4, pVG6, pVG8, pVG10, and pPF1 (Table 1), respectively. Plasmids containing mutant minC genes were screened for mutations by PCR amplification of minC_Ng with primers min10 and min29 (Table 2) and by PCR amplification of minC_Ec with primers EcminC-up and EcminC-down (Table 2). Purified amplicons were screened for mutations by digestion with appropriate enzymes since each site-directed mutation introduced a novel restriction site in the mutated gene that was not present in wild-type minC_Ec or minC_Ng (Table 2). It was thus possible to screen for minC mutants by examining the restriction endonuclease digestion patterns of plasmid-encoded minC genes from transformants and comparing them with the pattern of the wild-type minC gene. All mutant minC genes were sequenced to confirm point mutations, frame conservation, and gene integrity at the Biotechnology Research Institute, University of Ottawa.

Construction of recombinant MinCCh containing the N terminus of MinC_Ec and the C terminus of MinC_Ng.

Since no interactions of gonococcal MinC with itself or with MinD_Ng could be detected by using yeast two-hybrid methods, a chimeric MinC protein (MinCCh) was constructed to determine whether combined N- and C-terminal domains from MinC_Ec and MinC_Ng could restore these interactions. Plasmid pEGFP (Clontech) was used to make these constructs since it had suitable restriction sites needed for sequential cloning; however, the gfp gene was deleted, leaving only the chimeric minC gene (minCCh) cloned in the vector. The chimeric protein, MinCCh, comprised the N-terminal domain of MinC_Ec and the flexible linker and C-terminal domain of MinC_Ng. A 314-bp fragment which encoded amino acids 1 to 99 of MinC_Ec was PCR amplified with primers NEc-up and NEc-down, which contained PstI and SpeI restriction sites at their 5′ and 3′ ends, respectively (Table 2). This fragment replaced a PstI-SpeI fragment in pEGFP, deleting gfp and generating pSR23 (Table 1). A 425-bp fragment encoding amino acids 103 to 237 of MinC_Ng was PCR amplified with primers CNg-up and CNg-down containing SpeI and ApaI restriction sites at their 5′ and 3′ ends, respectively (Table 2). This partial minC_Ng fragment was cloned into pSR23 that had previously been digested with SpeI and ApaI, and pSR24 was generated with a chimeric minC gene encoding MinCCh (Table 1). minCCh was confirmed to be in frame by DNA sequencing at the Biotechnology Research Institute, University of Ottawa.

Morphological studies of E. coli transformants to determine MinC functionality.

To determine whether mutations in conserved glycine residues of MinC allowed the protein to retain its functionality, E. coli PB103 was transformed with plasmids containing each point mutation in minC_Ec (pSR32G135, pSR32G154, pSR32G161, pSR32G171, and pSR32P141), as well as with plasmids containing point mutations in gonococcal minC (pVG4, pVG6, pVG8, pVG10, and pPF1). Plasmids pSR2 (wild-type minC_Ng) and pSR32 (wild-type minC_Ec) were used as positive controls, and pUC18 was used as a negative control. Induction of filamentation due to the inability to form proper septa at the middle of the cell in E. coli has been used previously as an indicator of MinC functionality (5, 8, 12, 23). Similarly, to determine whether MinCCh retains functionality as a cell division inhibitor, E. coli PB103 was transformed with plasmid pSR24 encoding MinCCh. As no significant morphological differences were observed in cells with and without IPTG induction, all expression assays were conducted without induction and were repeated at least three times.

E. coli filamentation, defined as production of cells that were at least fourfold longer than wild-type rods (5), was assessed by both phase-contrast microscopy and flow cytometry. For phase-contrast microscopy, samples from each transformant were fixed to 0.1% polylysine-coated coverslips by using the protocol of Ramirez-Arcos et al. (23). Cells were visualized at a magnification of ×100 with oil immersion by phase-contrast microscopy by using a Zeiss Axioskop microscope. At least 10 microscope fields were examined for each transformant, and each field contained a minimum of 100 cells, which were analyzed to determine the percentage of filaments or normal-size short rods. Photographs were obtained with a Sony Power HAD 3CCD color video camera and Northern Eclipse software, version 6.0. Flow cytometry was used to determine the percentages of filamentous cells (length, >10 μm) and wild-type rods (length, 2 to 5 μm) in the whole population of E. coli cells. Cells were suspended in 1 ml of filter-sterilized phosphate-buffered saline to reduce the presence of background particles. Transformants were analyzed with a Beckman Coulter Epics XL-MCL flow cytometer at a voltage and gain of 50 V and 1.0, respectively, for side scatter and at a voltage and gain of 127 or 459 V and 1.0, respectively, for forward scatter. The event retrieval time was set to 300 s, and a maximum of 100,000 events were examined. Data retrieval and analysis were performed by using EXPO32 ADC XL 4 Color software and EXPO32 v.1.2 software, respectively. Gate A was created around wild-type rods by using PB103 transformed with pUC18 as a negative control. Gate B was created around filamentous cells by using PB103 transformed with pSR2 and pSR32 expressing MinC_Ng and MinC_Ec, respectively, as positive controls. The percentage of cells within each gate was calculated by determining the percentage of total gated cells within each gate and excluding background noise and particulate matter. A Student’s t test was used to determine significant differences in percentages of filamentation between wild-type and mutant proteins.

E. coli DR105 (minC) was used for complementation studies with gonococcal MinC having mutations at the conserved glycines, using previously described protocols (23).

Yeast two-hybrid assays.

Wild-type minC_Ng and minC_Ec, as well as minCCh, were PCR amplified individually by using primers incorporating EcoRI and BamHI restriction sites at the 5′ and 3′ ends, respectively (Table 2). Each gene was ligated in frame with the GAL4 DNA binding domain (BD) and the GAL4 activation domain (AD) of pGBT9 and pGAD424 (Clontech), respectively. The plasmids containing wild-type minC_Ng and minC_Ec were pGADminC (AD-MinC_Ng), pGBT9minC (BD-MinC_Ng), pSRAD-C (AD-MinC_Ec), and pSRBD-C (BD-MinC_Ec) (Table 1). The plasmids containing mutated minC_Ec were pSRAD-G135 (AD-MinC_Ec G135D), pSRBD-G135 (BD-MinC_Ec G135D), pSRAD-G154 (AD-MinC_Ec G154D), pSRBD-G154 (BD-MinC_Ec G154D), pSRAD-G161 (AD-MinC_Ec G161S), pSRBD-G161 (BD-MinC_Ec G161S), pHDAD-G171 (AD-MinC_Ec G171E), pHDBD-G171 (BD-MinC_Ec G171E), pSRAD-P141 (AD-MinC_Ec P141A), and pSRBD-P141 (AD-MinC_Ec P141A) (Table 1). Wild-type MinC_Ec interacted with MinD_Ec whether it was fused to the GAL4 DNA AD or the GAL4 DNA BD; however, the interaction was much stronger when MinC_Ec was fused to the GAL4 DNA BD. Hence, wild-type and mutant MinC_Ec fusions to this domain were used to examine their interactions with MinD_Ec, which was fused to the GAL4 DNA AD. The plasmids containing minCCh were pSRAD-Ch (AD-MinCCh) and pSRBD-Ch (BD-MinCCh) (Table 1). MinCCh interacted with MinD_Ng only when it was fused to the DNA BD of GAL4. Therefore, we constructed plasmids encoding mutant MinCCh proteins that were fused only to this domain. The gonococcal minC fragment encoding the C terminus of MinCCh in pSRBD-Ch (BD-MinCCh) was replaced with PCR fragments encoding the MinC_Ng G138D, G157D, G164S, G174E, and E144I mutants. These fragments were amplified from plasmids pVG4, pVG6, pVG8, pVG10, and pPF1 by using primers CNg-up and min29, containing SpeI and BamHI restriction sites at their 5′ and 3′ ends, respectively (Table 2). After SpeI and BamHI digestion of these fragments, they were ligated to a DNA fragment corresponding to pSRBD-Ch that had previously been digested with the same enzymes that excluded the region encoding the C terminus of wild-type MinC_Ng in this plasmid, and plasmids pSRBD-ChG135, pSRBD-ChG154, pSRBD-ChG161, pSRBD-ChG171, and pPF2 were generated (Table 1). These plasmids were transformed into E. coli PB103 for overexpression studies of the MinCCh mutant proteins. Filamentation was evaluated by phase-contrast microscopy. Plasmids pGADminD (AD-MinD_Ng), pGBT9minD (BD-MinD_Ng), pSRAD-D (AD-MinD_Ec), and pSRBD-D (BD-MinD_Ec) had been constructed previously in our laboratory (29) (Table 1).

All plasmids were transformed into S. cerevisiae SFY526 singly as negative controls or in pairs to test for protein-protein interactions by using the lithium acetate method (Clontech yeast two-hybrid manual). Yeast transformants were assayed for β-galactosidase activity by using the colony lift method with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) as a substrate and by liquid assays with o-nitrophenyl-d-galactopyranoside as a substrate as described in the Clontech yeast two-hybrid manual. Both colony lift and liquid assays were performed in triplicate at least twice for each interaction. Standard deviations were determined for the averages of the β-galactosidase activities. A Student’s t test was used to determine significant differences between β-galactosidase activities representing the strength of protein-protein interactions.

Protein analysis and Western blotting of bacterial and yeast extracts.

Protein extracts from E. coli PB103 expressing MinC_Ng, MinC_Ec, and MinCCh proteins with point mutations were prepared as described previously (23). Protein extracts from S. cerevisiae expressing mutant MinC_Ec and MinCCh fused to the GAL4 AD and the GAL4 BD were prepared by using the method described by Horváth and Riezman (9). Cells were grown to optical density at 600 nm of 1.5, harvested, washed in sterile ddH₂O, and resuspended in 100 μl of sample buffer (0.06 M Tris-HCl [pH 6.8], 10% glycerol, 2% sodium dodecyl sulfate, 0.0025% bromophenol blue). Samples were boiled for 5 min and then centrifuged at 14,000 rpm for 5 min (Sorvall MC 12V centrifuge) and the supernatants were designated the total-protein fractions. Bacterial and yeast protein fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and protein concentrations were standardized prior to membrane transfer by densitometry as described previously (23). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins were transferred to Immobilon-P membranes (Millipore Corporation), and Western blotting was performed by using methods described previously (23). The membranes were incubated with 1:100 anti-MinC_Ng antiserum overnight at 4°C. Differences in protein expression levels were calculated by densitometry.

Purification of His₆-MinC_Ng and circular dichroism (CD) analysis.

To purify wild-type and mutant MinC_Ng, the coding region of minC_Ng was PCR amplified with primers HLC2 and HLC3, which incorporated NdeI and HindIII restriction sites at the 5′ and 3′ ends, respectively (Table 2), by using pSR2 (wild-type MinC_Ng), pVG4 (MinC_Ng G138D), pVG6 (MinC_Ng G157D), pVG8 (MinC_Ng G164S), and pVG10 (MinC_Ng G174E) as templates. The genes were fused in frame to the C-terminal His₆ tag of pET30a (Novagen), generating pHLCC, pETminCG138, pETminCG157, pETminCG164, and pETminCG174, respectively (Table 1). The fusions were confirmed to be in frame by DNA sequencing. Log-phase cultures (500 ml) of E. coli C41(DE3) carrying these plasmids were induced with 0.4 mM IPTG for 0.5 h at 37°C with shaking at 250 rpm. Protein purification was performed as previously described (29), with a few modifications: proteins were washed with a buffer containing 30 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl (pH 7.9) and were eluted with the same buffer containing 250 mM imidazole, as described by Novagen. The protein concentration was determined by the Bradford method by using the Bio-Rad protein assay dye reagent.

CD spectroscopy was performed with a model 62DS CD spectrometer (AVIV Instruments, Inc., Lakewood, N.J.) with 1-mm-path-length quartz cell. The sample temperature was maintained at 20 ± 0.2°C. Far-UV CD spectra were recorded for both the wild-type protein and the mutants at protein concentrations between 0.1 and 0.2 mg/ml in 10 mM ammonium bicarbonate buffer (pH 8.0). All CD experiments were repeated three times to ensure spectral reproducibility. Following baseline subtraction, the CD spectra were normalized based on protein concentrations and were expressed in molar ellipticity per mean residue.

Protein alignment and modeling.

MinC sequences from 36 different prokaryotic microorganisms were obtained from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/). These sequences were aligned by using Clustal W software (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html).

Molecular modeling was performed by using SWISS-MODEL (http://www.expasy.org/swissmod/SWISS-MODEL.html). Briefly, we used MinC_Tm (PDB 1HF2) (4) and Pyrococcus furiosus MinD (MinD_Pf) (PDB 1G3R) (8) solved structures as templates for molecular modeling of a MinC_Tm-MinD_Pf complex. MinD_Pf has been previously modeled as a dimer by using the dimeric structure of the nitrogenase iron protein NifH (PDB 1N2C) (12, 21). Using this method, we superimposed MinD_Pf onto NifH to obtain a MinD_Pf dimer. We positioned this MinD_Pf dimer with the C terminus of a MinC_Tm monomer in a situation where a MinC_Tm-MinD_Pf interaction could occur between the MinD_Pf α-7 helix and the B-C surface junction of MinC_Tm. We calculated the overall exposure (fraction of solvent-accessible surface area) of the conserved glycine residues (G111, G129, G136, and G146) of MinC_Tm using the computer program VADAR (33). We also analyzed the structural flexibility that the conserved glycines conferred to MinC_Tm by measuring their specific temperature factors or B values (B factors) using Swiss-PDB (http://au.expasy.org/spdbv/). The B factors are linearly related to the mean square displacement of an atom and give an indication of atomic flexibility in the crystalline state of a protein.

RESULTS

Conserved C-terminal glycine residues in the MinC_Ng and MinC_Ec proteins are implicated in the function of MinC as a cell division inhibitor.

As of October 2003, 36 MinC sequences were available from the National Center for Biotechnology Information for comparison by protein alignment. Our analysis revealed that the C-terminal domain of MinC contains five completely conserved residues (one arginine and four glycines) (Fig. 1). The alignment also showed that the C terminus of MinC is more conserved than the N terminus and the flexible linker region of the protein (data not shown). A valine residue (corresponding to V154 of MinC_Ng) is conserved in all but 2 of the 36 species analyzed (Fig. 1). Due to the unique role that glycines may have in protein flexibility, protein stability, or protein-protein interactions, we investigated the functional effects of the conserved glycines in the C terminus of both MinC_Ng and MinC_Ec.

FIG. 1.

Multiple alignment of the conserved C terminus of prokaryotic MinC. Ng, Neisseria gonorrhoeae; Nm, Neisseria meningitidis; Ec, Escherichia coli; Sf, Shigella flexneri; St, Salmonella enterica serovar Typhi; Yp, Yersinia pestis; Pl, Photorabdus luminescens; Kp, Klebsiella pneumoniae; Bu, Buchnera sp.; Vc, Vibrio cholerae; So, Shewanella oneidensis; Xf, Xylella fastidiosa; Xc, Xanthomonas campestris; Pa, Pseudomonas aeruginosa; At, Agrobacterium tumefaciens; Sm, Sinorhizobium sp.; Bm, Brucella melitensis; Bj, Bradyrhizobium japonicum; Ml, Mesorhizobium sp.; Sy, Synechocystis sp.; Ta, Thermosynechococcus elongatus; Gv, Gleobacter violaceus; Bc, Bacillus cereus; Ba, Bacillus anthracis; Bs, Bacillus subtilis; Bh, Bacillus halodurans; Lm, Listeria monocytogenes; Li, Listeria innocua; Oh, Oceanobacillus iheyensis; Tt, Thermoanaerobacter tengcongensis; Cp, Clostridium perfringens; Dr, Deinococcus radiodurans; Tm, Thermotoga maritima; Lp, Lactobacillus plantarum; Hh, Helicobacter hepaticus; Hp, Helicobacter pylori. White type on a black background indicates residues that are conserved, while black type on a gray background indicates related amino acids. Asterisks indicate the four completely conserved glycine residues in the C terminus of MinC. The dagger, double dagger, and section sign indicate a completely conserved arginine, the position of the nonconserved residues selected for site-directed mutagenesis as controls, and a highly conserved valine, respectively. Predicted surfaces A, B, and C, as well as turns of the β-helix of the MinC C terminus, are labeled.

A number of single-point mutants were generated in these MinC proteins by site-directed mutagenesis, including G138D, G157D, G164S, and G174E MinC_Ng mutants and corresponding G135D, G154D, G161S, and G171E MinC_Ecmutants. The specific glycine substitutions selected in the present study were based on previous reports in which minC_Ec mutants were characterized after mutagenesis with nitrosoguanidine (12), ethyl methanesulfonate (18), or hydroxylamine (22). While many induced MinC_Ec mutants had nonsense and missense mutations, several mutations involved changes from glycine (G) to charged amino acids, such aspartic acid (G10D and G171D), glutamic acid (G161E), lysine (G161K), and arginine (G176R) (18, 22). In the present study, mutations at nonconserved (i.e., neutral) C-terminal residues (E144I mutation in MinC_Ng and P141A mutation in MinC_Ec) were also created to validate the functional significance of mutations in conserved glycine residues of MinC.

The functionalities of the different glycine mutants were determined by their abilities to inhibit cell division, as observed by filamentation, when they were overexpressed in wild-type E. coli PB103 (Table 1). Induction of filamentation in E. coli upon overexpression of MinC has often been used as an indicator of MinC functionality (5, 8, 12, 23). After transformation of E. coli PB103 with plasmids encoding the MinC_Ng or MinC_Ec mutants, phase-contrast microscopy and flow cytometry were used to assess the presence and extent of filamentation compared to the filamentation of wild-type rod-shaped cells.

In the analysis of MinC overexpression in E. coli by phase-contrast microscopy, at least 1,000 cells from random microscopic fields were counted. Approximately 90% of the E. coli cells overexpressing wild-type MinC_Ng from pSR2 (which encodes MinC_Ng cloned into pUC18) were filamentous, as were cells overexpressing the neutral MinC_Ng mutant E144I (Table 3). Most (∼99%) of the E. coli cells carrying pUC18, which served as a negative control, had a typical wild-type, short-rod morphology, indicating that normal cell division occurred. Mutant MinC_Ng proteins with mutations at each of the individual conserved glycines did not result in significant filamentation upon overexpression in E. coli, indicating that each mutant was not able to function as a cell division inhibitor (Table 3). The percentage of filamentation for the MinC_Ng glycine mutants varied from 0.07 ± 0.03 to 1.28 ± 0.38% (Table 3). Overexpression of the four glycine mutants of MinC_Ec also failed to produce filamentation in E. coli PB103, indicating that the mutations produced nonfunctional MinC_Ec proteins. The percentage of filamentation for these mutants varied between 0.75 ± 0.08 and 3.12 ± 0.39%, (Table 3). By contrast, overexpression of the MinC_Ec P141A mutant protein resulted in a filamentous phenotype similar to that of wild-type MinC_Ec, providing further evidence that the P141A mutation was neutral in affecting functionality (Table 3). Thus, the four conserved glycine residues in MinC_Ec or MinC_Ng are implicated in the function of MinC as a cell division inhibitor.

TABLE 3.

Summary of functionality of MinC_Ng, MinC_Ec, and MinCh mutants

Protein	Mutation	% Filamentation upon overexpression in E. coli PB103a	Protein interactions determined by yeast two-hybrid assays
			Self	MinD
MinCEc	Wild type	76.5 ± 0.71	+	+
	G135D	3.12 ± 0.39	+	−
	G154D	0.75 ± 0.08	+	−
	G161S	1.86 ± 0.15	−	+
	G171E	1.12 ± 0.49	Weak	−
	P141A	75.2 ± 0.08	+	+
MinCNg	Wild type	87.82 ± 13.61	−b	−b
	G138D	1.28 ± 0.38	NTc	NT
	G157D	0.14 ± 0.04	NT	NT
	G164S	0.09 ± 0.04	NT	NT
	G174E	0.07 ± 0.03	NT	NT
	E144I	86.25 ± 2.52	NT	NT
MinCCh	Wild type	75.5 ± 3.9	−d	+
	G135D	1.23 ± 0.09	NT	−
	G154D	4.1 ± 1.56	NT	−
	G161S	0.09 ± 0.03	NT	+
	G171E	0.07 ± 0.01	NT	−
	E141I	77.3 ± 5.46	NT	+

^aE. coli cells overexpressing wild-type and mutant MinC proteins were analyzed for the presence of filamentation by phase-contrast microscopy. At least 1,000 cells were counted for each transformant.

^bMinC_Ng did not interact with itself or with MinD_Ng. Therefore, MinC_Ng mutants were not tested for these interactions.

^cNT, not tested.

^dSince self-interaction of wild-type MinCCh was not detected, we did not test self-interactions of the MinCCh mutants.

Flow cytometry, an alternative to microscopic cell counting, permits analysis of larger numbers of bacterial cells (100,000 cells) in a short period of time (3). The phenotypic changes due to MinC overexpression as determined by flow cytometry were compared to the results obtained with phase-contrast microscopy in three independent assays. Positive and negative controls are shown in Fig. 2A and B, respectively. Flow cytometry dot plots showed that 97.44% of the E. coli cells transformed with pSR2 (wild-type MinC_Ng) were filamentous (Fig. 2A) and that a negligible percentage (0.43%) of cells carrying pUC18 were filamentous (Fig. 2B). As observed by phase-contrast microscopy, flow cytometry analysis demonstrated that overexpression of each of the MinC_Ng and MinC_Ec proteins bearing substitutions at conserved C-terminal glycines did not induce filamentation, in contrast to wild-type proteins (Fig. 2C). Mutations at nonconserved residues (MinC_Ng E144I and MinC_Ec P141A) did not significantly affect the abilities of the proteins to induce filamentation in E. coli (Fig. 2C), further supporting the premise that such mutations are functionally neutral.

FIG. 2.

Flow cytometry analysis of E. coli PB103 cells overexpressing MinC_Ec and MinC_Ng mutant proteins. (A and B) Flow cytometry dot plots of E. coli populations transformed with pSR2 (wild-type MinC_Ng) as a positive control (A) and with pUC18 as a negative control (B). The plots of side scatter (SS) versus forward scatter (FS) show two population sizes, the wild-type rods within gate A and long filaments within gate B. The percentages of cells are expressed as percentages of the gated events. (C) Graph showing the percentages of filamentation in a population of 100,000 cells expressing wild-type (WT) MinC_Ec and MinC_Ng proteins, as well as MinC_Ec mutants G135D, G154D, G161S, G171E, and P141A and MinC_Ng mutants G138D, G157D, G164S, G174E, and E144I. An unpaired t test analysis showed that the differences in the percentage of filamentation between wild-type MinC_Ng and MinC_Ng E144I and between wild-type MinC_Ec and MinC_Ec P141A were not significant (P = 0.063 and P = 0.097, respectively). The error bars indicate standard errors.

We previously demonstrated that wild-type MinC_Ng restored wild-type morphology to E. coli DR105 (minC) (23). None of the gonococcal MinC glycine mutants was able to complement E. coli DR105, and these cells retained the characteristic minicell phenotype (data not shown).

Although the abrogated function of MinC glycine mutants is consistent with a critical role for the glycine residues, it was possible that the mutants exhibited altered functions due to low levels of MinC expression. We determined the levels of expression of the wild-type and mutant MinC_Ec and MinC_Ng proteins in E. coli PB103 by Western blotting using antiserum to MinC_Ng, which cross-reacted with MinC_Ec (23). Densitometric analysis of MinC bands showed that wild-type MinC_Ec and MinC_Ec mutants G135D, G154D, and G161S had similar expression levels, which overall were ∼3.5-fold greater (Fig. 3A, lanes 2 to 5) than the expression levels of native MinC_Ec (Fig. 3A, lane 1). MinC_Ec mutants G171E and P141A had 2.5- and 3.0-fold-greater protein levels, respectively (Fig. 3A, lanes 6 and 7) than cells with native MinC_Ec (Fig. 3A, lane 1). Our results demonstrate that between 3.0-fold (MinC_Ec P141A) and 3.5-fold (wild-type MinC_Ec) overexpression of MinC_Ec is enough to cause filamentation in wild-type E. coli PB103 (Table 3). Since the levels of overexpression in MinC_Ec mutants G135D (Fig. 3A, lane 2), G154D (Fig. 3A, lane 3), and G161S (Fig. 3A, lane 4) were comparable to levels observed when the wild-type protein was overexpressed, the lost functionality was not due to altered levels of protein expression. It is still possible that the decreased expression of the MinC_Ec G171E mutant relative to the level of expression of wild-type MinC_Ec resulted in its inability to induce filamentation in E. coli PB103.

FIG. 3.

Expression of MinC mutant proteins in E. coli PB103: Western blots obtained with anti-MinC_Ng. (A) E. coli PB103 was transformed with pUC18 (control) (lane 1), pSR32 (wild-type minC_Ec control) (lane 2), pSR32G135 (minC_Ec encoding the G135D mutation) (lane 3), pSR32G154 (minC_Ec encoding the G154D mutation) (lane 4), pSR32G161 (minC_Ec encoding the G161S mutation) (lane 5), pSR32G171 (minC_Ec encoding the G171E mutation) (lane 6), and pSR32P141 (minC_Ec encoding the P141A mutation) (lane 7). (B) E. coli PB103 was transformed with pUC18 (control) (lane 1), pSR2 (wild-type minC_Ng control) (lane 2), pVG4 (minC_Ng encoding the G138D mutation) (lane 3), pVG6 (minC_Ng encoding the G157D mutation) (lane 4), pVG8 (minC_Ng encoding the G164S mutation) (lane 5), pVG10 (minC_Ng encoding the G174E mutation) (lane 6), and pPF1 (minC_Ng encoding the E144I mutation) (lane 7).

In the case of gonococcal MinC overexpression in E. coli PB103, wild-type and mutant G138D MinC_Ng proteins had similar overexpression levels (Fig. 3B, lanes 2 and 3). The levels of the MinC_Ng mutant G164S, G174E, and E144I proteins were 15, 33, and 14% less, respectively (Fig. 3B, lanes 5 to 7), than the level of overexpressed wild-type MinC_Ng (Fig. 3B, lane 2). Interestingly, the level of the gonococcal G157D MinC mutant protein (Fig. 3A, lane 4) was 30% higher than the level of the wild-type MinC_Ng (Fig. 3A, lane 2), and this mutant exhibited slightly retarded mobility in protein gels. It has been shown previously that overexpression of wild-type MinC_Ng from pSR2 induces filamentation in E. coli PB103 (23). Hence, it was expected that the MinC_Ng G138D mutant, which exhibited overexpression levels similar to those of the wild-type MinC_Ng, would not have altered functionality. It should be noted that the neutral mutant E144I could still cause cell division arrest despite 14% less expression than wild-type MinC_Ng. Thus, since the G164S mutant had a similar expression level, its nonfunctionality cannot be attributed to its expression level. However, it is possible that the MinC_Ng G174E mutant is nonfunctional because it does not reach a critical concentration required to arrest cell division in E. coli PB103. Therefore, with the exception of the MinC_Ec G171E and MinC_Ng G174E mutants, we are certain that the failure to cause filamentation of the MinC glycine mutants was not due to a lack of overexpression at adequate levels.

Specific MinC_Ec glycine mutants have altered interactions with themselves or MinD_Ec.

Interactions of MinC_Ec with itself and with MinD_Ec have been determined previously by using yeast two-hybrid methods (11). This method was therefore used to analyze the self-interactions of the four MinC_Ec glycine mutants, as well as their interactions with MinD_Ec (Table 3). MinC_Ec mutant G161S did not have the ability to self-interact (Fig. 4), in contrast to MinC_Ec mutants G135D, G154D, G171E, and P141A, which were capable of homodimer formation (although the self-interaction of the G171E mutant was significantly decreased compared to that of wild-type MinC_Ec[Fig. 4]). However, unlike MinC_Ec G161S, the MinC_Ec G135D, G154D, and G171E mutants were not able to interact with MinD_Ec, indicating that MinC homodimerization can be preserved while MinC-MinD interactions are selectively disrupted. It should be noted that the strength of the interaction of MinC_Ec G161S with MinD_Ec was significantly less than the strength of the wild-type interaction (Fig. 4). The neutral P141A mutation did not significantly alter the MinC_Ec-MinD_Ec interaction (Fig. 4).

FIG. 4.

Protein-protein interactions of MinC_Ec mutant proteins. The graph shows the interactions of wild-type (WT) and mutant E. coli MinC proteins (G135D, G154D, G161S, G171E, and P141A) with themselves (open bars) and with MinD_Ec (grey bars). An unpaired t test analysis showed that the differences in the strengths of self-interaction between wild-type MinC_Ec and the G135D, G154D, and P141A mutants were not significant (P = 0.128, P = 0.07, and P = 0.153, respectively). However, the difference in the strength of the self-interaction between wild-type MinC_Ec and the G171E mutant was significant (P = 0.005). The difference in the strength of the interaction between wild-type MinC_Ec or MinC_Ec G161S and MinD_Ec was significant (P = 0.005), whereas the interaction between the MinC_Ec P141A mutant and MinD_Ec was not significantly different (P = 0.091). The error bars indicate standard errors.

Creation of a recombinant MinC containing the N terminus of MinC_Ec and the C terminus of MinC_Ng (MinCCh) permitted detection of interactions between MinC_Ng and MinD_Ng.

Since yeast two-hybrid studies of MinC glycine mutants of E. coli distinguished specific glycine residues involved in homodimer formation or interactions with MinD, we proposed that MinC mutants with mutations in conserved glycines from N. gonorrhoeae should have identical interactions. However, interactions of MinC_Ng with itself or with MinD_Ng were not detected by yeast two-hybrid methods, in contrast to MinC_Ec interactions (Fig. 5). The failure to detect protein-protein interactions involving MinC_Ng could have been due either to low levels of expression or to the instability of the proteins in the yeast reporter strain. Western blot analysis of yeast extracts expressing MinC_Ng or MinD_Ng failed to detect these proteins, despite the fact that MinD_Ng showed positive self-association and interaction with MinE_Ng in the yeast two-hybrid system (29). This suggests that the expression levels of Min proteins in yeast extracts may not be high enough to be detected by polyclonal antisera to either MinC_Ng or MinD_Ng. In addition, the manufacturer of the system (Clontech) recognizes that the level of expression from the yeast two-hybrid vectors is inherently low in this particular yeast two-hybrid system.

FIG. 5.

Protein-protein interactions of MinC_Ng mutants. The graph shows the interactions of wild-type MinC_Ng and MinCCh with themselves and with MinD_Ng and the interactions of mutant MinCCh proteins with MinD_Ng. An unpaired t test analysis showed that the differences in the strengths of the interactions of wild-type MinCCh and mutant MinCCh G161S with MinD_Ng were significant (P = 0.000), while the interaction of the MinCCh E141I mutant with MinD_Ng was not significant (P = 0.155). The error bars indicate standard errors.

Since interactions involving MinC_Ec were detectable in this system, we hypothesized that this protein was more stably expressed than MinC_Ng in yeast cells and, furthermore, that the stability might be maintained by constructing a chimeric MinC protein. We therefore created a recombinant MinC protein (MinCCh) comprising the N terminus of MinC_Ec (for stability) and the C terminus of MinC_Ng (to examine the role of conserved glycines in the gonococcal protein). A similar approach was taken in studies with the β-galactosidase of Cellvibrio gilvus, which became stable once its C terminus was fused to the N terminus of the β-galactosidase from T. maritima (7).

It has been demonstrated previously that the N-terminal domain of MinC_Ec by itself does not interact with either MinC_Ec or MinD_Ec (11). Therefore, the MinCCh protein should impart MinC_Ng functionality since the interactions with itself and with MinD should be mediated by the C terminus. Plasmid pSR24, which encodes MinCCh, was constructed to contain the first 99 N-terminal residues of MinC_Ec and the flexible linker and C terminus of MinC_Ng (residues 103 to 237). Chimeric MinC mutants containing substitutions at the conserved glycines (G135, G154, G161, and G171) and at the nonconserved E141 residue were constructed. The residues of the C terminus of MinCCh were renumbered to correspond to the MinC_Ec numbering. As expected, overexpression of the wild-type MinCCh protein and overexpression of the neutral E141I mutant protein were able to induce filamentation in E. coli PB103 (Table 3). By contrast, overexpression of the G135D, G154D, G161S, and G171E chimeric mutants indicated that they were nonfunctional since they could not induce filamentation (Table 3). Western blotting revealed that the levels of expression of chimeric proteins in E. coli were similar to the level of expression of the wild-type MinC_Ng (data not shown).

Despite the inability of wild-type MinCCh to self-interact in yeast two-hybrid assays, this protein interacted with MinD_Ng (Fig. 5 and Table 3), and therefore interactions between MinCCh mutants and MinD_Ng were tested. As observed for the analogous MinC_Ec mutants, MinCCh G135D, G154D, and G171E mutants were not able to interact with MinD_Ng, in contrast to the MinCCh G161S mutant, which was able to interact with MinD_Ng (Fig. 5 and Table 3). As expected, the neutral E141I mutation did not significantly affect the interaction of MinCCh with MinD_Ng (Fig. 5 and Table 3). Western blotting was again ineffective in detecting the MinCCh mutants from yeast extracts with anti-MinC_Ng antiserum. These results show that the same three glycine residues that are important for an E. coli MinC-MinD interaction are implicated in N. gonorrhoeae MinC-MinD interactions as well.

MinC glycine residues residing in the B-C surface junction of the MinC C terminus are involved in the MinC-MinD interaction.

We positioned the conserved glycines in the C terminus of MinC_Tm (G111, G129, G136, and G146) using the crystal structure of this protein (4). Due to the high level of conservation of MinC C-terminal residues, it is likely that the homologous glycines of MinC_Ec (G135, G154, G161, and G171) and MinC_Ng (G138, G157, G164, and G174) are located at similar positions. The G111 and G129 glycines of MinC_Tm are in the first and second turns, respectively, of the β-helix, in the junction between the B and C surfaces (4) (Fig. 1). Using the software program VADAR (33), we found that the overall levels of exposure (fractions of the solvent-accessible surface area) for these two residues are 35 and 44%, respectively. G136 of MinC_Tm is probably not exposed since its overall level of exposure is only 2%. This residue is located in a turn in close proximity to the A surface, which was predicted to be the region of MinC dimerization (4) (Fig. 1). Residue G146 of MinC_Tm is located in the C surface in close proximity to the B-C surface junction, in the third turn of the β-helix (4) (Fig. 1), and it is not surface exposed. In MinC_Tm, the homologue of the nonconserved residues P141 of MinC_Ec and E144 of MinC_Ng is the H116 residue, which is located within the C surface, in close proximity to the A-C surface junction (4) (Fig. 1). Our results show that MinC_Ec G161, which is located in proximity to the A surface, is required for proper protein self-interaction. In addition, analysis of the B factor for this glycine revealed that it is not flexible and is completely buried in the dimer interface. However, the other three conserved glycine residues, which are located within or near the B-C surface junction in the MinC C terminus and which have B factors indicating that they are relatively flexible, are not involved in MinC homodimerization but are essential for the MinC-MinD interaction.

CD reveals that MinC_Ng glycine mutants have an unchanged secondary structure.

Glycine is a small residue that can adopt a wide range of backbone torsion angles and is often found in β-turns (1). Since the completely conserved residues G111, G129, G136, and G146 of MinC_Tm are found within or in close proximity to β-turns (4), the structural properties of these residues may be important for the overall MinC structure. Therefore, the folded state of the MinC_Ng glycine mutants was evaluated by far-UV CD. Analysis of these proteins indicated that the secondary structure of the mutant proteins was essentially identical to that of the wild-type protein (Fig. 6). Although this assay did not demonstrate that the tertiary structure of the proteins is unchanged, significant structural perturbations are unlikely since each glycine substitution in MinC selectively affects its interaction with either MinC or MinD but not its interaction with both proteins.

FIG. 6.

Far-UV CD spectra of wild-type MinC_Ng and its single-glycine mutants. All samples were dissolved in 10 mM ammonium bicarbonate buffer (pH 8.0), and the measurements were obtained at 20°C. [θ], molar ellipticity per mean residue; WT, wild type.

DISCUSSION

We have shown that four completely conserved glycine residues in the C-terminal domain of MinC are important and that they have a key role in the functioning of MinC as a cell division inhibitor. This is true irrespective of the bacterial origin of MinC since both MinC_Ec and MinC_Ng glycine mutants (MinC_Ec G135D, G154D, G161S, and G171E and the corresponding MinC_Ng G138D, G157D, G164S, and G174E proteins) were unable to induce filamentation upon overexpression in E. coli. By contrast, mutations at nonconserved amino acids (MinC_Ng E144I or MinC_Ec P141A) preserved the ability of the protein to inhibit septation. The lack of functionality of the mutant proteins, with the exception of MinC_Ec G171E and MinC_Ng G174E, could not be attributed to a diminution of the protein levels.

To investigate whether the conserved glycine residues of MinC are essential for protein function due to their critical structural locations, each glycine was mapped on the C-terminal surfaces (A, B, and C) of the crystal structure of MinC_Tm. Our analysis revealed that MinC_Ec G135 and G154 and the corresponding residues MinC_Ng G138 and G157 are in the first and second turns, respectively, of the β-helix, in the junction between the B and C surfaces. MinC_Ec G171 and the corresponding residue MinC_Ng G174 are located within the C surface near its junction with the B surface (4) (Fig. 1). Interestingly, our protein-protein interaction assays demonstrated that these glycine residues are involved in the interaction between MinC and MinD but not in MinC homodimerization. Therefore, the B-C surface junction of MinC is probably the region responsible for the interaction of MinC with MinD. By contrast, MinC_Ec G161 and the corresponding residue MinC_Ng G164 are located in a turn in close proximity to the A surface, the region of MinC dimerization (4) (Fig. 1). Yeast two-hybrid assays provided direct experimental evidence for the involvement of the A surface in MinC dimerization since the MinC_Ec G161S mutant was not able to self-interact. In addition, our results demonstrate that MinC dimerization is required for the protein function as a cell division inhibitor because the MinC_Ec G161 or MinC_Ng G164 mutant was also unable to inhibit cell division.

As determined by CD analyses, the mutated MinC proteins did not have gross structural changes in their secondary structures. In addition, the alteration of protein-protein interactions involving MinC was specific to certain glycine residues (i.e., interactions were lost with either MinC or MinD but not with both proteins simultaneously). Therefore, significant structural perturbations of the mutant proteins cannot explain their loss of function as cell division inhibitors. Another study, involving a mutation in rat insulin-like growth factor-binding protein 5, has also shown that replacement of a critical glycine with glutamine causes the protein to lose activity, although the mutation did not result in gross conformational changes in the protein (27).

It is still possible that local structural perturbations that are transparent to CD analysis but nevertheless affect protein-protein interactions are present in the MinC glycine mutants. Such structural changes include altered protein flexibility and/or stability or changes in the electrostatic nature of the mutant MinC proteins. Glycine, which is often present in β-turns, may confer flexibility to a protein backbone due to its single hydrogen atom side chain (1). Although the four conserved glycines of MinC are located within or in close proximity to the C-terminal β-helix turns, the B factors for the solved structure of MinC_Tm indicate that only G111, G129, and G146 (G138, G157, and G174 in MinC_Ng) are relatively flexible. Therefore, mutation of these residues may have resulted in reduced protein flexibility, thereby affecting the movement of the MinC C-terminal domain necessary for mediating interactions with MinD. By contrast, MinC_Tm G136 (G164 in MinC_Ng) is not flexible and remains buried in the homodimer interface. Alternatively, replacement of glycine with either aspartic acid (MinC_Ec G135D and G154D and MinC_Ng G138D and G157D) or glutamic acid (MinC_Ec G171E and MinC_Ng G174E) may have introduced electrostatic repulsion at the protein-protein interfaces.

Two mutant proteins, MinC_Ec G171E and MinC_Ng G174E, had decreased levels of expression compared to the levels of expression of wild-type proteins, suggesting that these proteins had decreased stability. Sen and Rothfield (26) have also shown that a G171S mutation of MinC_Ec led to protein instability. Therefore, low MinC_Ec G171E protein levels could explain why this protein exhibited decreased self-interaction compared to that of wild-type MinC_Ec and did not interact with MinD_Ec. Kong et al. (17) showed that a G145A substitution in the human glutathione transferase P1-1 resulted in loss of enzyme activity due to altered stability of the protein, despite crystallographic analyses which revealed that the structure of the mutant protein was identical to the structure of the wild-type protein.

In order to identify regions of MinD involved in its interaction with the C terminus of MinC, a MinC-MinD association was modeled by using the available structures of MinC_Tm (4) and MinD_Pf (8) (Fig. 7). The α-7 helix of MinD has been implicated previously in the interaction of this protein with MinC (8). We modeled MinD_Pf as a dimer and observed that part of the α-7 helix of each MinD_Pf monomer is exposed, suggesting that this region may be available for interaction with MinC. Our structural analysis showed that three hydrophobic amino acids of the α-7 helix of MinD_Pf (L148, M152, and I156) may remain surface exposed after formation of a MinD_Pf dimer. However, sequence alignments of MinD (8, 29) revealed that only L148 and I156 of MinD_Pf have homologous aliphatic amino acids at the same positions in other species. Most of the amino acids that align with L148 are valines (e.g., in the MinD_Ng and MinD_Ec sequences) or isoleucines. Similarly, the majority of MinD sequences, including the MinD_Ng and MinD_Ec sequences, have a glycine at the position that aligns with I156. We propose that the glycine residues corresponding to MinC_Tm G111 and G129, which are surface exposed and are located in the B-C surface junction of the C terminus, may participate in hydrophobic associations with the surface-exposed residues of the α-7 helix of MinD corresponding to MinD_Pf L148 and I156 (Fig. 7B and C). It has been reported previously that glycine residues can participate directly in protein-protein interactions (2, 6). Lutkenhaus and Sundaramoorthy (21) recently proposed that the interaction between MinC and MinD probably occurs in the cytoplasm and that the MinD-ATP-MinC complex persists because MinC does not stimulate the ATPase activity of MinD.

FIG. 7.

Molecular model of MinC-MinD interaction obtained by using the solved structures of MinC_Tm and MinD_Pf. (A) The MinD monomers in the MinD_Pf dimer are blue and green, while MinC_Tm is white. Yellow indicates α-7 helix residues of MinD_Pf. Residues present at the B-C surface junction of monomeric MinC_Tm are red. (B) Magnification of the putative region of interaction between the exposed glycines (G111 and G129) of MinC_Tm and the exposed residues (L148 and I156) of the α-helix of MinD_Pf. (C) 90° view of panel B, with a barrel showing the A, B, and C surfaces of the β-helix of the C terminus of MinC_Tm.

Several spontaneous MinC_Ec C-terminal mutants, including MinC_Ec G161E, G171D, and G171S, have been isolated and analyzed to determine their morphological effects when they are expressed in E. coli (14, 18, 22, 26). However, these chromosomal mutants were not tested for functionality by overexpression in E. coli or by yeast two-hybrid analyses. The present study is the first systematic investigation of the roles of completely conserved glycine residues in MinC function. We propose that the conserved glycines located within or near the B-C surface junction of the MinC C terminus are implicated in its interaction with MinD. We also provide experimental evidence that a conserved glycine, which is near the MinC C-terminal A surface, is essential for MinC self-interaction. The conserved MinC glycines are probably involved in protein flexibility, stability, and steric hindrance. Ultimately, these residues are responsible for specific topological arrangements of the secondary structure of MinC that are necessary for MinC dimerization and MinC-MinD interactions to occur.