The distinctive cell division interactome of Neisseria gonorrhoeae

Bacterial cell division is an essential process driven by the formation of a Z-ring structure, as a cytoskeletal scaffold at the mid-cell, followed by the recruitment of various proteins which form the divisome. The cell division interactome reflects the complement of different interactions between all divisome proteins. To date, only two cell division interactomes have been characterized, in Escherichia coli and in Streptococcus pneumoniae. The cell divison proteins encoded by Neisseria gonorrhoeae include FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsI, FtsW, and FtsN. The purpose of the present study was to characterize the cell division interactome of N. gonorrhoeae using several different methods to identify protein-protein interactions. We also characterized the specific subdomains of FtsA implicated in interactions with FtsZ, FtsQ, FtsN and FtsW. Using a combination of bacterial two-hybrid (B2H), glutathione S-transferase (GST) pull-down assays, and surface plasmon resonance (SPR), nine interactions were observed among the eight gonococcal cell division proteins tested. ZipA did not interact with any other cell division proteins. Comparisons of the N. gonorrhoeae cell division interactome with the published interactomes from E. coli and S. pneumoniae indicated that FtsA-FtsZ and FtsZ-FtsK interactions were common to all three species. FtsA-FtsW and FtsK-FtsN interactions were only present in N. gonorrhoeae. The 2A and 2B subdomains of FtsANg were involved in interactions with FtsQ, FtsZ, and FtsN, and the 2A subdomain was involved in interaction with FtsW. Results from this research indicate that N. gonorrhoeae has a distinctive cell division interactome as compared with other microorganisms.


Background
Cell division is essential for bacterial survival. In Escherichia coli (Ec), normal cell division is driven by the formation of an FtsZ-ring at the division site [1], followed by the recruitment of other essential proteins, which together form the divisome [2]. Genes encoding most cell division proteins are located in a conserved region, the division and cell wall (dcw) cluster [3]. dcw clusters have been identified in most bacterial species, including E. coli, Bacillus subtilis (Bs), Streptococcus pneumoniae (Sp), Caulobacter crescentus (Cc) and Neisseria gonorrhoeae (Ng) [4][5][6][7]. Although the gene organization of the dcw cluster varies in different bacteria species [8], proteins involved in the cell division process are relatively conserved [9,10].
Using a bacterial two-hybrid (B2H) assay, an E. coli cell division protein-protein interaction network, the cell division interactome, which included 16 interactions between 10 cell divison proteins, was identified [23,24]. The cell division interactome of S. pneumoniae was also characterized using a combination of B2H and coimmunoprecipitation assays [25]. A total of 17 interactions was observed among nine cell division proteins of S. pneumoniae which included FtsZ, FtsA, FtsK, DivlB, DivlC, FtsL, FtsW, and PBP2x [25]. To date, E. coli and S. pneumoniae are the only two organisms with characterized cell division interactomes [23][24][25].
To investigate the cell division interactome in N. gonorrhoeae, its cell division protein interactions were identified using a combination of B2H and glutathione S-transferase (GST) pull-down assays, as well as surface plasmon resonance (SPR). We identified nine interactions among the eight cell division proteins tested. We also identified the subdomains of FtsA Ng involved in its interaction with FtsQ Ng , FtsZ Ng , FtsN Ng , and FtsW Ng . Comparison of the cell division interactomes of E. coli, S. pneumoniae and N. gonorrhoeae indicates that N. gonorrhoeae possesses a distinctive cell division interactome.

DNA manipulations
N. gonorrhoeae CH811 genomic DNA was purified using a QIAamp® genomic DNA kit (Qiagen, Mississauga, Ontario, Canada). DNA samples were stored at −20°C. Oligonucleotides for polymerase chain reaction (PCR) amplifications were synthesized by Invitrogen (Table 2
The expression of ftsA Ng , ftsZ Ng and zipA Ng from B2H constructs was verified by Western blot analysis using appropriate antibodies prepared in our lab using previously described methods [33]. These proteins were expressed from the vectors under the conditions tested (data not shown). The expression of these proteins indicated that any negative B2H interactions involving them was not a function of lack of expression.
B2H assays were performed as described previously [24]. This assay is based on the reconstitution of a chimeric repressor that binds to the 434/P22 hybrid operator and represses the expression of a downstream lacZ gene in E. coli R721. Each gene tested for a potential interaction was cloned into pcI p22 and pcI 434 and recombinant constructs were transformed into E. coli R721 either singly or in combination. N.
gonorrhoeae FtsZ self-interaction was used as positive control. R721 without plasmids and single plasmid transformants were used as negative controls. R721 without plasmids had a β-galactosidase activity of 2504 ± 34 Miller units. The β-galactosidase activity of each combination was compared to that of R721. Values of less than 50% (<1250 Miller Units) indicate a positive interaction between two proteins, while values of more than 50% (>1250 Miller Units) indicate a negative interaction [24]. Statistical analyses were performed using the unpaired Student t-test. Standard deviations were determined for the mean value of Miller units where three independent experiments were performed.
Purified GST-fusion and His-fusion proteins were incubated with pre-equilibrated GST•Bind™ Resin in phosphate buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.5% Triton-X100, 1 mM DTT, pH 7.9) at 4°C overnight. Pre-purified GST was used as a negative control. The pre-bound resin was collected by centrifugation and washed in PBS three times. Bound proteins were dissociated from resin by adding 5X Laemmli buffer, separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), and identified by Western blot using polyclonal anti-GST or anti-6 × His antibodies (Thermo Scientific; Waltham, MA), sequentially.
All GST pull-down assays were performed minimally in duplicate.

FtsZ polymerization assays
FtsZ Ng polymerization was measured by 90°angle light scattering using a Dynapro-MS800 instrument (Wyatt Technology Corporation) with a wavelength of 310 nm and a slit width of 0.5 mm. MES buffer is optimal for FtsZ polymerization which is required to observe an FtsA-FtsZ interaction [34,35]. FtsZ Ng (~6 μM) in MES buffer (50 mM MES-NaOH, 50 mM KCl, 10 mM MgCl 2 , pH 7.5) was injected into a 45 ul quartz cuvette and warmed to 30°C, prior to the measurement. Data were collected, for 4 min, from unpolymerized FtsZ Ng to establish a baseline. GTP was then added to a final concentration of 2 mM and data were collected every 5 s for 25 min. Data were recorded and analyzed using Dynamics v5 software.
Negative stain electron microscopy was used to visualize FtsZ Ng polymers. 5 μl of FtsZ (6 μM) with, or without, GTP (final concentration 2 mM) was incubated, at 30°C, for 5 min. The mixture was placed on a carbon-coated copper grid (400 mesh size) for 2 min and then blot dried. The grid containing FtsZ Ng was stained with 1% uranyl acetate, blotted, and air-dried for 3 h. Polymers were visualized and photographed using a Hitachi transmission electron microscopy HT7700.

Surface plasmon resonance (SPR)
Protein interactions were examined by SPR using a Bio-Rad XPR36 (Bio-Rad Laboratories) instrument and a ProteOn™ HTE Sensor Chip (Bio-Rad Laboratories). The chip surface was regenerated by injection of 0.5% SDS, 50 mM NaOH, 100 mM HCl and 300 mM EDTA, at a flow rate of 30 μl/min, for 120 s. Activation was performed using 500 μM of NiSO 4 .
For FtsA Ng -FtsN Ng and FtsA Ng -FtsQ Ng SPR experiments, ligands (i.e. His-FtsN Ng for FtsA Ng -FtsN Ng , and His-FtsQ Ng for FtsA Ng -FtsQ Ng interactions) were immobilized onto the sensor chip at a concentration of 200 nM. A two-fold dilution series of the analyte (FtsA Ng ), in PBS buffer with Tween-20 (PBST; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.1% BSA, 0.05% Tween-20, pH 7.9), was injected at a flow rate of 30 μl/ min over the surface of the chip for 120 s. This was followed by an injection of PBST buffer for 300 s. Negative controls comprised a reference channel flowed with PBST buffer, and a chip surface immobilized with either FtsQ Ng or FtsN Ng flowed with GST in PBST.
For the FtsA Ng -FtsZ Ng interaction, the SPR binding assay was performed using MES buffer, supplemented with 0.1% BSA, 0.05% Tween-20, and 1 mM ATP were added with the pH adjusted to 7.5. FtsA Ng was immobilized on the chip surface as described above. Each 120-s injection of polymerized FtsZ Ng was followed by an injection of supplemented MES buffer for 300 s for dissociation. Negative controls included a reference channel which was flowed with MES buffer containing 2 mM GTP, and the FtsA Ngimmobilized chip surface flowed with GST in supplemented MES instead of polymerized FtsZ Ng .
All SPR data was analyzed using ProteOn Manager™ (Bio-Rad Laboratories). The sensorgram (i.e. a graph of the response unit versus time) was first subtracted by the response units (RU) of the reference channel, with no immobilized ligands, to reduce the non-specific binding signals between analyte and empty chip surface. Then, the sensorgram was subtracted with the RU signal with running buffer and ligand immobilized on the chip. Association and disassociation constants were obtained using the Langmuir 1:1 kinetic fit model, by nonlinear regression, using ProteOn Manager™. Each protein pair was tested minimally in duplicate.

GST pull-down of FtsA Ng -FtsQ Ng , FtsA Ng -FtsZ Ng and FtsA Ng -FtsN Ng interactions
To confirm the results of selected B2H assays, we examined several interactions (i.e. FtsQ Ng -FtsA Ng , FtsA Ng -FtsN Ng , FtsA Ng -FtsZ Ng ) using GST pull-down assays. GST pull-down results (Fig. 1a) showed that His-FtsQ Ng was pulled down by GST-FtsA Ng , but not GST itself (negative control), indicating an interaction between FtsA Ng and FtsQ Ng . Using similar evaluation criteria, we ascertained that His-FtsA Ng was pulled down by GST-FtsN Ng , indicating an interaction between these two proteins (Fig. 1b).
The interactions of FtsA Ng and FtsZ Ng from E. coli in vitro requires the presence of both ATP and GTP [35]. GTP promotes FtsZ polymerization, and ATP is necessary for FtsA to interact with FtsZ, but not for FtsZ polymerization [36,37]. The presence of FtsZ Ng polymers in MES buffer was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS; Additional file 2: Figure S2). GST pull-down assay did not detect an interaction between FtsA Ng and FtsZ Ng in the presence of 1 mM ATP and 2 mM GTP (Fig. 1c). This result was unexpected, given our B2H results and the commonality of FtsA-FtsZ interaction in other bacterial species [24,25,38,39], as ascertained by different in vivo assays (i.e. B2H, yeast two-hybrid, chemical cross-linking with co-immunoprecipitation).

Surface plasmon resonance evaluation of FtsA Ng -FtsQ Ng , FtsA Ng -FtsZ Ng and FtsA Ng -FtsN Ng interactions
Surface plasmon resonance (SPR) was used to confirm selected gonococcal cell division protein-protein interactions in real-time. SPR was used to evaluate the interactions of FtsA Ng with FtsZ Ng because of the conflicting results observed with B2H and GST pull-down assays. GTP was added to promote FtsZ Ng polymerization (Additional file 2: Figure S2). The sensorgram indicated that FtsZ Ng interacted with FtsA Ng at concentrations of 6 μM and 12 μM (Fig. 2a), but not at concentrations lower than 6 μM (data not shown). Kinetic analysis showed that the FtsA Ng -FtsZ Ng interaction had a slow association (ka = 3.56 × 10 2 M −1 s −1 ) and a significant Table 4 Interactions between eight cell division proteins in N. gonorrhoeae as determined by B2H assay By comparison to positive controls (E. coli R721 without plasmids), interactions with less than 50% of residual ß-galactosidase activity (framed) were considered as positive. FtsZ Ng self-interaction was used as a positive control. The numbers represent percentage of mean ß-galactosidase activity, ± standard deviation * Statistically significant (P ≤ 0.05); NS: not statistically significant (P > 0.05) disassociation activity (kd = 5.31 × 10 −3 s −1 ), giving a KD value of 14.9 μM. This suggested that the interaction between FtsA Ng and FtsZ Ng was likely transient. When GTP was absent from the FtsZ Ng protein solution, no binding was detected between FtsA Ng and FtsZ Ng (data not shown). The sensorgram of the interaction between FtsA Ng and the negative control (GST) also showed no binding activity (Fig. 2b), indicating the specificity of the SPR results for the interaction of FtsA Ng with FtsZ Ng .
For the SPR analysis of the FtsA Ng -FtsQ Ng interaction, FtsA Ng was tested using various concentrations (from 31.25 nM to 250 nM; Fig. 2c). At 0 s, the association of FtsA Ng and FtsQ Ng was observed immediately following injection of the FtsA Ng solution onto the FtsQ Ng -labeled chip surface, with a rapid increase of response units (ka = 2.72 × 10 5 M −1 s −1 ; Fig. 2c). This indicated a fast binding event between the two proteins. Disassociation between FtsA Ng and FtsQ Ng was not significant (kd = 4.09 × 10 − 3 s −1 ), suggesting this interaction was strong and stable (KD = 15.1 nM). The negative control, using non-interacting GST, did not cause any change in the response units (Fig. 2d).
The FtsA Ng -FtsN Ng interaction was observed with an increasing concentration of FtsA Ng (62.5 nM, 125 nM, 250 nM and 500 nM; Fig. 2e). His-FtsN Ng had a binding affinity (KD) of 53.3 nM with FtsA Ng . The association and disassociation constants were 1.15 × 10 5 M −1 s −1 , and 6.16 × 10 −3 s −1 , respectively (Fig. 2e), indicating a strong interaction between FtsA Ng and FtsN Ng . The injection of non-interacting GST onto the FtsN Ng immobilized chip surface did not cause any change in the response units (Fig. 2f ).

The 2A and 2B subdomains of FtsA Ng interacts with FtsZ Ng , FtsN Ng , FtsW Ng and FtsQ Ng
Since FtsA Ng interacted with FtsZ Ng , FtsQ Ng , FtsW Ng , and FtsN Ng , we further examined the interaction regions of FtsA Ng with these four proteins using B2H assays. Based on FtsA Ng homology modeling, six FtsA Ng truncations (T1-T6) were created (Additional file 1: Figure S1), which contained one or more FtsA Ng subdomains [33]. FtsZ Ng self-interaction was used as a positive control. And negative controls included E. coli R721 without plasmids or carrying each single recombinant B2H vector in which the gene of interest had been cloned. FtsA Ng truncations T3, T4, and T5 interacted with FtsZ Ng and FtsN Ng (Figs. 3 and 4, blue bars). FtsA Ng truncations T1, T2, and T6 did not show an interaction with these proteins (Figs. 3 and 4, green bars). The T4 and T5 truncations included the 2B and 2A 2 subdomains of FtsA Ng , suggesting that these subdomains of FtsA Ng interacted with both FtsZ Ng and FtsN Ng . The T3 construct contained also contained the 2A 1 subdomain of FtsA Ng , as compared to truncations T1 and T2, indicating that this subdomain was also involved in interactions with FtsZ Ng and FtsN Ng . FtsQ Ng interacted only with the T4 and T5 truncations of FtsA Ng (Fig. 5, blue bars), indicating that the 2B and 2A 2 subdomains, but not the 2A 1 subdomain, were required for the FtsA Ng -FtsQ Ng interaction. Only the T5 truncation of FtsA Ng interacted with FtsW Ng , suggesting that 2A 2 subdomain was involved in the interaction with FtsW Ng (Additional file 3: Figure S3). In summary, these results showed that the 2A 1 , 2A 2 and 2B subdomains of FtsA Ng are required for its interaction with FtsN Ng and FtsZ Ng . The FtsA Ng 2A 2 and 2B subdomains are required for interaction with FtsQ Ng , and the 2A 2 subdomain is involved in the interaction with FtsW Ng .

Discussion
The N. gonorrhoeae cell division interactome described in our study is the third cell division interaction network identified in bacteria, in addition to E. coli and S. pneumoniae (Fig. 6a) [23][24][25]. Compared to the other two interactomes (Fig. 6b and c), fewer interaction protein pairs are identified in N. gonorrhoeae (Fig. 6a). Only nine interactions are present among the eight divisome  [23,24], and c S. pneumoniae [25]. Red lines indicate common interactions; blue lines indicate unique interactions in N. gonorrhoeae proteins tested in N. gonorrhoeae, while E. coli and S. pneumoniae have 21 and 17 interactions among ten and eight divisome proteins, respectively [24,25].
The development of all three cell division interactomes was based on interaction data obtained from the same B2H system [24,25] The E. coli interactome was developed using B2H results exclusively while the S. pneumoniae study also applied co-immunoprecipitation to verify selected B2H positive interaction pairs [24,25]. In our study, we used a combination of GST pull-down and surface plasmon resonance to further study selected positive B2H interactions.
Two interactions, FtsA-FtsZ and FtsZ-FtsK, are conserved in the cell division interactomes of N. gonorrhoeae, E. coli and S. pneumoniae (Fig. 6, red lines). The FtsA-FtsZ interaction is a common interaction in prokaryotes [24,25,[39][40][41]. Both our B2H and SPR results confirmed this interaction in N. gonorrhoeae. A proper ratio between FtsA and FtsZ is crucial for the interaction in E. coli [42] and our SPR results support this finding; FtsA Ng interacts with FtsZ Ng only when its concentration is higher than 6 μM (Fig. 3b), indicating that the interaction requires a critical concentration threshold. Our SPR results further showed that interaction between FtsA Ng and FtsZ Ng was transient, a result warranting further study to fully understand its implications for divisome formation in N. gonorrhoeae. Unexpectedly, the GST pull-down assay, an in vitro assay, did not detect an FtsA Ng -FtsZ Ng interaction. We believe that this "false negative" in vitro result was caused by the requirement of a membrane/solid surface support for the interaction to anchor FtsA [35,43,44].
The interaction of FtsZ with FtsK has been observed in N. gonorrhoeae, E. coli, S. pneumoniae, B. subtilis and C. crescentus [24,25,45,46]. The C-terminus of FtsK is required for proper DNA segregation in E. coli [47]. The absence of an FtsZ-FtsK interaction in both E. coli and C. crescentus caused abnormal chromosome segregation and cell filamentation [45,48]. This suggests that the FtsZ-FtsK interaction connects the cell division process with chromosome segregation, by ensuring that the replicated chromosome is cleared from the division site.
The FtsA-FtsW interaction has been observed only in N. gonorrhoeae (Fig. 6, blue lines). Since FtsW is a membrane protein and difficult to purify, we did not verify the interaction by GST pull-down and SPR assays. However, we performed additional B2H assays to identify which subdomains of FtsA were involved in its interaction with FtsW (Additional file 3: Figure S3) and showed that the 2A 2 subdomain of FtsA strongly interacts with FtsW (Additional file 3: Figure S3). FtsW, an inner membrane protein, is required in E. coli for the recruitment of FtsI and the translocation of the cell well precursor, lipid II [20,21,49,50]. An FtsI-FtsW protein interaction has been observed in E. coli, Streptomyces coelicolor, and Mycobacterium tuberculosis [21,51,52]. Interestingly, we discovered that FtsI Ng only interacts with FtsW Ng , suggesting that its localization may depend on this protein.
The importance of the unique FtsK Ng -FtsN Ng interaction in N. gonorrhoeae, as determined by B2H, is not clear (Fig. 6, blue lines). In E. coli, FtsN is the last protein, of ten essential cell division proteins, recruited to the division site to initiate cell constriction [53,54]. A previous study suggested that E. coli FtsN and FtsK stabilize the Z-ring cooperatively, without direct interactions [55]. Since the FtsK-FtsN interaction is present in N. gonorrhoeae, their joint involvement in gonococcal cell division requires further investigation.
ZipA Ng did not interact with any other gonococcal cell division protein. In E. coli, ZipA only interacts with FtsZ, and is required for downstream protein recruitment, including FtsK, FtsQ, FtsL, and FtsN [24,56]. One report suggested that ZipA Ng is a homologue of the E. coli protein with high similarity in its key domains [57]. Although ZipA Ng complemented a conditional zipA mutant in E. coli, it did not fully restore a wild type phenotype in this strain [57]. Given these data, the role of ZipA in gonococcal cell division remains to be elucidated.
In N. gonorrhoeae, the existence of FtsL Ng is unclear due to its low homology with E. coli FtsL [58]. An open reading frame (ORF) located between mraW and ftsI in the dcw cluster of N. gonorrhoeae was reported by Francis et al. [7] and they reported that it was not a coding ORF. Snyder et al. [58] named the same ORF ftsL. Because this ORF shares only 17% amino acid similarity to its E. coli homologue, we considered that it was not a functional ORF and did not test its interaction with other gonococcal cell division proteins.
N. gonorrhoeae lacks FtsB [7]; thus, the protein complex FtsQ-B-L, present in other species, such as E. coli, S. pneumoniae and B. subtilis, would not be formed in N. gonorrhoeae [59][60][61]. This protein complex has been described as a bridge connecting FtsK and the FtsI-FtsW complex in E. coli [18]. A recent study suggests that the E. coli FtsQ-B-L complex acts as a signal transmitter for cell wall remodeling and constriction, which is mediated by direct interactions with the FtsI-W complex and FtsN [19]. In S. pneumoniae, the FtsQ homologue, DivIB, interacts with FtsK Sp , FtsL Sp, and FtsW Sp [25]. Interestingly, our B2H data show that FtsQ Ng only interacts with FtsA Ng , suggesting that the function of FtsQ Ng in cell division in N. gonorrhoeae may be distinct.
There are several models for bacterial cell constriction. One E. coli model suggests that the force that drives constriction comes from septal peptidoglycan synthesis [62]. In this model, the FtsA Ec -FtsN Ec interaction activates peptidoglycan synthesis by direct or indirect interaction with FtsI Ec [63]. Another E. coli model suggests that the energy generated from FtsZ-mediated GTP hydrolysis drives cell constriction [43]. We observed an FtsA Ng -FtsN Ng interaction in N. gonorrhoeae. However, there is no further evidence supporting either model of cell constriction in N. gonorrhoeae at this time.
The non-essential proteins, FtsE Ng and FtsX Ng , are also implicated in cell division in N. gonorrhoeae [64]. Similarly, in E. coli, FtsE and FtsX are non-essential for cell division under conditions of high osmotic pressure [65]. Gonococcal FtsE and FtsX have high similarity in amino acid sequence to known homologues in other species [64]. In E. coli, the interaction between FtsE and FtsZ has a regulatory effect on the Z-ring [65]. Future research could focus on revealing the effects of FtsE Ng and FtsX Ng on cell division in N. gonorrhoeae.
The major issue interpreting B2H assay results is the empirical cut-off of 50% residual ß-galactosidase activity used to discriminate positive and negative interactions. In particular, values close to the cut-off could be interpreted as either false positive or negative results. To validate our B2H results, we used other B2H interactions to test which subdomains of FtsA Ng interacted with FtsZ Ng , FtsN Ng , and FtsQ Ng . We determined that the 2A and 2B subdomains of FtsA Ng interacted with FtsZ Ng , FtsQ Ng , and FtsN Ng . We also evaluated some positive interactions obtained by B2H using SPR and GST pulldown assays. The SPR method detects and measures weak or transient interactions, in real-time, with high sensitivity [66]. The SPR method showed a transient FtsA Ng -FtsZ Ng interaction. GST pull-down assays, on the other hand, are ideal in detecting strong proteinprotein interactions, as weak interactions may dissociate during the assay [67]. We consider this to be a reasonable explanation for our failure to confirm when the interaction of FtsA Ng with FtsZ Ng when using a GST pull-down assay.
To date, most of studies on cell division have been focused on model organisms (i.e. the Gram-negative rod E. coli and the Gram-positive rod B. subtilis) due to the abundant availability of tools for genetic manipulation [62]. Research on cell division in nonmodel organisms is expanding, and this includes studies with N. gonorrhoeae [7,27]. For example, Chlamydia trachomatis, which lacks FtsZ, requires an actin-like protein, MreB, for cell division [68]. A gene cluster encoding three cell division proteins, named MldA, MldB, and MldC, was identified only in Clostridium difficile and its closely related bacteria [69]. Results from studies using non-model organisms suggest that cell division mechanisms are complex and vary in different organisms, reflecting vast biological diversity.

Conclusions
In our research, we discovered that nine interactions among eight cell division proteins defined the cell division interactome of N. gonorrhoeae. In comparison with the published cell division interactomes of E. coli and S. pneumoniae, FtsA-FtsZ and FtsZ-FtsK interactions were common to all three bacteria. FtsK-FtsN and FtsA-FtsW interactions were only present in N. gonorrhoeae, suggesting that they play different roles in the cell division of this microorganism. ZipA Ng did not interact with any other cell division proteins tested in this study, indicating that its role may differ as compared to its E. coli homologue. We also determined that the subdomains of FtsA Ng which interacted with FtsQ Ng , FtsZ Ng , FtsW Ng , or FtsN Ng , differed from its E. coli homologue. This suggests that N. gonorrhoeae possesses a distinctive cell division interactome, and likely a different mechanism of cell division as compared to E. coli and other organisms.