Protein interactions within and between two F‐type type IV secretion systems

Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F‐type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH‐TM bacterial two‐hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F‐plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F‐type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F‐type T4SS‐specific proteins. Furthermore, we demonstrate, for the first‐time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F‐type‐specific proteins and we confirmed two of them by co‐purification. The F‐type‐specific protein TraHN was found to localize to the outer membrane and the presence of significant amounts of TraHN in the outer membrane requires TraGN.

encodes protein homologs to most of the Vir core proteins (VirB2-VirB10 and VirD4) found in P-type T4SSs, but the F-plasmid additionally encodes several proteins that are conserved only in F-type T4SSs (Lawley et al., 2003). F-type T4SSs have been found on many conjugative plasmids and in genetic islands on the bacterial chromosome (Lawley et al., 2003). The genes encoding the N. gonorrhoeae T4SS proteins are located on a 59-kb genetic island (the Gonococcal Genetic Island, GGI) (Callaghan et al., 2017). 21 genes organized in 4 operons are important for secretion of ssDNA by the N. gonorrhoeae T4SS (Pachulec et al., 2014). The structural T4SS proteins encoded by 17 genes can be divided into three groups: (1) Proteins showing homology to proteins found in most type IV secretion systems, (2) proteins showing homology to proteins conserved only in F-type T4SSs, and (3) proteins specific to the N. gonorrhoeae T4SS or only found in GGI-like T4SSs (Hamilton et al., 2005;Pachulec et al., 2014) (For an overview, see Table 1 and Figure 1).
The energy-providing ATPases TraC (a VirB4 homolog) is found in F-type T4SSs while F-type T4SSs are missing a homolog to the ATPase VirB11 found in P-type T4SSs (Alvarez-Martines and Christie, 2009). N. gonorrhoeae encodes a TraC homolog required for DNA secretion (Hamilton et al., 2005;Pachulec et al., 2014).
Structural studies of the outer membrane core complex (OMCC) of two conjugative P-type T4SSs have been published (Chandran et al., 2009;Fronzes et al., 2009;Low et al., 2014). The OMCC consists of three proteins that can form a double membrane-spanning complex. The hub protein VirB10 inserts into both the outer and the inner membrane, spans the periplasm and has a short N-terminal end in the cytoplasm (Chandran Darbari and Waksman, 2015). The two other proteins in the OMCC, VirB7 and VirB9 are associated with the outer membrane (Chandran et al., 2009;Low et al., 2014). Structural (Hu et al., 2019) and two-hybrid (Harris et al., 2001) data suggest that the F-plasmid T4SS has a similar OMCC consisting of the VirB10 homolog TraB F , the VirB7 homolog TraV F , and the VirB9 homolog TraK F . TraL F , TraE F , and TraG F from the F-plasmid are proteins associated with the inner membrane with some homology to proteins from the P-type T4SSs (Lawley et al., 2003) (Table 1 and Figure 1b). The N. gonorrhoeae GGI encodes homologs of TraB F , TraV F , TraK F , as well as TraG F , TraL F , and TraE F homologs (Table 1, Figure 1a). Mutational analyses have shown that all of these proteins are important for DNA secretion by the N. gonorrhoeae T4SS (Hamilton et al., 2001;2005;Pachulec et al., 2014).
In addition to the proteins found in most other type IV secretion systems, F-type T4SSs have a group of periplasmic or peripheral membrane proteins (TraW F , TraU F , TraH F , TraF F , TrbC F , and TraN F ) that has been linked to assembly and extension of the conjugation pilus (Arutyunov and Frost, 2013). Although N. gonorrhoeae does not have pilus-dependent DNA secretion, the TraW N , TraU N , TraH N , TraF N , TrbC N , and TraN N homologs encoded by the N. gonorrhoeae GGI are essential for DNA secretion (Hamilton et al., 2001;2005;Pachulec et al., 2014).
F-type T4SSs generally encode periplasmic thiol-oxidoreductases that promote disulfide bond formation in the periplasm (Hemmis and Schildbach, 2013;Pachulec et al., 2014). Some plasmids with F-type T4SSs such as the F-plasmid encode the protein TrbB which has a redox-active site and a TraF protein without a redox-active site, while others such as the N. gonorrhoeae GGI and the plasmid R27 encode a periplasmic DsbC (disulfide bond) homolog often in combination with a TraF-like protein, both proteins having redox-active sites (Elton et al., 2005;Hemmis and Schildbach, 2013). DsbC N and TraF N are both essential for DNA secretion by the N. gonorrhoeae T4SS (Hamilton et al., 2005).
Lytic transglycosylases capable of peptidoglycan degradation are believed to play a role in the assembly of transport complexes in the cell envelope (Koraimann, 2003). Three of the proteins that are important for DNA secretion by N. gonorrhoeae AtlA N , Yag N , and LtgX N are thought to be associated with the peptidoglycan layer (Dillard and Seifert, 2001;Kohler et al., 2007;Pachulec et al., 2014). AtlA N and LtgX N are both lytic transglycosylases (Kohler et al., 2007).
LtgX N shows homology to Orf169, a lytic transglycosylase from the F-plasmid T4SS, while AtlA N is specific to the N. gonorrhoeae T4SS (Kohler et al., 2007).
The T4SS from N. gonorrhoeae shows some amino acid sequence similarity to the T4SS from the F-plasmid but the sequence identity is generally low, typically around 25% (see Table 2) (Hamilton et al., 2005;Ramsey et al., 2014), and while other F-type T4SSs are involved in contact-dependent DNA secretion, the T4SS from N. gonorrhoeae carries out contact-independent DNA secretion. Comparing the two systems could potentially be used to define generic features of TA B L E 1 Predicted localization of 17 T4SS proteins essential for DNA secretion by N. gonorrhoeae and the corresponding homologous proteins from the F-plasmid T4SS and P-type T4SSs was found in the outer membrane and total membrane fractions only ( Figure 2a). This localization pattern matched that of known outer membrane protein LtgA and was distinct from that of known inner membrane protein SecY and known soluble protein CAT ( Figure 2a).
The traHN, traGN, and atlAN genes are in an operon separate from the one encoding most of the structural proteins of the gonococcal T4SS (Pachulec et al., 2014). The transcript is found at significantly higher levels than that encoding the other T4SS structural proteins, and the translation of TraHN and TraGN, and possibly also AtlAN, is controlled by an RNA switch (Ramsey et al., 2015). The coregulation of these proteins suggested that they might work together for assembly of part of the T4SS. We hypothesized that perhaps TraHN requires the lytic transglycosylase AtlAN to make an opening in the cell wall for TraHN to pass through and that perhaps AtlAN might need TraGN in the inner membrane to access the periplasm.
We used an atlA N deletion mutant to test the necessity of atlA for TraH localization. Outer membrane preparations were examined for TraHN-FLAG3 by western blot. Contrary to our hypothesis, the deletion of atlAN did not significantly reduce TraHN-FLAG3 in the outer membrane ( Figure 2b).  Das et al. (1997) and Das and Xie (2000). OM, outer membrane, IM, inner membrane, PG, peptidoglycan. Proteins shared between the three systems are shown in red/violet colors while F-type-specific proteins are shown in green. Proteins specific to N. gonorrhoeae are shown in yellow while VirB11 found only in the P-type system is shown in blue TA B L E 2 Bioinformatics and literature data used to predict the localization of the N. gonorrhoeae T4SS proteins Proteins with homology to proteins from F-like T4SSs (Hamilton et al., 2005) TraW Amino acid identity between the protein from N. gonorrhoeae, and the corresponding protein from the F-plasmid. The amino acid identity is only calculated for the F-plasmid proteins used in this study.
of traKN had no effect ( Figure 2c). Thus TraGN, independent of AtlAN and TraKN, is needed for TraHN to be present at significant levels in the gonococcal outer membrane.
To obtain information about the localization of other N. gonorrhoeae T4SS proteins, we performed bioinformatic analyses as described in experimental procedures and compiled data from the literature. The outcome is summarized in Table 2.

| The BACTH and BACTH-TM systems
In the bacterial adenylate cyclase two-hybrid system (BACTH) (Karimova et al., 1998), the proteins of interest fused with the two fragments (T18 and T25) from the catalytic domain of Bordetella pertussis adenylate cyclase and interaction between the proteins result in functional complementation between T18 and T25 leading to cAMP synthesis and transcriptional activation of the lactose operon. The BACTH-TM system (Ouellette et al., 2014) inserts a transmembrane helix between the proteins of interest and the T18 and T25 fragments of the adenylate cyclase. While the BACTH system requires the proteins of interest to be located in the cytoplasm or the inner membrane (Karimova et al., 1998;2005), the BACTH-TM system enables the study of protein interactions in the periplasm (Ouellette et al., 2014). A combination of the BACTH and the BACTH-TM systems can be used to study interactions between inner membrane proteins with a cytoplasmic domain and a periplasmic protein.

| Interactions: N. gonorrhoeae genes cloned into the BACTH and the BACTH-TM system vectors
All proteins were fused with both T18 and T25 fragments. The BACTH vectors pUT18C and pKT25 put the T18 and the T25 fragments in the N-terminal end of the protein while pUT18 (Karimova et al., 2001) and p25N (Claessen et al., 2008)  According to, respectively, previous work (Ramsey et al., 2014) and our bioinformatics analysis (  (Kohler et al., 2013) and bioinformatics analyses indicate that the C-terminal ends of the proteins are likely to be in the cytoplasm while the N-terminal ends are likely to be in the periplasm ( Table 2). The genes encoding TraG N and TraL N were therefore cloned in pUT18 and p25N.
Because the BACTH-TM system (Ouellette et al., 2014) (Ramsey et al., 2014) were removed before cloning. For AtlA N and TraC N , we chose to put the T18 and T25 fragments at both ends of the protein, thus these proteins were cloned in all four BACTH vectors pUT18C, pUT18, pKT25, or p25N (Table 3). Note: +, -, and w indicate, respectively, interaction, no interaction, and weak interaction. Space indicates that the interaction was not tested. The placement of T18 and T25 relative to the protein name indicates N or C-terminal fusion. T18 or T25 indicate that the gene encoding the protein was cloned into the BACTH vectors. TM18 or TM25 indicates that the gene encoding the protein was cloned into the BACTH-TM vectors.

| Combinations of N. gonorrhoeae proteins tested
TraB N , TraG N , TraE N , and TraL N are believed to be transmembrane proteins with potential interaction partners in the cytoplasm, the inner membrane, the periplasm and in the case of TraB N also the outer membrane. Due to the possible interactions of these proteins with proteins in several cellular compartments, we tested all possible combinations of these proteins cloned in the BACTH system with all other proteins cloned in the BACTH or the BACTH-TM system by co-transformation into E. coli BTH10. Functional complementation was assayed as described in the method section (for an overview of the tested combinations of plasmids, see Table 3).
For the proteins cloned only in the BACTH-TM system (LtgX N ,   (Table 3).
As controls, we tested for interactions between the periplasmic proteins cloned in both the BACTH and the BACTH-TM systems (Table 3). Cloning the periplasmic proteins in the BACTH and the BACTH-TM vectors should result in protein expression in different cellular compartments, therefore no interactions should be observed between periplasmic proteins cloned in the two different vector systems. As expected, we did not observe any interactions between a periplasmic protein cloned in a BACTH vector and a periplasmic protein cloned in a BACTH-TM vector (Table 3).
For TraC N , we tested for both a TraC N /TraC N interaction and interactions with transmembrane proteins (Table 3).

| The detected interactions
To examine if the interactions observed for the N. gonorrhoeae T4SS proteins were specific for the N. gonorrhoeae T4SS proteins or could potentially be general for F-type T4SS proteins, we analyzed interactions among the corresponding F-plasmid proteins using the protein-adenylate fusions summarized in Table 4.
The observed interactions could be classified into three groups.  interactions, TrbC F is involved in 7 interactions, and TraK F is involved in 5 interactions (Figure 3). Some proteins with intrinsic tendency to interact with any protein, the so-called "sticky proteins," can give rise to false positives in two-hybrid screens (Battesti and Bouveret, 2012); however, all the proteins tested in this study showed selectivity with regard to interaction partners. Whether these interactions would be formed if several, possibly competing interaction partners were present at the same time is, however, a question that cannot be addressed by two-hybrid studies.
In some cases, for instance, for the TraB N /TraE N interaction, an interaction was only observed with one of the two possible combinations of T18 and T25. This has been observed in previous two-hybrid analyses, one possible reason being the different copy numbers of the T18 and T25 plasmids (Battesti and Bouveret, 2012

| Confirmation of selected interactions by copurification
Since two-hybrid systems can give both false-negative and falsepositive results, we aimed to further validate our findings with other methods. For many of the interactions observed in this study, some evidence for the interaction between the studied proteins or homologs from other T4SSs can be found in the literature (Das et al., 1997;Das and Xie, 2000;Gilmour et al., 2001;Harris et al., 2001;Ding et al., 2002;Harris and Silverman, 2004;Chandran et al., 2009;Fronzes et al., 2009;Sivanesan et al., 2010;Low et al., 2014;Ramsey et al., 2014;Casu et al., 2016;Oliveira et al., 2016;Shala-Lawrence et al., 2018;Hu et al., 2019). However, only limited evidence exists for interactions between OMCC proteins homologs (TraB, TraV, and TraK) and F-type-specific periplasmic proteins (Arutyunov et al., 2010), and the function of the F-type-specific periplasmic proteins is poorly understood. Therefore, we chose to concentrate on the confirmation of interactions between the OMCC protein homolog TraV and the periplasmic proteins TraW and TrbC. For TraV F and TraV N expression, the N-terminal lipobox was omitted. Instead, the proteins were equipped with an N-terminal pelB sequence for periplasmic expression and a C-terminal His-tag. For TraW N , the whole reading frame was expressed without a His-tag. TraW N was retained on Ni-NTA beads in the presence but not in the absence of TraV N -His, confirming that TraW N interacts with TraV N (Figure 4 and for further details see Figure S4). We were, however, unable to pull down TraW F with TraV F -His. TrbC F was expressed without a His-tag to confirm the F-plasmid-specific interaction between TrbC F and TraV F by copurification. Only a construct without the signal sequence gave a  Note: + and -indicate, respectively, interaction and no interactions. Space indicates that the interaction was not tested. The placement of T18 and T25 relative to the protein name indicates N or C-terminal fusion. T18 or T25 indicate that the gene encoding the protein was cloned into the BACTH vectors. TM18 or TM25 indicates that the gene encoding the protein was cloned into the BACTH-TM vectors.

F I G U R E 4 Co-purification of TraW
high level of expression and only this protein was used. for pull-down experiments with His-tagged TraV F . TrbC F was found to co-purify with TraV F (Figures 4 and S4). Two cysteine residues are found toward the C-terminal end of TrbC F , giving potentially different folding and protein interactions for TrbC F expressed in the cytoplasm compared to TrbC F expressed in the periplasm. E. coli Origami-2(DE3) is a protein expression strain with mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes. These alterations enhance disulfide bond formation in the cytoplasm. Similar results were obtained for TrbC F expressed in the E. coli BL21(DE3) and E. coli Origami-2(DE3) indicating that disulfide bond formation is not important for the pull-down result observed.

| Interactions between proteins from the F-plasmid T4SS and proteins from the N. gonorrhoeae T4SS
For interactions observed with proteins from the F-plasmid T4SS or with proteins from the N. gonorrhoeae T4SS, we examined if one of the proteins could be replaced by the corresponding F or N. gonorrhoeae T4SS proteins giving mixed F/N. gonorrhoeae T4SS interactions (Table 5). For all the interactions shared between the N.
gonorrhoeae T4SS and F-plasmid T4SS proteins (Figure 3a), either one or both proteins could be replaced by the corresponding protein from the other system (Table 5 and Figure S3). While the TraV N / TraK N and TraV F /TraK F interactions were only observed when the genes encoding the two proteins were cloned in the BACTH system, the two mixed interactions TM-TraV F /TM-TraK N and TM-TraV N /TM-TraK F were observed only for the BACTH-TM clones (Table 5).
Also, for groups 2 and 3 containing, respectively, N. gonorrhoeae-specific interactions and F-specific interactions, several mixed interactions were observed (Table 5, Figures S1 and S2). TraB N , TraB F , TraV N , TrbC F , and TraK F , which were able to form several non-mixed interactions, were also proficient in forming mixed interactions. Copurification confirmed mixed interactions between TraV F and TraW N and between TraV N and TrbC F (Figure 4).

| Evaluation of the BACTH-TM system
Since the BACTH two-hybrid system (Karimova et al., 1998)  coli OppB transmembrane domain is inserted between the protein of interest and the T18 or T25 fragment, exposing the protein of interest to the periplasmic environment but did not use the system to study interactions between periplasmic proteins. To evaluate the system, we cloned 13 proteins from the N. gonorrhoeae T4SS and 7 proteins from the F-plasmid T4SS system in the BACTH-TM system.
For comparison, 6 proteins from the N. gonorrhoeae T4SS and 2 proteins from the F-plasmid T4SS were cloned into both the BACTH and the BACTH-TM systems. For TraH N and TraU N , we only observed interactions when the proteins were cloned in the BACTH-TM system (Table 3). Since TraH N and TraU N are proteins rich in cysteine residues (9 and 16, respectively), it seems likely that these proteins are unable to fold correctly in the cytoplasm. For periplasmic proteins cloned only in the BACTH-TM system, we observed interactions between TraW and TrbC from both the N. gonorrhoeae T4SS and the F-plasmid T4SS. In several F-type T4SSs, TrbC proteins are fused to the N-terminus of TraW, and TrbC F and TraW F were found to co-purify, indicating that the TraW/TrbC interaction is important for F-type T4SSs (Shala-Lawrence et al., 2018). These results indicate that BACTH-TM system can be used successfully to study periplasmic protein interactions.
For the peripheral membrane proteins, TraV N and TraK N , an interaction in the cytoplasm after removal of signal sequences have previously been demonstrated using a bacterial two-hybrid system (Ramsey et al., 2014). Since the only cysteine in TraK N and TraK F is in the predicted signal peptide, the TraV/TraK interaction is unlikely to involve a disulfide bridge (Ramsey et al., 2014). TraV N , TraK N , TraV F , and TraK F were cloned in both the BACTH and the BACTH-TM systems. The TraV N /TraK N and the TraV F /TraK F interactions were only observed for the proteins cloned in the BACTH system (Table 3). It is possible that linking the proteins to the inner membrane interferes with the TraV/TraK interaction. We did, however, observe other interactions for TM-TraV N , TM-TraK N , TM-TraV F , and TM-TraK F .

| Interactions between F-type-specific T4SS periplasmic proteins
Two interaction groups for F-proteins have been defined by yeast two-hybrid screens (Harris et al., 2001;Harris and Silverman, 2004) -one consisting of the three proteins with homology to the outer membrane core proteins TraV F , TraK F , and TraB F (Harris et al., 2001) and one consisting of F-specific proteins (TrbB F , TrbI F , TraW F , TraU F , TraH F , and TraF F ) (Harris and Silverman, 2004). No interactions were observed between the two interaction groups. Our study using bacterial two-hybrid systems confirmed several of these previously observed interactions (Figure 3, Table 3). We have not included TrbB F and TrbI F in this study since the N. gonorrhoeae T4SS does not possess a TrbB homolog and the deletion of trbI N has been found not to affect DNA secretion by N. gonorrhoeae (Pachulec et al., 2014). For TraW F , TraH F , TraU F , and TraF F , the study carried out by Harris and Silvermann (Harris and Silverman, 2004) Harris and Silverman (Harris and Silverman, 2004) ( Figure 3b). The TraW F /TraU F interaction observed by Harris and Silvermann (Harris and Silverman, 2004) was identified in this study as an F-specific interaction (Figure 3c). Besides, we observed the  (Figure 3a,c) as well as some new interactions between the F-type-specific proteins (Figure 3b,c).
We demonstrate that TraH N is an outer membrane-associated protein in N. gonorrhoeae (Figure 2). Its F-plasmid homolog TraH F also associates with the outer membrane in the presence of other T4SS proteins (Arutyunov et al., 2010). TrbI F is required for correct TraH F localization (Arutyunov et al., 2010). Gonococci do not require TrbI N for DNA secretion (Pachulec et al., 2014). The transcriptomic study of Remmele, as well as the qRT-PCR results of Ramsey, indicated that the traH N -traG N -atlA N transcript was found at much higher levels than the long transcript containing most other T4SS genes (Remmele et al., 2014;Ramsey et al., 2015). Thus, we sought to determine if TraH N might work together with AtlA N or TraG N . TraG N was found to affect the localization of TraH N possibly by stabilizing TraH N or by facilitating the transport of TraH N to the gonococcal outer membrane.

| A possible biological implication of the interaction between TraV/TraK/TraB and F-type-specific proteins
In this study, we observed several interactions between the proposed OMCC protein TraB/TraV/TraK and F-type-specific proteins (Figures 3 and 4, Tables 3-5). The F-type-specific proteins have been assigned a function in pilus assembly/retraction and mating pair stabilization based on mutant studies (Arutyunov and Frost, 2013).
Until recently the physical and functional relationship of the T4SS apparatus and the F-pilus has been undefined. However, a recent CryoET study (Hu et al., 2019) indicates that the F-pilus is connected to the T4SS outer membrane complex (OMC). Further, the study indicates that the F pilus nucleates assembly at the outer membrane in a process leading to a structural change in the OMC (Hu et al., 2019). It is tempting to speculate that the F-type-specific proteins are involved in this structural change. Although the N. gonorrhoeae T4SS lacks a pilus, a structural change of the OMC mediated by the F-type-specific proteins might still be needed to allow for substrate transfer.

| Interactions between TraV and F-type-specific proteins
TraV F is an outer membrane lipoprotein (Doran et al., 1994). TraH F , TraF F , TraU F , and TraW F have been shown to localize to the outer membrane when in the context of the complete transfer apparatus, probably with TraV F as the anchor protein (Arutyunov et al., 2010). We observed several interactions between TraV N and TraV F and F-type-specific periplasmic proteins using BACTH studies (Figure 3a-c). The TraW N /TraV N and the TrbC F /TraV F interactions were confirmed by co-purification (Figure 4). The results indicate that TraV can anchor F-type-specific periplasmic proteins to the outer membrane both for the N. gonorrhoeae T4SS and the F-plasmid T4SS. The TraV homolog VirB7 is a small lipoprotein that helps to stabilize the outer membrane complex at the outer membrane (Christie, 2016). VirB7 from the A. tumefaciens is only 55 amino acids; however, longer forms of VirB7 with additional functions have been described (Christie, 2016). TraV N and TraV F are, respectively, 193 and 171 amino acids with only the N-terminal part of the proteins showing weak homology to VirB7 (Ramsey et al., 2014). It is, therefore possible that the C-terminal part of TraV could be involved in interactions with F-type-specific proteins.

| The TraB-TraE interaction
Due to the high divergence of the primary sequence, some VirB8 homologs have been identified only upon structural analysis (Goessweiner-Mohr et al., 2013). A bioinformatic study placed TraE F in a universally present group of VirB8 homologs (Guglielmini et al., 2014) and secondary structure predictions also indicate that TraE F and TraE N are VirB8-like proteins (Goessweiner-Mohr et al., 2013). CryoEM of a P-type T4SS from the conjugative R388 plasmid shows that the inner membrane complex consists of the N-terminal part of VirB10 in connection with a set of other inner membraneassociated proteins including VirB8 (Low et al., 2014). Interactions between VirB8 homologs and VirB10 homologs have been demonstrated using the BACTH system (Casu et al., 2016) as well as other two-hybrid systems (Das and Xie, 2000;Ding et al., 2002). TraE F has been shown to associate with the inner membrane (Arutyunov et al., 2010) and is essential for conjugation (Lawley et al., 2003), but the function of the protein is unknown. We observed the interaction between TraB and TraE for both proteins from the N. gonorrhoeae T4SS and proteins from the F-plasmid ( Figure 3a) and a mixed TraE N / TraB F interaction (Table 5 and Figure S3). This result indicates that in addition to inner membrane localization, TraE shares with VirB8 the ability to interact with the VirB10 homolog TraB.

| Cross-system interchangeability of T4SS proteins
In this study, we observed several interactions between proteins from the F-plasmid T4SS and proteins from the N. gonorrhoeae T4SS system, indicating a high degree of cross-system interchangeability of homologous T4SS proteins despite low sequence homology (Table 5, Figures S1-S3). This phenomenon has been observed in several other studies (Gillespie et al., 2015;Casu et al., 2016;Carraro et al., 2017;Gordon et al., 2017). For P-type T4SS, there are indications for cross-system interchangeability between VirB8, VirB10, and VirB5 homologs (Schmidt-Eisenlohr et al., 1999;Gillespie et al., 2015;Casu et al., 2016;Gordon et al., 2017). In nature, this crosssystem interchangeability might be important for bacteria carrying more than one T4SS (Gillespie et al., 2015). With regard to F-type T4SSs, it is not unusual for multidrug-resistant enterobacteria to carry both IncF and IncA/C plasmids (Rayamajhi et al., 2011;Silva et al., 2015). Like IncF plasmids, IncA/C plasmids encode F-type T4SSs (Harmer and Hall, 2015). An interesting example of crosstalk occurs between an IncA/C plasmid and Salmonella genomic island 1 (Carraro et al., 2017). While the IncA/C plasmid encodes an F-type T4SS, the Salmonella genomic island 1 only encodes homologs of TraN, TraH, and TraG with amino acid identity between 37% and 78% to the plasmid proteins (Carraro et al., 2017). The Tra subunits of the genomic island can complement their plasmid counterpart in mutant studies; however, the outcome of the conjugation is shifted toward the spread of the genomic island rather than the IncA/C plasmid (Carraro et al., 2017). The presence of an IncF plasmid increases the conjugation rate of co-residing IncA/C plasmids by an unknown mechanism (Gama et al., 2017). Our data support cross-system interchangeability between F-type T4SS proteins. This interchangeability might be a way different co-residing conjugative plasmids can interact in processes that could influence the spread of antibiotic resistance.
In conclusion, our results indicate that the T4SSs from the F-plasmid and N. gonorrhoeae share an overall architecture, especially with regard to conserved T4SS protein homologs (Figure 3a).
However, interactions between F-type-specific proteins and between F-type-specific proteins and conserved T4SS proteins (TraV, TraK, and TraB) exhibit more variation between systems (Figure 3b,c).
We present maps of the protein interactions that build these two F-type T4SSs and demonstrate that multiple protein components are likely interchangeable within these interaction networks.

| Bacterial strains and growth conditions
All bacterial strains are listed in supplementary material Table S1.

| Construction of plasmids
Chromosomal DNA from N. gonorrhoeae MS11 was isolated from liquid overnight cultures using the GenElute Bacterial Genomic DNA kit from Sigma following the recommendation of the manufacturer except that approx. 8-9 ml of overnight culture was used for each preparation (rather than 1.5 ml) to compensate for a low OD 600 in the overnight cultures. coli JM101 as a template using primers 44-64 and 75-78 (Table S2).

| Construction of N. gonorrhoeae mutants, subcellular fractionation, and western blotting
The traH N gene from N. gonorrhoeae strain MS11 was cloned into pMR100 to add the 3x-FLAG tag in-frame with the TraH N coding sequence, creating the intermediate pAY25. The resulting traH N -FLAG3 gene was subcloned from pAY25 into pMR68 to place it under transcriptional control by the anhydro-tetracycline-inducible promoter and locate it between gonococcal genes iga and trpB. The resulting plasmid, pAY27, was then used to insert the traH N -FLAG3 construct onto the gonococcal chromosome in wild-type N. gonorrhoeae strain MS11 or its derivatives lacking traK N (MR535, Ramsey et al., 2014), Kohler et al., 2013), or atlA N (PK127, Kohler et al., 2007). To FLAG3-tag TraH N at the native locus, a fragment of DNA downstream of the traH native site was cloned in pAY25, creating pAY28. pAY28 was used to transform MS11, generating AY529.
Gonococci were transformed as previously described (Ramsey et al., 2015). The expression of TraH N -FLAG3 was induced with 0.2 ng/ml anhydro-tetracycline. Subcellular fragmentation and western blots were performed essentially as described before (Ramsey et al., 2014;. For the western blots approximately 5 µg of protein from each fraction was subject to SDS-PAGE.

| Determination of interactions and measurements of β-galactosidase activity
The initial interaction screening was done after the co-transforma- For β-galactosidase assays, cells were grown overnight at 30°C in LB with appropriate antibiotics and 0.5 mM IPTG and β-galactosidase activities were measured as described by Miller (1972). TraV F pellets were resuspended in buffer I or buffer II and frozen at −20°C. After one round of freezing and thawing, partial binding of TraV N and TraV F to nickel resin was observed ( Figure S4) and samples treated this way were used for co-purification and control experiments. For control experiments, cells carrying expression plasmids were replaced with cells carrying pCOLADuet-1 or pET22b. For resin binding, samples were applied to 0.5 ml of washed and equilibrated Ni-NTA agarose beads (QIAGEN, Hilden, Germany) and incubated overnight at 4°C, with mixing. The beads were washed with 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole at pH 8. Subsequently, bound proteins were stepwise eluted with 1 ml of 25 mM NaH 2 PO 4 , 150 mM NaCl, 125 mM imidazole at pH 8 and 1 ml of 50 mM NaH 2 PO 4 , 300 mM NaCl, and 250 mM imidazole ( Figure S4). Equal amounts of the eluted sample from copurification and control experiments were analyzed by SDS-PAGE.

ACK N OWLED G M ENTS
This work was supported by EPSRC Grant Nos EP/L001489/2, EP/ J004111/2, EP/N031926/1 and a Royal Academy of Engineering Chair in Emerging Technology to N.K and NIH grant R01AI047958 to J.P.D. We thank Scot P. Ouellette for providing plasmids pUTM18C and pSTM25. NK thanks S. Heeb for bringing N. gonorrhoeae's type IV secretion system to his attention many years ago.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in the supplementary material of this article. In addition, we have created version-controlled cell repositories (cellrepo) as recommended in Tellechea-Luzardo et al. (2020) to facilitate reproduction and derivative work from this paper. These repositories focus on the TraV proteins and describe the plasmids containing them. The cellrepo for our data is available here: