Cell division interactome

While the gene arrangement in the dcw cluster varies in different bacteria species, key cell division proteins are relatively conserved (Pucci et al., 1997). For examples, proteins like FtsZ, FtsA, ZipA, FtsQ/DivIB, FtsL, FtsW, FtsB/DivIC, FtsI and FtsK are highly conserved in almost all cell-walled Eubacteria (Margolin, 2000; Harry et al., 2006). But additional proteins like Min proteins, ZipA, ZapA, EzrA, FtsN or SepF, may or may not be present depending on the bacterial species (Margolin, 2000). All these proteins interact with one another to form one large multicomponent complex spanning the cytoplasmic membrane. Using in vivo and in vitro biochemical techniques such as bacterial two-hybrid (B2H) assay, GST-pull down assay, Co-immunoprecipitation (Co-IP) and Surface Plasmon Resonance (SPR) cell division protein-protein interaction networks have been established for only four bacterial species i.e. E. coli (Di Lallo et al., 2003; Karimova et al., 2005), N. gonorrhoeae (Zou et al., 2017), S. aureus (Steele et al., 2011) and S. pneumoniae (Fadda et al., 2007; Maggi et al., 2008).

1.9.1. Cell division interactome in Gram-positive bacteria

Maggi et al. (2008) tested 11 streptococcal cell division proteins for interactions using a B2H assay and co-immunoprecipitation from S. pneumoniae. A total of 37 homo- and/or hetero-dimeric interactions were observed where each protein interacted with at least two or more interacting partners except for PBP1A which had only one interacting partner (Maggi et al., 2008). There were 7 unique interactions i.e. FtsASp–FtsKSp, FtsASp–FtsLSp, FtsZSp–FtsWSp, FtsZSp–FtsQSp/DivIBSp, FtsZSp–FtsLSp, FtsKSp–FtsWSp, FtsLSp–FtsISp/PBP2XSp, when compared with the E. coli interactome (Maggi et al., 2008). Using co-immunoprecipitation, seventeen confirmed interactions (i.e. FtsZSp with FtsASp, FtsKSp, FtsQSp, FtsBSp, FtsLSp, and FtsWSp; FtsASp with FtsKSp, FtsLSp, and FtsLSp; FtsKSp with FtsQSp, FtsISp, and FtsWSp; FtsQSp

with FtsLSp, and FtsWSp; FtsBSp-FtsWSp; and FtsLSp with FtsISp, and FtsWSp) were observed among nine cell division proteins that included FtsZSp, FtsASp, FtsKSp, DivlBSp, DivlCSp, FtsLSp, FtsWSp, and PBP2xSp (Maggi et al., 2008).

In S. aureus, the potential interactions between thirteen divisome proteins (i.e. FtsZSa, FtsASa, EzrASa, GpsBSa, SepFSa, Pbp1Sa, Pbp2Sa, Pbp3Sa, DivIBSa, DivICSa, FtsLSa, FtsWSa and RodASa) were mapped using a B2H assay by Steele et al. (2011). Around 49 homo-and/or hetero-dimeric protein interactions were identified and almost all proteins were found to interact with multiple interacting partners except for SepFSa and GpsBSa which interacted with

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only EzrASa (Steele et al., 2011). SepFSa interaction with FtsZSa has been well-studied in B.

subtilis (Hamoen et al., 2006) but was not observed in S. aureus (Steele et al., 2011). When compared with the interactome of S. pneumoniae, following interactions were observed to be conserved: FtsASa with FtsZSa, all division-specific PBPs, FtsWSa, DivICSa and FtsLSa; FtsWSa

with FtsLSa and all division-specific PBPs; DivICSa, DivIBSa and FtsLSa with all division-specific PBPs; and FtsLSa with DivICSa. EzrASa interacted with all thirteen cell division proteins (Fig. 1.5.) (Steele et al., 2011).

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Figure 1.5. Characterized cell division interactomes from A) S. pneumoniae (Maggi et al., 2008), and B) S. aureus (Steele et al., 2011).

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1.9.2. Cell division interactome in Gram-negative bacteria

Di Lallo et al. (2003) were first to use B2H assay to deduce the cell division interactome network in E. coli using 9 divisome proteins (i.e. FtsZEc, FtsAEc, ZipAEc, FtsKEc, FtsQEc, FtsLEc, FtsIEc, FtsWEc, and FtsNEc). Karimova et al. (2005) later expanded on this knowledge using their own version of a B2H assay i.e. the bacterial adenylate cyclase two-hybrid (BACTH) system, which relies on the reconstruction of a cyclic AMP (cAMP) signalling cascade upon interaction (Karimova et al., 1998). They reconfirmed all the interactions showed by Di Lallo et al. (2003) and included FtsB for testing possible interactions with other cell division proteins.

Collectively in E. coli, 16 interactions (i.e. FtsZEc with FtsAEc, ZipAEc, FtsKEc; FtsAEc with FtsIEc, FtsNEc, FtsQEc; FtsKEc with FtsIEc, FtsQEc; FtsQEc with FtsBEc, FtsLEc, FtsIEc, FtsNEc, FtsWEc; FtsBEc with FtsLEc, FtsIEc; FtsLEc with FtsIEc, FtsWEc; FtsIEc with FtsWEc, FtsNEc; and FtsWEc with FtsNEc) between ten cell division proteins (i.e. including FtsZEc, FtsAEc, ZipAEc, FtsKEc, FtsQEc, FtsBEc, FtsLEc, FtsIEc, FtsWEc, and FtsNEc) were identified (Di Lallo et al., 2003;

Karimova et al., 2005). Maggi et al. (2008) compared S. pneumoniae interactome with E. coli and observed 8 unique interactions that were absent in E. coli interactome which was a reflection of distinct cell division mechanisms in these two organisms (Di Lallo et al., 2003;

Karimova et al., 2005; Maggi et al., 2008).

Zou et al. (2017) characterized cell division interactome from Neisseria gonorrhoeae, another Gram-negative coccal bacterium, using B2H and GST-pull down assays. Nine positive interactions (i.e. FtsZNg-FtsANg, FtsZNg-FtsKNg, FtsZNg-FtsWNg, FtsANg-FtsKNg, FtsANg -FtsQNg, FtsANg-FtsWNg, FtsANg-FtsNNg, FtsINg-FtsWNg, and FtsKNg-FtsNNg) were observed among 8 cell division proteins i.e. FtsZNg, FtsANg, ZipANg, FtsKNg, FtsQNg, FtsINg, FtsWNg, and FtsNNg, that defined the cell division interactome. FtsANg did not homodimerize or interact with FtsZEc but interacted with FtsNNg which is unlike E. coli interactome (Fig. 1.6) (Di Lallo et al., 2003; Karimova et al., 2005; Zou et al., 2017).

1.9.3. Conserved cell divisome interactions

When cell division interactomes from E. coli (Di Lallo et al., 2003; Karimova et al., 2005), N. gonorrhoeae (Zou et al., 2017), S. aureus (Steele et al., 2011) and S. pneumoniae (Fadda et al., 2007; Maggi et al., 2008) were compared, the interaction between FtsZ and FtsA was found to be conserved in all four interactomes. The interaction between FtsZ and FtsK was positive in E. coli, N. gonorrhoeae, and S. pneumoniae but S. aureus was not tested.

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Figure 1.6. Characterized cell division interactomes from Gram-positive organisms: A) Neisseria gonorrhoeae (Zou et al., 2017); B) E. coli (Karimova et al., 2005; Di Lallo et al., 2003 ).

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