Role of FtsA in cell division in Neisseria gonorrhoeae
Bacterial cell division is an essential process, which is initiated by forming the Z-ring as a cytoskeletal scaffold at the midcell site, followed by the recruitment of a series of divisome proteins. In Escherichia coli (Ec), at least 15 divisome proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsW, FtsN, FtsE, FtsX, ZapA, AmiC, EnvC) have been implicated in this process. The components of the cell division machinery proteins in Neisseria gonorrhoeae (Ng) differs from E. coli. N. gonorrhoeae possesses FtsA, but lacks FtsB. ZipA and FtsL in N. gonorrhoeae have low identity to ZipA and FtsL from E. coli. Our laboratory has studied the central division protein FtsZ in N. gonorrhoeae. Thus, my research investigated the role of N. gonorrhoeae FtsA in cell division and investigated the interactions between divisome proteins from N. gonorrhoeae to understand divisome assembly. This study determined the association of FtsANg with FtsZNg and other divisome proteins in N. gonorrhoeae and identified the functional domains of FtsANg involved in these interactions using a bacterial two-hybrid (B2H) assay. FtsANg interacted with FtsZNg, FtsKNg, FtsWNg, FtsQNg, and FtsNNg. Self-interactions of FtsANg and FtsZNg were also detected. FtsINg, FtsENg and FtsXNg did not interact with FtsANg. The 2A1, 2A2 and 2B domains of FtsANg were sufficient to interact with FtsZNg independently. Domain 2A1 interacted with FtsKNg and FtsNNg. Domain 2B of FtsANg interacted with FtsKNg, FtsQNg, and FtsNNg. Domain 2A2 of FtsANg interacted with FtsQNg, FtsWNg, and FtsNNg. These data suggest that FtsA in N. gonorrhoeae plays a key role in interactions with FtsZ and other divisome proteins. The potential interactions between divisome proteins in N. gonorrhoeae were examined using B2H assays. The comparisons between the N. gonorrhoeae divisome protein interaction network and those of E. coli and S. pneumoniae indicates that the divisome protein interactome of N. gonorrhoeae is more similar to that of S. pneumoniae and differs from that of E. coli. The comparisons revealed that compared to the interactions in E. coli and S. pneumoniae, more interactions between divisome proteins upstream of FtsANg (including FtsANg) and downstream of FtsANg were observed in N. gonorrhoeae while fewer interactions between divisome proteins downstream of FtsANg were observed in N. gonorrhoeae. Possible reasons for this include the inability of ZipANg to interact with other divisome proteins and the absence of FtsL and FtsB in N. gonorrhoeae, resulting in the lack of an FtsQ-FtsB-FtsL complex in N. gonorrhoeae. These results indicate a possibly different divisome assembly in N. gonorrhoeae from that proposed models for E. coli. A model for FtsANg structure was predicted based on structural homology modeling with the resolved crystal structure of Thermotoga maritima FtsA. Four domains on the molecule were identified, designated 1A, 1C, 2B and 2A (including 2A1 and 2A2). Domains 2A and 2B of FtsA were highly conserved based on multi-sequence alignments of FtsAs from 30 bacteria. FtsANg located to the division site in N. gonorrhoeae cells and the ratio of FtsA to FtsZ ranged from 1:24 to 1: 33 in three N. gonorrhoeae strains, which gave a lower cellular concentration of FtsA compared to other organisms. I also determined that overexpression of FtsANg in E. coli led to cell filamentous in rod-shaped E. coli and cell enlargement and aggregation in mutant, round E. coli. FtsANg failed to complement an ftsAEc-deletion E. coli strain although the overexperssion of FtsANg disrupted E. coli cell division. In addition, overexpression of FtsANg only affected cell division in some cells and its localization in E. coli was independent of interaction with E. coli FtsA or FtsZ. These results indicate that FtsANg exhibits a species-specific functionality and E. coli is not a suitable model for studying FtsANg functionality. This is the first study to characterize FtsA from N. gonorrhoeae in cell division. I identified novel functional domains of FtsANg involved in interactions with other divisome proteins. The N. gonorrhoeae divisome protein interaction network determined by B2H assays provides insight into divisome assembly in N. gonorrhoeae.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorDillon, Jo-Anne R.
CommitteeTodd, Chris; Wang, Hong; Howard, Peter; Wilson, Ken; Shafer, William M.
Copyright DateJanuary 2011
bacterial two-hybrid assay