A unique cell division protein critical for the assembly of the bacterial divisome

Identification of unique essential bacterial genes is important for not only the understanding of their cell biology but also the development of new antimicrobials. Here, we report a previously unrecognized core component of the Acinetobacter baumannii divisome. Our results reveal that the protein, termed Aeg1 interacts with multiple cell division proteins, including FtsN, which is required for components of the divisome to localize to the midcell. We demonstrate that the FtsAE202K and FtsBE65A mutants effectively bypassed the need of Aeg1 by A. baumannii, as did the activation variants FtsWM254I and FtsWS274G. Our results suggest that Aeg1 is a cell division protein that arrives at the division site to initiate cell division by recruiting FtsN, which activates FtsQLB and FtsA to induce the septal peptidoglycan synthase FtsWI. The discovery of the new essential cell division protein has provided a new target for the development of antibacterial agents.

Introduction specific conditions, including commonly used rich media such as LB broth (22). Using a 242 conditional gene deletion method, we have revealed that from 10 putative essential genes 243 identified by this method, three previously annotated as hypotheticals are essential for A. 244 baumannii viability, suggesting that assignment of essentiality to genes identified by 245 methods of this kind need to be individually verified. 246 Our assignment of aeg1 as an essential gene is consistent with a recent study which 247 revealed its essentiality (termed advA) by transposon insertion sequencing, which also 248 observed cell elongation upon the depletion of this gene (29), Our results provided at least 249 two additional lines of evidence to support the notion that Aeg1 is a core cell division protein in cell division ( Fig. 4A-B). 255 In the process of bacterial cell division, over 30 distinct proteins are assembled into a 256 complex typically referred to as "divisome" (40). Ten of these are normally essential for both 257 cell division and survival, and they are now mainly suggested to be the core of the divisome assembly of the divisome is consistent with the fact that the requirement of Aeg1 can be 291 bypassed by constitutively active mutants of FtsA E202K (Fig. 3), FtsB (FtsB E65A ), FtsL 292 (FtsL Q70K ) and of FtsW (FtsW M254I and FtsW S274G ) (Fig. 5). 293 It is worth noting that that although homologs of Aeg1 are present in many 294 Gram-negative bacteria, including some recent isolate of E. coli, it appears to be absent in 295 more extensively studied model strains such as K12 (Fig. S2). It is possible that in strain 296 K12, its activity is conferred by a yet unidentified protein that employs a conserved 297 mechanism in cell division. Future study aiming at elucidating the biochemical and structural 298 basis of the activity of Aeg1 and its homologs, and their relationship with other components 299 of the divisome may provide not only insights into the mechanism of bacterial cell division 300 but also leads for the design of antibiotics with novel mechanism of action.

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Bacterial strains, growth conditions, and plasmids construction 303 All bacterial strains and plasmids used in this study are listed in were harvest by centrifugation (4000g) for 5 min. Mixed cells washed twice with 1 mL of 338 sterile water were spotted onto LB agar and the mating was allowed to proceeded for 4-6 h 339 at 37 o C before plating onto selective medium. 341 To delete the putative essential genes, we first introduced a plasmid expressing the 342 gene of interest into A. baumannii and the resulting strains were used to construct 343 chromosomal deletion mutants using a method based on the suicide plasmid pSR47s (25).

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Briefly, the coding region of the gene was inserted into pJL03, which allows expression of 345 Flag-tagged protein from the P BAD promoter (24) and the resulting plasmid was introduced 346 into A. baumannii by electroporation.

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To construct the plasmid used for in-frame deletion of A. baumannii genes, 1.0 kilobase 348 DNA fragment was amplified from the upstream and downstream of the gene to delete, 349 respectively, digested with appropriate restriction enzymes and inserted into similarly 350 digested pSR47s (25). The primers were designed so that in each case the gene to be  harvested by centrifugation were washed twice with buffer A prior to being plated onto LB 367 agar supplemented with 5% glucose. Colonies appeared were purified and tested by 368 spotting diluted cells onto LB agar with or without ara, respectively to confirm the ability to 369 grown in the absence Aeg1. To eliminate the possibility that the ara-independent growth 370 phenotype was due to mutations in the P BAD promoter, the plasmid from each mutant 371 candidate was rescued and the promoter region was sequenced. Only mutants harboring an 372 intact P BAD promoter were retained for further study.   Proteins separated by SDS-PAGE were transferred onto nitrocellulose membranes, which 401 were first blocked with 5% nonfat milk for 1 h followed by incubation with appropriate primary 402 antibodies for 14 h. Washed membranes were incubated with the appropriate IRDye-labeled 403 secondary antibodies and signals were detected using an Odyssey CLx system (LI-COR).

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Fluorescence imaging 405 To determine the cellular localization of Aeg1 and its colocalization with members of the 406 divisome, we constructed pJL03::Aeg1-mCherry, which expressed the fusion from the P BAD 407 promoter (23). GFP chimera of Fts proteins were expressed from pJL05 driven by the P TAC 408 promoter inducible by IPTG. Each of the GFP-Fts fusion was introduced into strain 409 Δaeg1(P BAD ::mCherry-Aeg1). All strains were maintained in media containing 1% ara to 410 ensure their viability.      Each of the 10 genes predicted to be essential were deleted by the method described in A 618 and the resulting bacterial strains were tested for growth by spotting serially diluted cells on 619 medium with or without 1% arabinose. Images were acquired after incubation at 37 o C for 18 h.