International Journal of Biological Macromolecules 106 (2018) 763–767 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Identification of functional interactome of a key cell division regulatory protein CedA of E.coli Pankaj Sharma, Anil Kumar Tomar, Bishwajit Kundu ∗ Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India a r t i c l e i n f o Article history: Received 21 July 2017 Received in revised form 9 August 2017 Accepted 10 August 2017 Available online 14 August 2017 Keywords: CedA-binding proteins Chromosomal DNA over-replication Pull down assay a b s t r a c t Cell division is compromised in DnaAcos mutant Escherichia coli cells that results in filamentous cell morphology. This is countered by over-expression of CedA protein that induces cytokinesis and thus, regular cell morphology is regained; however via an unknown mechanism. To understand the process systematically, exact role of CedA should be deciphered. Protein interactions are crucial for functional organization of a cell and their identification helps in revealing exact function(s) of a protein and its bind- ing partners. Thus, this study was intended to identify CedA binding proteins (CBPs) to gain more clues of CedA function. We isolated CBPs by pull down assay using purified recombinant CedA and identified nine CBPs by mass spectrometric analysis (MALDI-TOF MS and LC–MS/MS), viz. PDHA1, RL2, DNAK, LPP, RPOB, G6PD, GLMS, RL3 and YBCJ. Based on CBPs identified, we hypothesize that CedA plays a crucial and multifaceted role in cell cycle regulation and specific pathways in which CedA participates may include transcription and energy metabolism. However, further validation through in-vitro and in-vivo experi- ments is necessary. In conclusion, identification of CBPs may help us in deciphering mechanism of CedA mediated cell division during chromosomal DNA over-replication. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Cell division and chromosomal DNA replication in bacteria occur with high accuracy, co-ordination and regulation. In Escherichia coli (E. coli), DnaA protein initiates and regulates the process of chro- mosomal DNA replication [1,2]. Basically, it binds to the origin of replication (oriC) and initiates formation of the DNA replication ini- tiation complex. Previously, several studies have been performed with DnaA mutants in an attempt to decipher the mechanism(s) of chromosome replication and cell division [3–5]. One such mutant is DnaAcos, a cold sensitive DnaA mutant that carries four amino acid substitutions. It induces excessive initiation from E. coli oriC at 30 ◦ C and thus, causes over-replication of chromosomal DNA. Consequently, the process of cell division is compromised in these cells which undergo excessive karyokinesis without cytokinesis and attain a filamentous morphology [1,6,7]. A multi-copy suppressor gene cedA, that encodes a cell divi- sion activator protein (CedA), is expressed in dnaAcos mutants and interestingly, its expression controls the cell division. It is an 80 amino acids long DNA-binding protein. Katayama et al. reported ∗ Corresponding author at: Room No. 204, Kusuma School of Biological Sciences, Indian Institute of Technology Delhi (IIT Delhi), Hauz Khas, New Delhi, 110016, India. E-mail address: bkundu@bioschool.iitd.ac.in (B. Kundu). that CedA over-expression in DnaAcos mutants started septation, cell division and regular colony formation and thus, inhibited the formation of filamentous morphology at 30 ◦ C; however, chro- mosomal DNA over-replication continued [1]. It is evident from their study that though CedA is important for the initiation of cell division, it does not inhibit dnaAcos to bind at oriC to initi- ate chromosomal replication. CedA was also identified as one of the components of RNA polymerase complex in E. coli [8]. So, it becomes interesting to decipher how CedA in case of DnaAcos mutants main- tains a balance between two entirely integrated events, cell division and chromosomal replication. Protein–protein interactions are crucial for understanding structural and functional organization of a cell. Their identifi- cation helps us in decoding exact physiological function(s) of a specific protein and its binding partners. In addition, interaction network analysis plays a decisive role in exposing underlying mech- anism(s) of various related biological processes. Thus, this study was designed to identify functional partners of CedA as a first step to reveal its putative role in cell division associated events. Here, we have cloned, expressed and purified E. coli CedA and then, iden- tified its binding proteins by pull down assay followed by mass spectrometric analysis. http://dx.doi.org/10.1016/j.ijbiomac.2017.08.073 0141-8130/© 2017 Elsevier B.V. All rights reserved.