The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites

Abstract

SummaryBacterial cytokinesis is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. DivIB and its orthologue FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. A molecular dissection approach was used to determine which of these domains are essential for recruitment of DivIB to incipient division sites and for its cell division functions. We show that DivIB has three molecular epitopes that mediate its localization to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and we have used this information to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localization epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually non-essential.

Central Domain of DivIB Caps the C-terminal Regions of the FtsL/DivIC Coiled-coil Rod

“…Also, the complete POTRA domain can be deleted from B. subtilis DivIB (23) without compromising growth. In contrast, mutations in the POTRA domains have been found to affect the recruitment of FtsQ and DivIB to the division site of E. coli and B. subtilis (23,24). Could the POTRA domain intervene in the function of FtsL and DivIC(FtsB)?…”

“…Factors that could stabilize the structure of the POTRA domain in vivo include the pH, which may be different close to the membrane than in the bulk water, the ionic strength, the charges at the membrane surface, or the presence of interacting protein partners. Indeed, epitopes required for the recruitment of DivIB(FtsQ) to the division site have been found in the POTRA domain, suggesting interactions with other proteins (23,24).…”

“…Localization epitopes have been identified in the transmembrane segment, the ␣-domain, and a region encompassing the C-terminal part of the ␤-domain and ␥-tail of DivIB from B. subtilis (23). Likewise in E. coli, a region in the ␣-domain is required for localization of FtsQ, whereas the C-terminal region of the ␤-domain and the last ␣-helix are required for recruitment of FtsL and FtsB (24).…”

“…Refs. 20 and 21)), although the transmembrane segment also contributes to the localization (22,23). The extracellular part is organized in three regions termed ␣, ␤, and ␥.…”

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

“…We constructed a series of plasmids in which zapA was present as a translational fusion to either full-length divIB, or various domain deletions thereof, with a C-terminal Myc3 tag (i.e., ZapA-DivIB Myc3 ). The divIB fragments for these plasmids were obtained by PCR amplification from plasmids encoding DivIB domain deletion mutants that we constructed previously (38). PCR products were purified, digested using EcoRI and XbaI, and ligated into plasmid pCR27 (30) to generate Myc3-tagged DivIB constructs.…”