Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.Cell division is a fundamental process in all organisms. In Escherichia coli, it requires the coordinated constriction of the three layers of the gram-negative cell envelope: the invagination of the inner membrane and the biosynthesis of septal peptidoglycan, accompanied by the invagination of the outer membrane. The inner membrane invagination is mediated by the divisome, an assembly of proteins that forms a ring-like structure at midcell. In E. coli, the divisome includes at least 10 essential proteins (FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsB, FtsW, FtsI, and FtsN) and a set of nonessential proteins (ZapA, ZapB, FtsE, FtsX, FtsP [SufI], AmiC, and EnvC) (19,34,45,55,56). The cell divisome formation starts with the polymerization of the FtsZ protein at midcell to form the so-called Z ring. This structure marks the future division site and provides a scaffold for the recruitment of the other cell division proteins (3,7,43,52,53). Numerous studies showed that the divisome proteins are sequentially recruited to the septum in E. coli. First, two FtsZ binding proteins, FtsA and ZipA, almost simultaneously localize at midcell independently of each other, presumably to stabilize the Z-ring structure. The nonessential accessory proteins ZapA and ZapB that are able to directly associate with FtsZ in vivo (27, 28, 32) may also participate in stabilizing the Z ring (51). Then, a series of integral membrane proteins are recruited to the septum in a sequential manner in the following order: FtsK, FtsQ, FtsL/ FtsB, FtsW, FtsI, and FtsN (for reviews, see references 34, 45, 54, and 56). The assembly of these proteins with the divisome is essential for the progression and completion of cytokinesis. Several nonessential prot...