Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

Abstract

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...

ATP-Binding Site Lesions in FtsE Impair Cell Division

“…First, we have not investigated whether the mutant FtsE proteins support recruitment of several nonessential proteins to the septal ring (6,7,25,32,50). Although failure to recruit any one of these proteins would not account for the division defects reported here, failure to recruit several of them might.…”

“…We also constructed a new ftsEX null mutant, named EC1215, that has a markerless deletion extending from codon 6 of ftsE to codon 297 of ftsX. This deletion leaves intact the major promoter for 32 embedded in the 3Ј end of ftsX (22). We constructed EC1215 because the mutant used in our previous study, RG60, has a nonexcisable Kan r determinant in ftsE, making it incompatible with our P lac ::ftsEX plasmids, which confer Kan r (20,47).…”

Identification ofEscherichia coliZapC (YcbW) as a Component of the Division Apparatus That Binds and Bundles FtsZ Polymers

“…In the second stage, the Z ring matures into a constriction-competent SR that includes all known essential division proteins. The third stage starts at initiation of the constriction process, after which many of the nonessential SR proteins join the division apparatus (4,10,29,30,46,80). Temporally, there is a substantial delay between assembly of the Z ring and recruitment of the later-assembling components (2,26).…”

“…The number of known nonessential protein components of the SR has increased steadily in the last few years. Though these are individually dispensable for cell fission and viability, many appear to have overlapping roles in important aspects of cell constriction, and their study is required for a satisfactory understanding of the whole process (4,8,10,11,22,23,29,30,33,46,70,72,80,82).…”

Role of Leucine Zipper Motifs in Association of the Escherichia coli Cell Division Proteins FtsL and FtsB

“…These modifications should destabilize the hydrophobic interface formed between the two LZ motifs without affecting the overall alpha-helical structure of the coiled coil, thus avoiding a dramatic alteration of the proteins that could lead to their instability and degradation (7,18). The impact of these modifications was assessed by using the BACTH assay that has been previously used to analyze interactions between Fts proteins (2,10,11,21,23). In this assay, the proteins of interest are fused to two complementary fragments, T25 and T18, from the adenylate cyclase of Bordetella pertussis and expressed in an E. coli cya strain.…”