Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an Nlinkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position ؉2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with ␤-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.Over the past 25 years, several techniques and tools have been developed to express and purify recombinant proteins for protein structure-function studies, for the development of new drugs, or simply for the manufacture of enzymes. The most frequently used method for isolating recombinant protein from a cell lysate in a single purification step is immobilized metal ion affinity chromatography (IMAC). In the simplest application of this method, the target protein is tagged with a polyhistidine sequence (typically 6ϫHis), which mediates chelation to immobilized divalent metal ions such as nickel or cobalt. Other studies have demonstrated that peptides with nonconsecutive histidines are also capable of chelation to immobilized divalent metal ions (5) (U.S. patent 7,176,298 [41] and U.S. patent application 2006/0030007 A1).Escherichia coli is the most commonly used host for highyield expression of recombinant protein, usually by exploiting the high promoter specificity and transcriptional activity of bacteriophage T7 RNA polymerase. However, several E. coli host proteins also contain nonconsecutive histidine residues exposed to the surface of their ternary structure. In addition, metal binding motifs often mediate binding to nickel-and/or cobalt-containing purification resins. Such host proteins are routinely copurified during IMAC procedures and are therefore referred to as "contaminants." Several metal binding proteins that behave as IMAC contaminants have been identified in recent years. For example, Bolanos-Garcia et al. reviewed this issue in detail by classifying the E. coli metal binding proteins according to their affinity for Ni...

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...

FtsL and FtsB are two inner-membrane proteins that are essential constituents of the cell division apparatus of Escherichia coli. In this study, we demonstrate that the leucine zipper-like (LZ) motifs, located in the periplasmic domain of FtsL and FtsB, are required for an optimal interaction between these two essential proteins.

FtsN, the last essential protein in the cell division localization hierarchy in Escherichia coli, has several peculiar characteristics, suggesting that it has a unique role in the division process despite the fact that it is conserved in only a subset of bacteria. In addition to suppressing temperature-sensitive mutations in ftsA, ftsK, ftsQ, and ftsI, overexpression of FtsN can compensate for a complete lack of FtsK in the cell. We examined the requirements for this phenomenon. We found that the N-terminal terminal region (cytoplasmic and transmembrane domains) is critical for suppression, while the C-terminal murein-binding domain is dispensable. Our results further suggest that FtsN and FtsK act cooperatively to stabilize the divisome.