Role of leucine zipper motifs in association of the Escherichia coli cell division proteins FtsL and FtsB.

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.

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

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.

Evidence from artificial septal targeting and site-directed mutagenesis that residues in the extracytoplasmic ß domain of DivIB mediate its interaction with the divisomal transpeptidase PBP 2B.

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the ß domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the ß domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.

Characterization of YmgF, a 72-residue inner membrane protein that associates with the Escherichia coli cell division machinery.

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.

Artificial septal targeting of Bacillus subtilis cell division proteins in Escherichia coli: an interspecies approach to the study of protein-protein interactions in multiprotein complexes.

Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the "bait") to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the "prey") to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

Contribution of the FtsQ transmembrane segment to localization to the cell division site.

The Escherichia coli cell division protein FtsQ is a central component of the divisome. FtsQ is a bitopic membrane protein with a large C-terminal periplasmic domain. In this work we investigated the role of the transmembrane segment (TMS) that anchors FtsQ in the cytoplasmic membrane. A set of TMS mutants was made and analyzed for the ability to complement an ftsQ mutant. Study of the various steps involved in FtsQ biogenesis revealed that one mutant (L29/32R;V38P) failed to functionally insert into the membrane, whereas another mutant (L29/32R) was correctly assembled and interacted with FtsB and FtsL but failed to localize efficiently to the cell division site. Our results indicate that the FtsQ TMS plays a role in FtsQ localization to the division site.

Role for the nonessential N terminus of FtsN in divisome assembly.

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.

Depletion of apolipoprotein N-acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia coli.

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage 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 beta-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 (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.

An intramolecular disulphide bond reduces the efficacy of a lipoprotein plasma membrane sorting signal.

To study lipoprotein sorting in Escherichia coli, we devised a novel screen in which sensitivity or resistance to bacteriophage T5 and colicin M reflects the membrane localization of the bacteriophage T5-encoded lipoprotein Llp, which inactivates the outer membrane (OM) T5 receptor (FhuA). When processed by lipoprotein signal peptidase, Llp has a serine at position +2, immediately after the fatty acylated N-terminal cysteine. As predicted by the '+2 lipoprotein sorting rule' that determines the localization of lipoproteins in the cell envelope, Llp is located in the OM. However, contrary to expectations, when serine +2 was replaced by aspartate, the canonical plasma membrane lipoprotein retention signal, Llp was still > or =40% targeted to the OM and protected cells against colicin M and phage T5. OM association of this Llp derivative was abolished when a peptide spacer was inserted between the aspartate and the rest of Llp or when the formation of an intramolecular disulphide bond in Llp was prevented by substituting one or other of the cysteines involved. Furthermore, analysis of a MalE-Llp hybrid protein with or without a lipid moiety demonstrated that fatty acylation of Llp is essential for its OM association and for protection against colicin M and bacteriophage T5. These data suggest (i) that phage-encoded Llp uses the endogenous E. coli Lol pathway for lipoprotein sorting to the OM and (ii) that the conformation of a lipoprotein can affect its sorting within the cell envelope.