Specificity of the transport of lipid II by FtsW in Escherichia coli.

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg(145) and Lys(153)) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane.

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

Channel properties of the translocator domain of the autotransporter Hbp of Escherichia coli.

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming ß-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.

Pbp, a cell-surface exposed plasminogen binding protein of Bacteroides fragilis.

The Gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine, but is also frequently found in severe intra-abdominal infections. Several B. fragilis virulence factors have been implicated in the development of these infections. A B. fragilis protein of circa 60-kDa was identified as a putative plasminogen binding protein (Pbp). The corresponding gene was located, cloned, sequenced and the subcellular localization of the protein was investigated. Pbp was both determined in the outer membrane of B. fragilis and of E. coli that expressed the cloned protein. Protease accessibility studies showed that the protein is expressed at the cell surface. Importantly, we demonstrated that Pbp is sufficient and required for plasminogen binding to whole cells in both E. coli and B. fragilis. Pbp-like proteins were also detected in some other Bacteroides subspecies. The role of this potential B. fragilis virulence factor in pathogenicity is discussed.

Transmembrane transport of peptidoglycan precursors across model and bacterial membranes.

Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.

Crystal structure of hemoglobin protease, a heme binding autotransporter protein from pathogenic Escherichia coli.

The acquisition of iron is essential for the survival of pathogenic bacteria, which have consequently evolved a wide variety of uptake systems to extract iron and heme from host proteins such as hemoglobin. Hemoglobin protease (Hbp) was discovered as a factor involved in the symbiosis of pathogenic Escherichia coli and Bacteroides fragilis, which cause intra-abdominal abscesses. Released from E. coli, this serine protease autotransporter degrades hemoglobin and delivers heme to both bacterial species. The crystal structure of the complete passenger domain of Hbp (110 kDa) is presented, which is the first structure from this class of serine proteases and the largest parallel beta-helical structure yet solved.

Characterization of an iron-regulated alpha-enolase of Bacteroides fragilis.

This study describes the identification, cloning and molecular characterization of the alpha-enolase P46 of Bacteroides fragilis. The gram-negative anaerobic bacterium B. fragilis is a member of the commensal flora of the human intestine but is also frequently found in severe intra-abdominal infections. Several virulence factors have been described that may be involved in the development of these infections. Many of these virulence factors are upregulated under conditions of iron- or heme-starvation. We found a major protein of 46 kDa (P46) that is upregulated under iron-depleted conditions. This protein was identified as an alpha-enolase. Alpha-enolases in several gram-positive bacteria and eukaryotic cells are located at the cell surface and function as plasminogen-binding proteins. Localization studies demonstrated that P46 is mainly located in the cytoplasm and partly associated with the inner membrane (IM). Under iron-restricted conditions, however, P46 is localized primarily in the IM fraction. Plasminogen-binding to B. fragilis cells did occur but was not P46 dependent. A 60-kDa protein was identified as a putative plasminogen-binding protein in B. fragilis.

Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein.

Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.

RNA polymerase II complexes in the very early phase of transcription are not susceptible to TFIIS-induced exonucleolytic cleavage.

TFIIS is a transcription elongation factor for RNA polymerase II (pol II), which can suppress ribonucleotide misincorporation. We reconstituted transcription complexes in a highly purified pol II system on adenovirus Major-Late promoter constructs. We noted that these complexes have a high propensity for read-through upon GTP omission. Read-through occurred during the early stages at all registers analyzed. Addition of TFIIS reversed read-through of productive elongation complexes, which indicated that it was due to misincorporation. However, before register 13 transcription complexes were insensitive to TFIIS. These findings are discussed with respect to the structural models for pol II and we propose that TFIIS action is linked to the RNA:DNA hybrid.