K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells. Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD). FaeC is the tip component of the K88 fimbriae. We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher. In the absence of the chaperone or the usher, FaeC could not be detected in E. coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator. The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found. The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD. Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality. Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided. The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting. FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD. This binding was not observed when control outer membranes without FaeD were used. No other K88 specific proteins were required for this interaction. The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments. FaeE was not required for this interaction. Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E. coli periplasm, but directly binds to the outer membrane molecular usher FaeD.
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.
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 β-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.
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.
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 β-helical structure yet solved.
Summary 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.
