Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking.

The envelope of Escherichia coli contains approximately 100 different species of lipoproteins, most of which are localized to the inner leaflet of the outer membrane. The localization of lipoprotein (Lol) system, consisting of five Lol proteins, is responsible for the trafficking of lipoproteins to the outer membrane. LolCDE binds to lipoproteins destined for the outer membrane and transfers them to the periplasmic chaperone LolA.

An unusual case of fatal hypothermia involving topical diphenhydramine.

Purpose: Diphenhydramine is an antihistamine drug widely used to alleviate symptoms caused by allergies and the common cold. Diphenhydramine-involved fatalities have been reported in the past but usually involving overdose by ingestion. We report a peculiar case of fatal hypothermia during non-winter season involving topical diphenhydramine.

Reversible autoinhibitory regulation of metallopeptidase BepA for selective β-barrel protein degradation.

periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated.

Structural Basis for the Function of the β-Barrel Assembly-Enhancing Protease BepA.

The β-barrel assembly machinery (BAM) complex mediates the assembly of β-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of β-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed.

The TPR domain of BepA is required for productive interaction with substrate proteins and the β-barrel assembly machinery complex.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β-barrel assembly machinery (BAM) driving integration of β-barrel proteins into the outer membrane.

Bacterial lipoproteins; biogenesis, sorting and quality control.

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane.

Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σE in Escherichia coli.

Unlabelled: σ(E), an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth of Escherichia coli not only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of the hicB gene has been reported to suppress the lethality caused by the loss of σ(E). hicB encodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane.

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes.

Protease homolog BepA (YfgC) promotes assembly and degradation of β-barrel membrane proteins in Escherichia coli.

Gram-negative bacteria are equipped with quality-control systems for the outer membrane (OM) that sense and cope with defective biogenesis of its components. Accumulation of misfolded outer membrane proteins (OMPs) in Escherichia coli leads to activation of σ(E), an essential alternative σ factor that up-regulates transcription of multiple genes required to preserve OM structure and function. Disruption of bepA (formerly yfgC), a σ(E)-regulated gene encoding a putative periplasmic metalloprotease, sensitizes cells to multiple drugs, suggesting that it may be involved in maintaining OM integrity.

Functional differentiation of structurally similar membrane subunits of the ABC transporter LolCDE complex.

A photo-sensitive amino acid analogue was introduced into an outer membrane lipoprotein, Pal, and then subjected to photo-crosslinking with the lipoprotein-specific ABC transporter LolCDE. Pal crosslinked to LolE but not LolC in vivo despite that both are structurally similar membrane subunits. LolCDE liganded with Pal containing the photo-sensitive amino acid analogue was isolated and subjected to in vitro photo-crosslinking.

Defective lipoprotein sorting induces lolA expression through the Rcs stress response phosphorelay system.

The Escherichia coli LolA protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative LolA mutant, LolA(I93C/F140C), in which both (93)Ile and (140)Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced lolA gene transcription.

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase.

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively.

ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria.

The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane.

Novel mutations of the LolCDE complex causing outer membrane localization of lipoproteins despite their inner membrane-retention signals.

In Gram-negative bacteria, lipoproteins are targeted to either the inner or outer membrane depending on their sorting signals. An ABC transporter LolCDE complex in Escherichia coli releases outer membrane-specific lipoproteins. Inner membrane-specific lipoproteins remain in the inner membrane because they each have a LolCDE-avoidance signal and therefore are not released by LolCDE.

Biogenesis and Membrane Targeting of Lipoproteins.

Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane.

A periplasmic LolA derivative with a lethal disulfide bond activates the Cpx stress response system.

LolA accommodates the acyl chains of lipoproteins in its hydrophobic cavity and shuttles between the inner and outer membranes through the hydrophilic periplasm to place lipoproteins in the outer membrane. The LolA(I93C/F140C) derivative, in which Cys replaces Ile at position 93 and Phe at position 140, strongly inhibited growth in the absence of a reducing agent because of the lethal intramolecular disulfide bond between the two Cys residues. Expression of I93C/F140C was found to activate the Cpx two-component system, which responds to cell envelope stress.

Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa.

The Phi CTX-based integration vector pYM101 harboring a tightly controlled modified phage T7 early gene promoter/LacI(q) repressor (T7/LacI) system was constructed for the generation of unmarked conditional mutants in Pseudomonas aeruginosa. Promoter activity of the T7/LacI system was demonstrated to be dependent on the presence of the inducer isopropyl -beta-D-1-thiogalactopyranoside (IPTG), as evaluated by measuring beta-galactosidase activity. In the absence of the inducer, the promoter was silent as its activity was lower than those of a promoter-less lacZ control.

Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.

Membrane topology and functional importance of the periplasmic region of ABC transporter LolCDE.

The LolCDE complex is an ATP-binding cassette transporter that mediates the release of newly synthesized lipoproteins from the cytoplasmic membrane of gram-negative bacteria, which results in the initiation of outer-membrane sorting of lipoproteins through the Lol pathway. LolCDE is composed of one copy each of membrane subunits LolC and LolE, and two copies of nucleotide-binding subunit LolD. In this study, we examined the membrane topology of LolC and LolE by PhoA fusion analysis.

Real time analysis of lipoprotein transfer from LolA to LolB by means of surface plasmon resonance.

Lipoproteins of Escherichia coli are sorted to the outer membrane through a pathway composed of five Lol proteins. LolA transports lipoproteins released from the inner membrane by LolCDE to LolB on the outer membrane via the periplasm. Interaction between LolA and LolB was speculated to be strong when LolA binds lipoprotein.

Dissection of LolB function--lipoprotein binding, membrane targeting and incorporation of lipoproteins into lipid bilayers.

Escherichia coli cells express at least 90 species of lipoprotein. LolB is one of the essential outer membrane lipoproteins, being involved in the last step of lipoprotein sorting. It accepts lipoproteins from a periplasmic molecular chaperone, LolA, and mediates the outer membrane anchoring of lipoproteins through a largely unknown mechanism.

Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides.

Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits.