A major role of the outer membrane (OM) of Gram-negative bacteria is to provide a protective permeability barrier for the cell, and proper maintenance of the OM is required for cellular viability. OM biogenesis requires the coordinated assembly of constituent lipids and proteins via dedicated OM assembly machineries. We have previously shown that, in Escherichia coli, the multicomponent YaeT complex is responsible for the assembly of OM ␤-barrel proteins (OMPs). This complex contains the OMP YaeT and three OM lipoproteins. Here, we report another component of the YaeT complex, the OM lipoprotein small protein A (SmpA). Strains carrying loss-of-function mutations in smpA are viable but exhibit defects in OMP assembly. Biochemical experiments show that SmpA is involved in maintaining complex stability. Taken together, these experiments establish an important role for SmpA in both the structure and function of the YaeT complex.

The outer membrane of most Gram-negative bacteria is made up of LPS, and in nearly all bacteria that contain LPS it is essential for the life of the organism. The lipid portion of this molecule, lipid A, also known as endotoxin, is a potent activator of the innate immune response. More than 50 genes are required to synthesize LPS and assemble it at the cell surface. Enormous progress has been made in elucidating the structure and biosynthesis of LPS, but until recently the cellular components required for its transport from its site of synthesis in the inner membrane to its final cellular location at the cell surface remained elusive. Here we describe the identification of a protein complex that functions to assemble LPS at the surface of the cell. This complex contains two proteins: Imp, already identified as an essential outer-membrane protein implicated in LPS assembly; and another protein, RlpB, heretofore identified only as a rare lipoprotein. We show that RlpB is also essential for cell viability and that the Imp͞RlpB complex is responsible for LPS reaching the outer surface of the outer membrane.essential lipoprotein ͉ Gram-negative bacteria ͉ outer-membrane biogenesis T he cell envelope of Gram-negative bacteria such as Escherichia coli is composed of the inner (cytoplasmic) membrane (IM), the outer membrane (OM), and the periplasmic space in between, where the bacterial peptidoglycan (cell wall) is located. The OM is an asymmetric lipid bilayer with phospholipids forming the inner leaf let and LPS forming the outer leaf let (1). ␤-Barrel OM proteins (OMPs) are inserted into the OM whereas lipoproteins are anchored to the inner leaf let of the OM through posttranslationally attached lipid moieties. The OM serves as a permeability barrier that protects the cells against toxic compounds such as antibiotics and detergents in their environment (2).The components of the OM (proteins and lipids) are synthesized inside the cell or at the inner leaflet of the IM. They need to be transported to and assembled at the OM in the correct orientation to maintain this barrier function during cell growth and division. Proteins involved in transporting these components across the IM have been identified and characterized (3-5). Much less is known, however, about how the OM components are transported across the aqueous periplasmic space and inserted into the OM. The recently identified Lol system targets lipoproteins to the OM through a periplasmic carrier protein, LolA, and an OM receptor, LolB (6, 7). Periplasmic factors involved in OMP folding have also been identified (8, 9). However, the mechanism(s) of how these factors facilitate transport of the OMPs across the periplasmic space is not known. Recently, a multiprotein complex involved in OMP assembly was identified by using a combination of genetic and biochemical approaches (10-12). This complex contains an OMP, YaeT, and three previously uncharacterized lipoproteins, YfgL, YfiO, and NlpB.An essential gene imp was recently shown to be involved in OM biogenesis. In ...

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.ABC transporter ͉ bioinformatics ͉ endosymbiont ͉ membrane biogenesis T he hallmark of Gram-negative bacteria is the presence of two extracytoplasmic membranes: the inner and outer membranes. The inner membrane (IM), which surrounds the cytoplasm, is separated from the outer membrane (OM) by an aqueous compartment known as the periplasm (1, 2). The IM is composed of phospholipids, integral transmembrane (TM) proteins that span the IM with ␣-helical TM domains, and lipoproteins (3, 4). In contrast, the OM is typically an asymmetric lipid bilayer where the inner leaflet is composed of phospholipids and the outer leaflet is composed mainly of lipopolysaccharide (LPS) (5). In addition, the OM contains lipoproteins and integral outer membrane proteins (OMPs), most of which span the OM via antiparallel ␤-sheets that fold into ␤-barrels (4, 6). Although some Gram-negative bacteria lack LPS and in others LPS is not essential (7), LPS is essential in Escherichia coli, Salmonella, and probably many other bacteria.In E. coli, the main function of the OM is to serve as a selective permeability barrier against many toxic chemicals, such as detergents and antibiotics (1). Porin proteins in the OM control permeability to hydrophilic molecules, but unlike typical phospholipid bilayers, the OM is quite impermeable to hydrophobic molecules, mainly because of LPS (1). For the OM to serve as a barrier, all OM components must be assembled properly. None of the components of the OM are synthesized in situ, so they must be transported to the OM from their site of synthesis. All OMPs and lipoproteins are made in the cytoplasm and cross the IM through the Sec translocon (3). After their signal sequence is removed, OMPs are thought to travel across the periplasm aided by chaperones that deliver them to the OM Bam complex (␤-barrel a...

The viability of Gram-negative organisms is dependent on the proper placement of lipopolysaccharide (LPS) in the outer leaflet of its outer membrane. LPS is synthesized inside the cell and transported to the surface by seven essential Lpt proteins. How these proteins cooperate to transport LPS is unknown. We show that these Lpt proteins can be found in a membrane fraction that contains inner and outer membranes, and that they co-purify. This constitutes the first evidence that the Lpt proteins form a trans-envelope complex. We suggest that this protein bridge provides a route for LPS transport across the cell envelope.

The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.outer membrane | Bam complex | β-barrel | protein folding T he assembly of β-barrel membrane proteins into the outer membrane (OM) of Gram-negative bacteria, mitochondria, and chloroplasts is facilitated by conserved cellular machinery (1-4). The β-barrel assembly machine (Bam) folds and inserts integral membrane proteins into the OM of Gram-negative organisms (5). Bam is a five-protein complex consisting of the essential protein BamA, a β-barrel itself, and four lipoproteins, BamB, -C, -D, and -E, of which only BamD is essential (4-8). The Bam complex recognizes a large number of different substrates, but how each component catalyzes the folding and insertion of such structurally diverse substrates is unclear.How β-barrels are assembled into membranes is not obvious. Where and how folding occurs is unclear because intermediates could contain both exposed polar amides and hydrophobic residues until the barrel has completed its fully hydrophobic exterior. By contrast, α-helical membrane proteins have internally satisfied hydrogen bonds, making stepwise assembly from stable secondary structural elements possible. Although Bam has been shown to accelerate membrane β-barrel assembly (9-11), the transient nature of folding intermediates has made accumulating such discrete species for characterization difficult (12-15). If structurally defined folding intermediates were to exist long enough for characterization, they could reveal crucial aspects of the folding process.Here, we studied the assembly of an essential, slow-folding β-barrel, LptD. LptD is one of two components of the OM translocon that transports lipopolysaccharide to the cell surface (16-18). The other component, LptE, is a lipoprotein that forms a plug inside the LptD barrel (19)(20)(21)(22). LptD also contains two disulfide bonds (23), and its assembly involves the formation of consecutive disulfide bonds that after barrel folding rearrange to form nonconsecutive disulfide bonds (24). The assembly of LptD is orders-ofmagnitude slower (∼20 min versus seconds) than that of other barrel substrates (24-26). Because of the slow rate of folding and our ability to use oxidation sta...