Domain activities of PapC usher reveal the mechanism of action of an Escherichia coli molecular machine

P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesin-bound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTD-Plug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.     

Insights into pilus assembly and secretion from the structure and functional characterization of usher PapC

Ushers constitute a family of bacterial outer membrane proteins responsible for the assembly and secretion of surface organelles such as the pilus. The structure at 3.15-Å resolution of the usher pyelonephritis-associated pili C (PapC) translocation domain reveals a 24-stranded kidney-shaped β-barrel, occluded by an internal plug domain. The dimension of the pore allows tandem passage of individual folded pilus subunits in an upright pilus growth orientation, but is insufficient for accommodating donor strand exchange. The molecular packing revealed by the crystal structure shows that 2 PapC molecules in head-to-head orientation interact via exposed β-strand edges, which could be the preferred dimer interaction in solution. In vitro reconstitution of fiber assemblies suggest that PapC monomers may be sufficient for fiber assembly and secretion; both the plug domain and the C-terminal domain of PapC are required for filament assembly, whereas the N-terminal domain is mainly responsible for recruiting the chaperone–subunit complexes to the usher. The plug domain has a dual function: gating the β-pore and participating in pilus assembly.     

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.The outer membrane of gram-negative bacteria is decorated with a potent endotoxin (called lipid A), which plays a significant role in bacterial pathogenicity and immune evasion (1). It also acts as a physical barrier protecting the cell from chemical attack and represents a significant obstacle for the effective delivery of numerous antimicrobial agents (2, 3). The outer membrane is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet (4). Phospholipids consist of a glycerol molecule, a phosphate group, and two fatty acid moieties (except for cardiolipins) (5) (see reviews (5, 6) and SI Appendix for the biosynthesis and regulation of phospholipids). LPS, on the other hand, contains three distinct components: lipid A, core oligosaccharides, and O-antigen (7, 8). Lipid A is the sole essential component of LPS, and its biosynthesis involves nine enzyme-catalyzed reactions (8). The lipid A pathway has been widely investigated, and we recently produced a pathway model that incorporates all of the known regulatory mechanisms (9). Briefly, the first reaction step catalyzed by LpxA is highly unfavorable, which makes the proceeding enzyme, LpxC, the first committed enzyme (10). LpxC is regulated by the protease FtsH (11, 12), and we recently postulated that the negative feedback signal arises from lipid A disaccharide, the substrate for LpxK (9). Furthermore, FtsH regulates WaaA (formerly called KdtA), an enzyme downstream of LpxC (13). The exact rationale for WaaA regulation remains unknown.A wealth of research exists for either LPS or phospholipids biosynthesis; however, our current understanding on the crosstalk between both pathways is limited at the moment. Because both pathways are synchronized to ensure a proper balance of membrane components (11, 14), studies underpinning the underlying mechanisms would appear valuable. There are a number of experimental findings that indicate the existence of strong links between both biosynthetic pathways (11, 15, 16). Thus, in the context of outer membrane biogenesis, the role involving phospholipids cannot be ignored in the study of LPS regulation. Furthermore, during membrane synthesis, ∼20 million molecules of fatty acids are synthesized in Escherichia coli (8). Yu et al. (17) reconstituted an in vitro steady-state kinetic system of fatty acid biosynthesis using purified enzymes and observed that the maximum fatty acid production rate obtainable was 100 µM/min. This production rate falls far below the amount of fatty acids required by a cell in vivo [if one assumes a cell volume of 6.7 × 10⁻¹⁶ L (18) and a generation time of 30 min (19)]. Therefore, to test the consistency of reported in vitro parameters and investigate the role of the biosynthetic enzymes on fatty acids turnover rate, a “systems” approach is necessary. Similarly, ever since the regulation of WaaA by FtsH was first reported (13), no study has investigated the underlying regulatory mechanism to date. This would also appear important because under wild-type conditions, WaaA catalyzes a step that is required for the endotoxic activity of lipid A (20).In this work, we present a detailed picture of the crosstalk between the LPS and phospholipids biosynthetic machinery. Our work involves a computational kinetic model spanning 81 chemical reactions and involving 90 chemical species. Additionally, we used a series of E. coli fatty acid biosynthesis mutants to investigate the effect of substrate flux into the saturated and unsaturated fatty acid pathway on LpxC stability. Our complete model agrees qualitatively with published datasets and with our own experiments. Our results imply that the catalytic activation of LpxK is dependent on unsaturated fatty acids. Furthermore, our experimental investigations have implicated a secondary protease involved in LpxC regulation. Finally, we have provided experimental evidence to explain the rationale for WaaA regulation.     

Structural basis of chaperone self-capping in P pilus biogenesis

PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits.     

Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous assemblies composed of multiple copies of ∼30 different proteins called nucleoporins. To unravel the basic scaffold underlying the NPC, we have characterized the species-specific scaffold nucleoporin Nup37 and ELY5/ELYS. Both proteins integrate directly via Nup120/160 into the universally conserved heptameric Y-complex, the critical unit for the assembly and functionality of the NPC. We present the crystal structure of Schizosaccharomyces pombe Nup37 in complex with Nup120, a 174-kDa subassembly that forms one of the two short arms of the Y-complex. Nup37 binds near the bend of the L-shaped Nup120 protein, potentially stabilizing the relative orientation of its two domains. By means of reconstitution assays, we pinpoint residues crucial for this interaction. In vivo and in vitro results show that ELY5 binds near an interface of the Nup120–Nup37 complex. Complementary biochemical and cell biological data refine and consolidate the interactions of Nup120 within the current Y-model. Finally, we propose an orientation of the Y-complex relative to the pore membrane, consistent with the lattice model.     

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.     

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular (“passenger”) domain that folds into a β-helical structure and a C-terminal β-barrel (“β”) domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.The survival of bacterial pathogens in a host environment requires the transport of virulence factors to the cell surface and beyond. One of the most commonly used secretion pathways in Gram-negative pathogens is the autotransporter (type Va) pathway (1). Autotransporters consist of a signal peptide, an N-terminal extracellular (“passenger”) domain that typically mediates a virulence function, and a C-terminal β-barrel (“β”) domain that resides in the outer membrane (OM). Passenger domains are highly divergent in sequence but generally adopt a characteristic β-helical structure (2). The size of passenger domains is also variable, but they often exceed 100 kDa. β-domains are ∼30 kDa in size, and although they are also divergent in sequence, they fold into nearly superimposable 12-stranded β-barrel structures (3–5). After autotransporters are translocated across the inner membrane (IM) via the Sec pathway they interact with chaperones that presumably keep them in an assembly-competent state in the periplasm (6–8). Subsequently they interact with the Bam complex, an essential heterooligomer that catalyzes the insertion of β-barrel protein into the OM (7–12). Following their translocation across the OM, many passenger domains are released from the cell surface by one of several distinct proteolytic processing mechanisms (13).Although it has been established that passenger domain translocation proceeds in a C- to N-terminal direction (7, 14), the mechanism of translocation has remained enigmatic. It was originally proposed that autotransporters contain all of the functional elements needed to mediate passenger domain secretion (15). At first glance, this hypothesis seems to be supported by X-ray crystallographic studies showing that the passenger and β-domains are linked by an α-helical segment that traverses the β-barrel pore (3–5). The same studies, however, show that the β-domain pore is only ∼10 Å in diameter. Given that the directionality of translocation presumably requires the formation of a C-terminal hairpin followed by the progressive sliding of more N-terminal segments past a static strand, both strands of the hairpin would need to be in a fully extended conformation. Small folded polypeptides, however, have been shown to be secreted efficiently by the autotransporter pathway, and some naturally occurring passenger domains acquire at least limited tertiary structure in the periplasm (16, 17). Furthermore, an α-helical segment likely resides inside the β-domain pore before the completion of translocation (18, 19). Finally, a component of the Bam complex (BamA) has been shown to interact with the passenger domain during the translocation reaction (7). Although the β-domain does appear to play a role in translocation (20, 21), available evidence suggests that the Bam complex promotes the membrane insertion of the β-domain and the secretion of the passenger domain in a concerted reaction (7, 8).The energy source for passenger domain translocation has likewise remained unclear. Although protein translocation reactions generally require the input of external energy in the form of either ATP hydrolysis or a membrane potential, there is no ATP in the periplasm, electrochemical gradient across the OM, or obvious connection to the protonmotive force across the IM. To explain the energetics of autotransporter secretion it has been proposed that the progressive folding of small segments of the passenger domain in the extracellular space drives translocation forward while preventing retrograde movement into the periplasm (2, 22). This vectorial folding model is supported by the finding that mutations that impair the folding of C-terminal segments of two different autotransporter passenger domains significantly reduce the efficiency of secretion (19, 23). It should be noted, however, that an examination of the energetics of other types of protein translocation reactions has shown that moving proteins across membranes is often extremely costly. The equivalent of >10,000 ATP molecules is required to move a protein from the chloroplast stroma into the thylakoid lumen (24), and in the absence of a protonmotive force ∼5,000 ATP molecules are required to move a protein through the bacterial Sec machinery (25). Even the transport of proteins across the chloroplast envelope, which is considerably less costly, requires ∼650 ATP molecules (26). Given that even this lower value represents a ΔG = 30,000 kJ/mol whereas the ΔG associated with protein folding is typically in the ∼20–60 kJ/mol range (27), it seems likely that the folding of the passenger domain does not fully account for the energetics of secretion.In this study, we tested the vectorial folding hypothesis by examining the fate of a conditionally disordered chimeric passenger domain. We fused the receptor-binding domain (RD) of the Bordetella pertussis toxin CyaA to a C-terminal fragment of EspP, an Escherichia coli O157:H7 autotransporter whose passenger domain undergoes proteolytic processing. CyaA is an ∼177-kDa toxin that is normally secreted through a type I pathway (28). The 675-residue RD consists of ∼40–45 glycine- and aspartate-rich repeat in toxin (RTX) motifs that are involved in calcium binding (29). In the absence of calcium the CyaA RD does not fold into a well-defined 3D structure, but instead exists in a premolten globule ensemble of conformations (30). Like other intrinsically disordered polypeptides it contains a high content of random coils and is highly hydrated (30–32). Upon binding calcium, the CyaA RD folds into a stable β-roll structure that facilitates interaction with a cell surface receptor and the translocation of an N-terminal adenylate cyclase domain across the plasma membrane (32–36). We found that the chimeric passenger domain was secreted efficiently even in the absence of calcium and thereby showed that translocation can proceed efficiently without passenger domain folding. Furthermore, site-directed mutagenesis experiments together with an analysis of naturally occurring passenger domain sequences unmasked a potentially important role of protein charge in the translocation reaction. Finally, by examining the secretion of a truncated version of the chimeric passenger domain in the presence of calcium we confirmed that small folded polypeptides can be secreted via the autotransporter pathway.     

Phase separation in the outer membrane of Escherichia coli

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Diderm bacteria, such as Escherichia coli, are surrounded by an outer membrane (OM) that protects cells against the immune systems of plants and animals, contributes to the mechanical stability of the cell, and excludes many classes of antibiotics, thereby contributing to antimicrobial resistance (1, 2). The OM is comprised of an asymmetric bilayer of phospholipids in the inner leaflet, lipopolysaccharides (LPS) in the outer leaflet, and many outer-membrane proteins (OMPs). OMPs are hugely diverse β-barrel proteins that can be present at hundreds to hundreds of thousands of copies per cell (3). They have been shown to be relatively static (4), probably due to promiscuous protein–protein interactions and binding of LPS that exists in a slow-moving, liquid-crystalline state (5, 6). Using fluorescent labels, some OMPs have been shown to cluster into supramolecular islands of ∼0.3- to 0.5-μm sizes (4, 7–9). However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane.To address this fundamental question, we have imaged the entire surface of live and metabolically active bacteria at nanometer resolution using atomic force microscopy (AFM). Applying such large-scale, high-resolution imaging on engineered E. coli strains and complementing it by specific labeling of abundant OMPs, we identify large-scale and near-static protein-rich networks interspersed with nanoscale domains that are enriched in LPS. Key components of the protein-rich networks are abundant trimeric porins such as OmpF, in addition to (the monomeric) OmpA, which forms noncovalent interactions to the underlying cell wall (10). By contrast, no significant protein content is detected in the LPS-rich domains, which are also found to grow and merge with other patches during cell elongation. When the LPS–phospholipid asymmetry of the OM is perturbed by mislocalization of phospholipids to the outer leaflet (11), we find deformation of the membrane rather than expansion of LPS patches, indicating the appearance of a new, phospholipid-enriched phase at the bacterial surface.     

Regulation of cell size in response to nutrient availability by fatty acid biosynthesis in Escherichia coli

Cell size varies greatly among different types of cells, but the range in size that a specific cell type can reach is limited. A long-standing question in biology is how cells control their size. Escherichia coli adjusts size and growth rate according to the availability of nutrients so that it grows larger and faster in nutrient-rich media than in nutrient-poor media. Here, we describe how, using classical genetics, we have isolated a remarkably small E. coli mutant that has undergone a 70% reduction in cell volume with respect to wild type. This mutant lacks FabH, an enzyme involved in fatty acid biosynthesis that previously was thought to be essential for the viability of E. coli. We demonstrate that although FabH is not essential in wild-type E. coli, it is essential in cells that are defective in the production of the small-molecule and global regulator ppGpp. Furthermore, we have found that the loss of FabH causes a reduction in the rate of envelope growth and renders cells unable to regulate cell size properly in response to nutrient excess. Therefore we propose a model in which fatty acid biosynthesis plays a central role in regulating the size of E. coli cells in response to nutrient availability.     

Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea, morbidity, and mortality in low- and middle-income countries (1), while enterohemorrhagic E. coli (EHEC) is a major cause of food poisoning in industrial countries (2). The ability of EPEC and EHEC to colonize the intestinal epithelium is encoded on a pathogenicity island named the locus of enterocyte effacement, which encodes gene regulators, the outer membrane adhesin intimin, structural components of a type III secretion system (T3SS), translocon components, chaperones, effectors, and an ATPase, which energizes protein translocation (3).T3SS is a common virulence factor among Gram-negative pathogens of humans, animals, and plants, including Salmonella enterica serovars, Shigella, Chlamydia, and Yersinia spp., Pseudomonas aeruginosa, and Pseudomonas syringae (4). The overall architecture of the multisubunit T3SS injectisome, which spans the entire cell envelope, is highly conserved among the different pathogens. It comprises several substructures, including a cytosolic C-ring and an ATPase complex (EscN in EPEC), a basal body consisting of a series of ring structures embedded in the bacterial inner and outer membranes (including a Secretin, EscC) and a periplasmic rod (EscI) which connects the inner membrane rings with a hollow extracellular needle projection (EscF). In EPEC, the EscF needle is 8–9 nm in diameter and 23 nm in length (5–7). In most pathogenic bacteria having a T3SS, the function of the needle is to connect the basal body to a translocation pore in the plasma membrane of the eukaryotic cell (3, 4).Electron microscopic observations of EPEC and EHEC have shown an ∼12-nm-diameter helical tube, made of the secreted translocator protein EspA (8–10), which serves as an extension to the needle, enclosing a central channel of ∼25 Å diameter (11, 12). Around 12 EspA filaments are elaborated on individual EPEC bacteria (8); when grown in vitro, EspA can vary in length and can reach 600 nm (7). Our current understanding is that the EPEC and EHEC EspA filaments evolved as an adaptation to their environment, where the needle alone would not be long enough to traverse the intestinal mucus layer. EspA filaments, like the needle, share similar helical symmetry parameters with flagellar structures and are elongated by addition of EspA subunits to the tip of the growing filament, the same mode of elongation that occurs in flagella filaments (13). Functionally, the filaments form a long flexible helical conduit which connects the tip of the needle with the translocation pore (made in EPEC by EspB and EspD), thus mediating effector translocation (3). Indeed, EspA has been shown to interact with both EspB and EspD (14, 15). In addition to their protein translocation activity, EspA filaments are important adhesins, mediating binding to both epithelial cells and edible leaves (16, 17). In the absence of EspA filaments, effectors can be secreted but not translocated; accordingly, the virulence of an espA deletion is highly attenuated in animal models (18). Moreover, EspA filaments are major antigens in vivo; antibodies against EspA were found in both human colostrum of mothers in Brazil and in serum from culture-positive patients infected with EHEC (19, 20). In animal models, IgG antibodies against EspA play a major role in clearing the pathogen (21).EspA alone is sufficient to form filamentous structures (22). Similarly to flagellar biosynthesis (23), EspA coiled-coil interactions between N- and C-terminal α-helical segments are required for assembly of the filament (24, 25). Monomeric EspA subunits are maintained in the cytosol via interactions with the chaperone CesAB (26), which has also been called CesA (22). CesAB is essential for stability of EspA within the bacterial cell prior to secretion. A cesAB deletion cannot secrete EspA or assemble EspA filaments (26). Crystallographic analysis of the CesAB–EspA complex at 2.8 Å resolution (22) showed that the EspA α-helices are also involved in extensive coiled-coil interactions with CesAB (22). Due to disorder in the parts of EspA not directly interacting with CesAB, only 72 of the 192 EspA residues were visualized in this complex. Importantly, the ATPase EscN selectively interacts with the CesAB–EspA complex; abrogation of this interaction attenuates EspA secretion and infection (27).Like flagella, EspA filaments show antigenic polymorphism, as EspA from different EPEC and EHEC clones show no immunological cross-reactivity (10). We have previously identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope (28). By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments (26). As with the Salmonella flagellin D3 domain (29), which is known to tolerate insertions of natural and artificial amino acid sequences (30), we were able to insert short peptides into the surface-exposed, hypervariable region of EspA (28).While EspA was first identified in 1996 (31), and the EspA filaments were first described in 1998 (8), low-resolution structures (at ∼15–25 Å resolution) were reported about 15 y ago (11, 12). The aim of this study was to obtain a high-resolution cryoelectron microscopy (cryo-EM) structure of the EPEC EspA filament.     

Structural basis for substrate specificity in the Escherichia coli maltose transport system

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK₂ bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK₂ despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK₂ contribute to the overall substrate selectivity of the system.The ATP-binding cassette (ABC) transporter family contains more than 2,000 members sharing a common architecture of two transmembrane domains (TMDs) that form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that hydrolyze ATP (1). Importers found in prokaryotes require additional soluble proteins that bind substrates with high affinity and deliver them to the TMDs. Some ABC transporters recognize only a single substrate, whereas others are more promiscuous. For example, ABC transporters that secrete toxins, hydrolytic enzymes, and antibiotic peptides are dedicated to one specific substrate (2), but in contrast, the multidrug transporter P-glycoprotein interacts with more than 200 chemically diverse compounds (3). MRP1, ABCG2, and TAP also have broad substrate spectra (2).Regardless of substrate specificity, the ATPase activity of ABC transporters is regulated by the presence of substrates. Thus, substrate binding must generate a signal that enables ATP hydrolysis. Understanding how ABC transporters interact with their substrates has been a major challenge in the field.A controversial issue in the ABC transporter field is whether the transmembrane components contain a well-defined substrate-binding site. It has been suggested that for binding protein-dependent ABC transporters, substrate specificity is defined exclusively by the binding protein, which interacts with the substrate with high affinity. The transmembrane components act as a nonspecific pore for substrate to diffuse through the membrane (4). However, for the Escherichia coli maltose transporter, it has been well established that the selectivity of sugar binding to the maltose-binding protein (MBP) does not fully account for the selectivity of sugar transport. For example, cyclic maltodextrins, maltodextrins containing more than seven glucosyl units, and maltose analogs with a modified reducing end are not transported despite their high-affinity binding to MBP (5, 6). Further evidence for selectivity through the ABC transporter MalFGK₂ itself comes from mutant transporters that function independently of MBP. In the absence of MBP, these mutants constitutively hydrolyze ATP and specifically transport maltodextrins (7, 8).In this study, we determined the crystal structures of the maltose transport complex MBP-MalFGK₂ bound with large maltodextrin in two conformational states. The determination of these structures, along with previous studies of maltoporin and MBP, allow us to define how overall substrate specificity is achieved for the maltose transport system.     

Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic Escherichia coli

Signal peptides (SPs) are critical for protein transport across cellular membranes, have a highly conserved structure, and are cleaved from the mature protein upon translocation. Here, we report that naturally occurring mutations in the SP of the adhesive, tip-associated subunit of type 1 fimbriae (FimH) are positively selected in uropathogenic Escherichia coli. On the one hand, these mutations have a detrimental effect, with reduced FimH transport across the inner membrane, fewer FimH and fimbriae expressed on the bacterial surface, and decreased bacterial adhesion under flow conditions. On the other hand, the fimbriae expressed by the mutants are significantly longer on average, with many fimbriae able to stretch to >20 μm in length. More surprisingly, the SP mutant bacteria display an increased ability to resist detachment from the surface upon a switch from high to low flow. This functional effect of longer fimbriae highlights the importance of the nonadhesive fimbrial rod for adhesive function. Also, whereas bacterial adhesion to bladder epithelial cells was preserved in most mutants, binding to and killing by human neutrophils was decreased, providing an additional reason the SP mutations are relatively common among uropathogenic strains. Thus, this study demonstrates how mutations in an SP, while decreasing transport function and not affecting the final structure of the translocated protein, can lead to functional gains of the extracellular organelles that incorporate the protein and overall adaptive changes in the organism's fitness.     

Periplasmic orientation of nascent lipid A in the inner membrane of an Escherichia coli LptA mutant

The core-lipid A domain of Escherichia coli lipopolysaccharide (LPS) is synthesized on the inner surface of the inner membrane (IM) and flipped to its outer surface by the ABC transporter MsbA. Recent studies with deletion mutants implicate the periplasmic protein LptA, the cytosolic protein LptB, and the IM proteins LptC, LptF, and LptG in the subsequent transport of nascent LPS to the outer membrane (OM), where the LptD/LptE complex flips LPS to the outer surface. We have isolated a temperature-sensitive mutant (MB1) harboring the S22C and Q111P substitutions in LptA. MB1 stops growing after 30 min at 42°C. ³²P_i and [³⁵S]methionine labeling show that export of newly synthesized phospholipids and proteins is not severely impaired, but export of LPS is defective. Using the lipid A 1-phosphatase LpxE as a periplasmic IM marker and the lipid A 3-O-deacylase PagL as an OM marker, we show that core-lipid A reaches the periplasmic side of the IM at 42°C in MB1 but not the outer surface of the OM. Electron microscopy of MB1 reveals dense periplasmic material and a smooth OM at 42°C, consistent with a role for LptA in shuttling LPS across the periplasm.     

Organization and coordinated assembly of the type III secretion export apparatus

Type III protein secretion systems are unique bacterial nanomachines with the capacity to deliver bacterial effector proteins into eukaryotic cells. These systems are critical to the biology of many pathogenic or symbiotic bacteria for insects, plants, animals, and humans. Essential components of these systems are multiprotein envelope-associated organelles known as the needle complex and a group of membrane proteins that compose the so-called export apparatus. Here, we show that components of the export apparatus associate intimately with the needle complex, forming a structure that can be visualized by cryo-electron microscopy. We also show that formation of the needle complex base is initiated at the export apparatus and that, in the absence of export apparatus components, there is a significant reduction in the levels of needle complex base assembly. Our results show a substantial coordination in the assembly of the two central elements of type III secretion machines.     

A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium

Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.     

Conformational pathways in the gating of Escherichia coli mechanosensitive channel

The pathway of the gating conformational transition of Escherichia coli mechanosensitive channel was simulated, using the recently modeled open and closed structures, by targeted molecular dynamics method. The transition can be roughly viewed as a four-stage process. The initial motion under a lower tension load is predominantly elastic deformation. The opening of the inner hydrophobic pore on a higher tension load takes place after the major expansion of the outer channel dimension. The hypothetical N-terminal S1 helical bundle has been confirmed to form the hydrophobic gate, together with the M1 helices. The sequential breaking of the tandem hydrophobic constrictions on the M1 and S1 helices makes the two parts of the gate strictly coupled, acting as a single gate. The simulation also revealed that there is no significant energetic coupling between the inner S1 bundle and the outer M2 transmembrane helices. The molten-globular-like structural features of the S1 bundle in its intermediate open states may account for the observed multiple subconductance states. Moreover, the intermediate open states of mechanosensitive channels are not symmetric, i.e., the opening does not follow iris-like motion, which sharply contrasts to the potassium channel KcsA.     

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.     

Escherichia coli as a cause of diarrhea

Escherichia coli is the best-known member of the normal microbiota of the human intestine and a versatile gastrointestinal pathogen. The varieties of E. coli that cause diarrhea are classified into named pathotypes, including enterotoxigenic, enteroinvasive, enteropathogenic and enterohemorrhagic E. coli. Individual strains of each pathotype possess a distinct set of virulence-associated characteristics that determine the clinical, pathological and epidemiological features of the diseases they cause. In the present brief review, we summarize the key distinguishing features of the major pathotypes of diarrheagenic E. coli. Knowledge of the pathogenic mechanisms of these bacteria has led to the development of rational interventions for the treatment and prevention of E. coli-induced diarrhea. In addition, investigations into E. coli virulence are providing useful insights into the origins and evolution of bacterial pathogens more generally.     

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.Although proteins localized to biological membranes constitute about a quarter of the products of an organism’s ORFs, only 0.1% of known protein structures are membrane proteins (1, 2). The lag in our knowledge of the physiology and structural biology of membrane proteins is due to many factors including the low expression levels of membrane proteins and the complex biophysical nature of their interactions at the water/lipid interface. Membrane proteins, which have one or more segments that traverse the lipid bilayer, require dedicated cellular machineries for their targeting to and integration into the membranes (3). Even though these cellular machineries have maintained conserved features in all domains of life, efforts to express membrane proteins in foreign organisms have often been unsuccessful. In many cases, the overexpression of indigenous membrane proteins leads to mis-targeting and inclusion body formation, protein degradation, or cell death (4).Promoting the correct orientation of proteins within the membrane provides an additional challenge to the insertion machinery. Proper assembly is a crucial step in the folding pathway of a membrane protein because a wrong orientation could be detrimental to functioning of the protein and to the viability of the cell. The overall architecture of a membrane protein, including its final membrane topology, is achieved by topogenic signals embedded in the amino acid sequence. Such signals include long stretches of mostly hydrophobic amino acids (around 20) that determine the portion of the protein to be embedded in the membrane. Furthermore, the abundance of positively charged amino acids in cytoplasmic domains of membrane proteins, generally referred to as the positive-inside rule, is a major contributor to membrane protein topology (5, 6).In Escherichia coli, the direct insertion of proteins into the inner membrane is mediated by the translocon protein complex SecYEG and/or the insertase YidC (7). Although the majority of inner membrane proteins tested require SecYEG for assembly, fewer are dependent on YidC for integration into the membrane (8). Nevertheless, both integration machineries are essential for E. coli viability. A previous study presented evidence that some proteins dependent on the YidC protein for proper assembly are ones that exhibit violations of the positive-inside rule (9). More recent work has also suggested that both decreasing transmembrane helix (TMH) hydrophobicity and increasing polarity, particularly the positive charge of the periplasmic loop of a model protein, increases dependence on YidC (10).In this paper, we describe a genetic selection for mutants of E. coli that promote the efficient membrane integration and expression of a foreign membrane protein, one that is ordinarily barely expressed in a wild-type E. coli. These studies arose out of our interest in protein disulfide bond formation in bacteria. The formation of disulfide bonds in E. coli depends on two enzymes: DsbA that directly catalyzes disulfide bond formation in protein substrates in the bacterial cell envelope and DsbB that maintains DsbA in the oxidized state (11). In contrast to E. coli, many bacteria (e.g., mycobacteria) use the membrane protein VKOR for oxidation of DsbA instead of DsbB (Fig. 1) (12). Despite the lack of homology of Mycobacterium tuberculosis VKOR (MtbVKOR) with DsbB, MtbVKOR, when cloned into E. coli, can substitute for the enzyme EcDsbB (12). Bacterial VKORs are so named because they were first identified as homologs of the mammalian integral membrane protein vitamin K epoxide reductase (VKORc1) involved in blood coagulation (12). VKORc1 is also the target of the widely used anticoagulant warfarin (13). Because we are carrying out high-throughput screening to identify compounds that might serve as leads for antibiotics against M. tuberculosis (14) we sought to express mammalian VKORc1 in E. coli as a simple counter-screen to exclude compounds that might be toxic to humans due to anticoagulant activity. However, when the mammalian VKORc1 was expressed from efficient E. coli promoters, it failed to substitute for EcDsbB. Therefore, we devised for this work a genetic selection for mutants that allowed functioning of VKORc1 in the E. coli disulfide bond-forming pathway and enhanced the expression of the protein VKORc1. The first mutations obtained resulted in amino acid substitutions that reduced the positive charge in a hydrophilic loop of VKORc1 (SI Appendix, Fig. S1) and gave measurable although weak restoration of disulfide bond formation. Then, after mutagenesis of an E. coli dsbB⁻ strain expressing one of these VKORc1 mutations, we identified and characterized chromosomal mutations that further enhanced the expression of VKORc1 function in disulfide bond formation. Mutations in the genes for the YidC protein and for the protease HslV were repeatedly isolated in independent selections. The yidC mutations mapped to a putative substrate-binding site in the insertase YidC, and the hslV mutations mapped to essential catalytic residues in HslV. The results reported here suggest that YidC may play a role in quality control of membrane proteins at the level of insertion. Alteration of this function can greatly enhance functional overexpression of other membrane proteins. Our findings shed light on the problems leading to the dysfunctional expression of at least some foreign membrane proteins in E. coli and on the mechanism of YidC-mediated membrane protein insertion and quality control. Our results also allow new potential approaches to studying the mechanism of action of VKORc1 and to obtaining inhibitors of this blood coagulation protein.Open in a separate windowFig. 1.Disulfide bond formation in E. coli is promoted by EcDsbB and MtbVKOR but not by vertebrate VKORc1.