The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs).
UNLABELLED: The seven human sirtuins are a family of ubiquitously expressed and evolutionarily conserved NAD(+)-dependent deacylases/mono-ADP ribosyltransferases that regulate numerous cellular and organismal functions, including metabolism, cell cycle, and longevity. Here, we report the discovery that all seven sirtuins have broad-range antiviral properties. We demonstrate that small interfering RNA (siRNA)-mediated knockdown of individual sirtuins and drug-mediated inhibition of sirtuin enzymatic activity increase the production of virus progeny in infected human cells. This impact on virus growth is observed for both DNA and RNA viruses. Importantly, sirtuin-activating drugs inhibit the replication of diverse viruses, as we demonstrate for human cytomegalovirus, a slowly replicating DNA virus, and influenza A (H1N1) virus, an RNA virus that multiplies rapidly. Furthermore, sirtuin defense functions are evolutionarily conserved, since CobB, the sirtuin homologue in Escherichia coli, protects against bacteriophages. Altogether, our findings establish sirtuins as broad-spectrum and evolutionarily conserved components of the immune defense system, providing a framework for elucidating a new set of host cell defense mechanisms and developing sirtuin modulators with antiviral activity.
IMPORTANCE: We live in a sea of viruses, some of which are human pathogens. These pathogenic viruses exhibit numerous differences: DNA or RNA genomes, enveloped or naked virions, nuclear or cytoplasmic replication, diverse disease symptoms, etc. Most antiviral drugs target specific viral proteins. Consequently, they often work for only one virus, and their efficacy can be compromised by the rapid evolution of resistant variants. There is a need for the identification of host proteins with broad-spectrum antiviral functions, which provide effective targets for therapeutic treatments that limit the evolution of viral resistance. Here, we report that sirtuins present such an opportunity for the development of broad-spectrum antiviral treatments, since our findings highlight these enzymes as ancient defense factors that protect against a variety of viral pathogens.
RpoS (σ(38)) is required for cell survival under stress conditions, but it can inhibit growth if produced inappropriately and, consequently, its production and activity are elaborately regulated. Crl, a transcriptional activator that does not bind DNA, enhances RpoS activity by stimulating the interaction between RpoS and the core polymerase. The crl gene has two overlapping promoters, a housekeeping, RpoD- (σ(70)) dependent promoter, and an RpoN (σ(54)) promoter that is strongly up-regulated under nitrogen limitation. However, transcription from the RpoN promoter prevents transcription from the RpoD promoter, and the RpoN-dependent transcript lacks a ribosome-binding site. Thus, activation of the RpoN promoter produces a long noncoding RNA that silences crl gene expression simply by being made. This elegant and economical mechanism, which allows a near-instantaneous reduction in Crl synthesis without the need for transacting regulatory factors, restrains the activity of RpoS to allow faster growth under nitrogen-limiting conditions.
RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.
In gram-negative bacteria, integral outer membrane β-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam) complex. The essential components of this complex are the OMP BamA [which contains a carboxyl-terminal β-barrel and an amino-terminal periplasmic module composed of five polypeptide transport associated (POTRA) domains] and the lipoprotein BamD. In Escherichia coli, the Bam complex also contains three nonessential lipoproteins (BamBCE), all of which require the barrel-proximal POTRA domain (P5) for stable interactions with BamA. We have previously reported that the BamA β-barrel assumes two different conformations. A method for conformation-specific labeling of BamA described here reveals that these conformers reflect the degree of surface exposure of the conserved sixth extracellular loop (L6). L6 is surface accessible in one conformation but not in the other, likely because it occupies the lumen of the BamA β-barrel in the latter case. A gain-of-function mutation that promotes Bam activity (bamDR197L) and a loss-of-function mutation that decreases the activity of Bam (ΔbamE) both favor surface exposure of BamA L6, suggesting that BamD and BamE normally act to control L6 exposure through opposing functions. These results, along with the synthetic lethality of the bamDR197L ΔbamE double mutant, imply a cyclic mechanism in which the Bam lipoproteins regulate the conformation of BamA during the OMP assembly reaction. Our results further suggest that BamDE controls L6 exposure via conformational signals transmitted through P5 to L6.
It has recently been suggested that bactericidal antibiotics, including aminoglycoside antibiotics (AGAs), and toxic small molecules, such as hydroxyurea (HU), kill bacteria the same way, namely, by generating reactive oxygen species (ROS) via a process requiring activation of the Cpx stress response. We suggest an opposite, protective role for Cpx. We have confirmed the initial finding that cpxA null mutations confer resistance to HU. However, the two-component sensor CpxA is both a kinase and a phosphatase, and previous work from our lab has shown that removing CpxA can activate the stress response owing to buildup of the phosphorylated response regulator (CpxR∼P) that occurs in the absence of the phosphatase activity. We show that a dominant cpxA* mutation that constitutively activates the Cpx stress response confers a high level of resistance to both HU and AGAs in a CpxR-dependent manner. In contrast, inactivating the CpxR response regulator by mutating the phosphorylation site (D51A) or the putative DNA-binding motif (M199A) does not increase resistance to HU or AGAs. Taken together, these results demonstrate that activation of the Cpx stress response can protect cells from HU and AGAs. However, the Cpx response does not increase resistance to all classes of bactericidal antibiotics, as the cpxA* mutants are not significantly more resistant to fluoroquinolones or β-lactams than wild-type cells. Thus, it seems unlikely that all bactericidal antibiotics kill by the same mechanism.
Lipopolysaccharide (LPS) is the major outer leaflet constituent of the Gram-negative outer membrane (OM) bilayer. A bipartite protein complex of LptD and LptE assembles LPS into the OM. It has been established that LptE assists folding and assembly of its β-barrel partner LptD, yet reported biochemical evidence suggested additional LptE functions. Here, we isolated dominant negative lptE mutations, seeking to inform these functions. The lptE14 mutation increased OM permeability to erythromycin, even when the wild-type lptE gene was present. We show that the lptE14 mutation does not cause a defect in either LptD assembly or LPS export. A spontaneous IS1 insertion in secA suppressed lptE14 erythromycin sensitivity by removing the C-terminal SecB-binding domain of SecA. While this suppressor mutation broadly impeded SecB-dependent secretion of preproteins, we show that suppression was a direct and specific consequence of reduced LptD levels in the OM. We suggest that lptE14 causes poor plugging of the LptD β barrel and that a reduction of ineffectively plugged LptD-LptE14 complexes in the OM decreases permeability to erythromycin. Hence, lptE14 supports a proposed plug-and-barrel LptE-LptD arrangement.
The essential outer membrane β-barrel protein BamA forms a complex with four lipoprotein partners BamBCDE that assembles β-barrel proteins into the outer membrane of Escherichia coli. Detailed genetic studies have shown that BamA cycles through multiple conformations during substrate assembly, suggesting that a complex network of residues may be involved in coordinating conformational changes and lipoprotein partner function. While genetic analysis of BamA has been informative, it has also been slow in the absence of a straightforward selection for mutants. Here we take a bioinformatic approach to identify candidate residues for mutagenesis using direct coupling analysis. Starting with the BamA paralog FhaC, we show that direct coupling analysis works well for large β-barrel proteins, identifying pairs of residues in close proximity in tertiary structure with a true positive rate of 0.64 over the top 50 predictions. To reduce the effects of noise, we designed and incorporated a novel structured prior into the empirical correlation matrix, dramatically increasing the FhaC true positive rate from 0.64 to 0.88 over the top 50 predictions. Our direct coupling analysis of BamA implicates residues R661 and D740 in a functional interaction. We find that the substitutions R661G and D740G each confer OM permeability defects and destabilize the BamA β-barrel. We also identify synthetic phenotypes and cross-suppressors that suggest R661 and D740 function in a similar process and may interact directly. We expect that the direct coupling analysis approach to informed mutagenesis will be particularly useful in systems lacking adequate selections and for dynamic proteins with multiple conformations.
The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in Escherichia coli. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in E. coli, SurA, results in synthetic lethality. However, a Δskp mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a Δskp ΔfkpA double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the ΔsurA mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in E. coli. Other OMPs, including LamB, are less affected in the Δskp ΔfkpA and Δskp bamB::kan double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.
UNLABELLED: SurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) in Escherichia coli. Although SurA contains two well-conserved proline isomerase (PPIase) domains, the contribution of these domains to SurA function is unclear. In the present work, we show that defects in OMP assembly caused by mutation of the β-barrel assembly factors BamA or BamB can be corrected by gain-of-function mutations in SurA that map to the first PPIase domain. These mutations apparently bypass the requirement for a stable interaction between SurA and the Bam complex and enhance SurA chaperone activity in vivo despite destabilization of the protein in vitro. Our findings suggest an autoinhibitory mechanism for regulation of SurA chaperone activity through interdomain interactions involving a PPIase domain. We propose a model in which SurA activity is modulated by an interaction between SurA and the Bam complex that alters the substrate specificity of the chaperone.
IMPORTANCE: The dominant surA mutations described here alter amino acid residues that are highly conserved in eukaryotic homologs of SurA, including Pin 1, the human proline isomerase (PPIase) implicated in Alzheimer's disease and certain cancers. Consequently, a mechanistic description of SurA function may enhance our understanding of clinically important PPIases and their role(s) in disease. In addition, the virulence of Gram-negative bacterial pathogens, such as Salmonella, Shigella, and Escherichia coli O157:H7, is largely dependent on SurA, making this PPIase/chaperone an attractive antibiotic target. Investigating the function of SurA in outer membrane (OM) biogenesis will be useful in the development of novel therapeutic strategies for the disruption of the OM or the processes that are essential for its assembly.
The outer membrane (OM) of gram-negative bacteria such as Escherichia coli contains lipoproteins and integral β-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of β-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wild-type levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.
The bacterial outer membrane (OM) is an exceptional biological structure with a unique composition that contributes significantly to the resiliency of Gram-negative bacteria. Since all OM components are synthesized in the cytosol, the cell must efficiently transport OM-specific lipids and proteins across the cell envelope and stably integrate them into a growing membrane. In this review, we discuss the challenges associated with these processes and detail the elegant solutions that cells have evolved to address the topological problem of OM biogenesis. Special attention will be paid to the Bam machine, a highly conserved multiprotein complex that facilitates OM β-barrel folding. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Biogenesis of the outer membrane (OM) is an essential process in gram-negative bacteria. One of the key steps of OM biogenesis is the assembly of integral outer membrane beta-barrel proteins (OMPs) by a protein machine called the Bam complex. In Escherichia coli, the Bam complex is composed of the essential proteins BamA and BamD and three nonessential lipoproteins, BamB, BamC, and BamE. Both BamC and BamE are important for stabilizing the interaction between BamA and BamD. We used comprehensive genetic analysis to clarify the interplay between BamA and the BamCDE subcomplex. Combining a ΔbamE allele with mutations in genes that encode other OMP assembly factors leads to severe synthetic phenotypes, suggesting a critical function for BamE. These synthetic phenotypes are not nearly as severe in a ΔbamC background, suggesting that the functions of BamC and BamE are not completely overlapping. This unique function of BamE is related to the conformational state of BamA. In wild-type cells, BamA is sensitive to externally added proteinase K. Strikingly, when ΔbamE mutant cells are treated with proteinase K, BamA is degraded beyond detection. Taken together, our findings suggest that BamE modulates the conformation of BamA, likely through its interactions with BamD.
The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.
The master regulator of stationary phase in Escherichia coli, RpoS, responds to carbon availability through changes in stability, but the individual steps in the pathway are unknown. Here we systematically block key steps of glycolysis and the citric acid cycle and monitor the effect on RpoS degradation in vivo. Nutrient upshifts trigger RpoS degradation independently of protein synthesis by activating metabolic pathways that generate small energy molecules. Using metabolic mutants and inhibitors, we show that ATP, but not GTP or NADH, is necessary for RpoS degradation. In vitro reconstitution assays directly demonstrate that ClpXP fails to degrade RpoS, but not other proteins, at low ATP hydrolysis rates. These data suggest that cellular ATP levels directly control RpoS stability.
The lipoprotein Lpp is the most numerically abundant protein in Escherichia coli, has been investigated for over 40 years, and has served as the paradigmatic bacterial lipoprotein since its initial discovery. It exists in two distinct forms: a 'bound-form', which is covalently bound to the cell's peptidoglycan layer, and a 'free-form', which is not. Although it is known that the carboxyl-terminus of bound-form Lpp is located in the periplasm, the precise location of free-form Lpp has never been determined. For decades, it has been widely assumed that free-form Lpp is associated with bound-form. In this work, we show that the free and bound forms of Lpp are not largely associated with each other, but are found in distinct subcellular locations. Our results indicate that free-form Lpp spans the outer membrane and is surface-exposed, whereas bound-form Lpp resides in the periplasm. Thus, Lpp represents a novel example of a single lipoprotein that is able to occupy distinct subcellular locations, and challenges models in which the free and bound forms of Lpp are assumed to be associated with each other.
Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two-amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the β-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the β-barrel assembly machine.
The bacteria cell envelope is a complex multilayered structure that serves to protect these organisms from their unpredictable and often hostile environment. The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the gram-negatives. Threading through these layers of peptidoglycan are long anionic polymers, called teichoic acids. The composition and organization of these envelope layers and recent insights into the mechanisms of cell envelope assembly are discussed.
The Gram-negative bacterial envelope is bounded by two membranes. Disulfide bond formation and isomerization in this oxidizing environment are catalyzed by DsbA and DsbC, respectively. It remains unknown when and how the Dsb proteins participate in the biogenesis of outer membrane proteins, which are transported across the cell envelope after their synthesis. The Escherichia coli protein LptD is an integral outer membrane protein that forms an essential complex with the lipoprotein LptE. We show that oxidation of LptD is not required for the formation of the LptD/E complex but it is essential for function. Remarkably, none of the cysteines in LptD are essential because either of two nonconsecutive disulfide bonds suffices for function. Oxidation of LptD, which is efficiently catalyzed by DsbA, does not involve the isomerase DsbC, but it requires LptE. Thus, oxidation is completed only after LptD interacts with LptE, an interaction that occurs at the outer membrane and seems necessary for LptD folding.
Poly(A) polymerase I (PAP I) is the enzyme responsible for the addition of poly(A) tails onto RNA molecules in Escherichia coli. Polyadenylation is believed to facilitate the destruction of such RNAs by the mRNA degradosome. Recently, it was discovered that the stationary-phase regulatory protein SprE (RssB) has a second function in the control of polyadenylation that is distinct from its known function in the regulated proteolysis of RpoS. In the work presented herein, we used a targeted proteomic approach to further investigate SprE's involvement in the polyadenylation pathway. Specifically, we used cryogenic cell lysis, immunopurifications on magnetic beads, and mass spectrometry to identify interacting partners of PAP I-green fluorescent protein. We provide the first in vivo evidence that PAP I interacts with the mRNA degradosome during both exponential and stationary phases and find that the degradosome can contain up to 10 different proteins under certain conditions. Moreover, we demonstrate that the majority of these PAP I interactions are formed via protein-protein interactions and that SprE plays an important role in the maintenance of the PAP I-degradosome association during stationary phase.