Journal articles on the topic 'OMP biogenesis'
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Costello, Shawn M., Ashlee M. Plummer, Patrick J. Fleming, and Karen G. Fleming. "Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins." Proceedings of the National Academy of Sciences 113, no. 33 (August 1, 2016): E4794—E4800. http://dx.doi.org/10.1073/pnas.1601002113.
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Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.
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Albrecht, Reinhard, Monika Schütz, Philipp Oberhettinger, Michaela Faulstich, Ivan Bermejo, Thomas Rudel, Kay Diederichs, and Kornelius Zeth. "Structure of BamA, an essential factor in outer membrane protein biogenesis." Acta Crystallographica Section D Biological Crystallography 70, no. 6 (May 30, 2014): 1779–89. http://dx.doi.org/10.1107/s1399004714007482.
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Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. InEscherichia colithis function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of theE. coliBamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA fromHaemophilus ducreyiandNeisseria gonorrhoeaeand are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation,E. coliBamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer.E. coliBamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.
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Konovalova, Anna, Marcin Grabowicz, Carl J. Balibar, Juliana C. Malinverni, Ronald E. Painter, Daniel Riley, Paul A. Mann, et al. "Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins." Proceedings of the National Academy of Sciences 115, no. 28 (June 25, 2018): E6614—E6621. http://dx.doi.org/10.1073/pnas.1806107115.
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The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a β-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.
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Volokhina, Elena B., Frank Beckers, Jan Tommassen, and Martine P. Bos. "The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis." Journal of Bacteriology 191, no. 22 (September 18, 2009): 7074–85. http://dx.doi.org/10.1128/jb.00737-09.
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ABSTRACT The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.
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Hart, Elizabeth M., Angela M. Mitchell, Anna Konovalova, Marcin Grabowicz, Jessica Sheng, Xiaoqing Han, Frances P. Rodriguez-Rivera, et al. "A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier." Proceedings of the National Academy of Sciences 116, no. 43 (October 7, 2019): 21748–57. http://dx.doi.org/10.1073/pnas.1912345116.
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The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K. BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.
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Horne, Jim E., and Sheena E. Radford. "A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis." Biochemical Society Transactions 44, no. 3 (June 9, 2016): 802–9. http://dx.doi.org/10.1042/bst20160020.
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Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.
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Weirich, Johanna, Cornelia Bräutigam, Melanie Mühlenkamp, Mirita Franz-Wachtel, Boris Macek, Ina Meuskens, Mikael Skurnik, et al. "Identifying components required for OMP biogenesis as novel targets for antiinfective drugs." Virulence 8, no. 7 (February 6, 2017): 1170–88. http://dx.doi.org/10.1080/21505594.2016.1278333.
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Tata, Muralidhar, Santosh Kumar, Sarah R. Lach, Shreya Saha, Elizabeth M. Hart, and Anna Konovalova. "High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function." Proceedings of the National Academy of Sciences 118, no. 32 (August 4, 2021): e2100369118. http://dx.doi.org/10.1073/pnas.2100369118.
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The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494L and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.
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Soltes, Garner R., Jaclyn Schwalm, Dante P. Ricci, and Thomas J. Silhavy. "The Activity of Escherichia coli Chaperone SurA Is Regulated by Conformational Changes Involving a Parvulin Domain." Journal of Bacteriology 198, no. 6 (January 4, 2016): 921–29. http://dx.doi.org/10.1128/jb.00889-15.
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ABSTRACTThe periplasmic chaperone SurA is critical for the biogenesis of outer membrane proteins (OMPs) and, thus, the maintenance of membrane integrity inEscherichia coli. The activity of this modular chaperone has been attributed to a core chaperone module, with only minor importance assigned to the two SurA peptidyl-prolyl isomerase (PPIase) domains. In this work, we used synthetic phenotypes and covalent tethering to demonstrate that the activity of SurA is regulated by its PPIase domains and, furthermore, that its activity is correlated with the conformational state of the chaperone. When combined with mutations in the β-barrel assembly machine (BAM), SurA mutations resulting in deletion of the second parvulin domain (P2) inhibit OMP assembly, suggesting that P2 is involved in the regulation of SurA. The first parvulin domain (P1) potentiates this autoinhibition, as mutations that covalently tether the P1 domain to the core chaperone module severely impair OMP assembly. Furthermore, these inhibitory mutations negate the suppression of and biochemically stabilize the protein specified by a well-characterized gain-of-function mutation in P1, demonstrating that SurA cycles between distinct conformational and functional states during the OMP assembly process.IMPORTANCEThis work reveals the reversible autoinhibition of the SurA chaperone imposed by a heretofore underappreciated parvulin domain. Many β-barrel-associated outer membrane (OM) virulence factors, including the P-pilus and type I fimbriae, rely on SurA for proper assembly; thus, a mechanistic understanding of SurA function and inhibition may facilitate antibiotic intervention against Gram-negative pathogens, such as uropathogenicEscherichia coli,E. coliO157:H7,Shigella, andSalmonella. In addition, SurA is important for the assembly of critical OM biogenesis factors, such as the lipopolysaccharide (LPS) transport machine, suggesting that specific targeting of SurA may provide a useful means to subvert the OM barrier.
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Marx, Dagan C., Ashlee M. Plummer, Anneliese M. Faustino, Taylor Devlin, Michaela A. Roskopf, Mathis J. Leblanc, Henry J. Lessen, et al. "SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins." Proceedings of the National Academy of Sciences 117, no. 45 (October 22, 2020): 28026–35. http://dx.doi.org/10.1073/pnas.2008175117.
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The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.
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Horne, Jim E., David J. Brockwell, and Sheena E. Radford. "Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria." Journal of Biological Chemistry 295, no. 30 (June 4, 2020): 10340–67. http://dx.doi.org/10.1074/jbc.rev120.011473.
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β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.
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Rollauer, Sarah E., Moloud A. Sooreshjani, Nicholas Noinaj, and Susan K. Buchanan. "Outer membrane protein biogenesis in Gram-negative bacteria." Philosophical Transactions of the Royal Society B: Biological Sciences 370, no. 1679 (October 5, 2015): 20150023. http://dx.doi.org/10.1098/rstb.2015.0023.
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Gram-negative bacteria contain a double membrane which serves for both protection and for providing nutrients for viability. The outermost of these membranes is called the outer membrane (OM), and it contains a host of fully integrated membrane proteins which serve essential functions for the cell, including nutrient uptake, cell adhesion, cell signalling and waste export. For pathogenic strains, many of these outer membrane proteins (OMPs) also serve as virulence factors for nutrient scavenging and evasion of host defence mechanisms. OMPs are unique membrane proteins in that they have a β-barrel fold and can range in size from 8 to 26 strands, yet can still serve many different functions for the cell. Despite their essential roles in cell survival and virulence, the exact mechanism for the biogenesis of these OMPs into the OM has remained largely unknown. However, the past decade has witnessed significant progress towards unravelling the pathways and mechanisms necessary for moulding a nascent polypeptide into a functional OMP within the OM. Here, we will review some of these recent discoveries that have advanced our understanding of the biogenesis of OMPs in Gram-negative bacteria, starting with synthesis in the cytoplasm to folding and insertion into the OM.
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Fardini, Yann, Jérôme Trotereau, Elisabeth Bottreau, Charlène Souchard, Philippe Velge, and Isabelle Virlogeux-Payant. "Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella." Microbiology 155, no. 5 (May 1, 2009): 1613–22. http://dx.doi.org/10.1099/mic.0.025155-0.
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In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the ΔbamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in ΔbamC and ΔdegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As ΔsurA and ΔbamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD.
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Masi, Muriel, Guillaume Duret, Anne H. Delcour, and Rajeev Misra. "Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli." Microbiology 155, no. 6 (June 1, 2009): 1847–57. http://dx.doi.org/10.1099/mic.0.027219-0.
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TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.
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Noinaj, Nicholas, Adam Kuszak, Curtis Balusek, JC Gumbart, Petra Lukacik, Hoshing Chang, Nicole Easley, Trevor Lithgow, and Susan Buchanan. "The role of BamA in the biogenesis of beta-barrel membrane proteins." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C578. http://dx.doi.org/10.1107/s2053273314094212.
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Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.
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Wu, Si, Xi Ge, Zhixin Lv, Zeyong Zhi, Zengyi Chang, and Xin Sheng Zhao. "Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism." Biochemical Journal 438, no. 3 (August 26, 2011): 505–11. http://dx.doi.org/10.1042/bj20110264.
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The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone–substrate interaction may be essential for the quality control of the biogenesis of OMPs
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Luthra, Amit, Arvind Anand, Kelly L. Hawley, Morgan LeDoyt, Carson J. La Vake, Melissa J. Caimano, Adriana R. Cruz, Juan C. Salazar, and Justin D. Radolf. "A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326." Journal of Bacteriology 197, no. 11 (March 30, 2015): 1906–20. http://dx.doi.org/10.1128/jb.00086-15.
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ABSTRACTWe recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) inTreponema pallidumand that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intactT. pallidum. Using the solved structure ofNeisseria gonorrhoeaeBamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop inT. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments usingEscherichia coliexpressing OM-localized TP_0326 as aT. pallidumsurrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu593→ Gln593) in the L4 sequences ofT. pallidumstrains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of aT. pallidumOMP within the context of syphilitic infection.IMPORTANCEPreviously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) inTreponema pallidumand that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis ofT. pallidum's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of aT. pallidumrare OMP within the context of syphilitic infection.
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Chen, Ching-ju, Deborah M. Tobiason, Christopher E. Thomas, William M. Shafer, H. Steven Seifert, and P. Frederick Sparling. "A Mutant Form of the Neisseria gonorrhoeae Pilus Secretin Protein PilQ Allows Increased Entry of Heme and Antimicrobial Compounds." Journal of Bacteriology 186, no. 3 (February 1, 2004): 730–39. http://dx.doi.org/10.1128/jb.186.3.730-739.2004.
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ABSTRACT A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry.
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Friedrich, V., C. Gruber, I. Nimeth, S. Pabinger, G. Sekot, G. Posch, F. Altmann, P. Messner, O. Andrukhov, and C. Schäffer. "Outer membrane vesicles of Tannerella forsythia : biogenesis, composition, and virulence." Molecular Oral Microbiology 30, no. 6 (June 16, 2015): 451–73. http://dx.doi.org/10.1111/omi.12104.
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Xu, R., Q. Hu, Q. Ma, C. Liu, and G. Wang. "The protease Omi regulates mitochondrial biogenesis through the GSK3β/PGC-1α pathway." Cell Death & Disease 5, no. 8 (August 2014): e1373-e1373. http://dx.doi.org/10.1038/cddis.2014.328.
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Karakas, Umit, Ozlem Izci Ay, Mustafa Ertan Ay, Wei Wang, Mehmet Ali Sungur, Kenan Çevik, Gurbet Dogru, and Mehmet Emin Erdal. "Regulating the Regulators in Attention-Deficit/Hyperactivity Disorder: A Genetic Association Study of microRNA Biogenesis Pathways." OMICS: A Journal of Integrative Biology 21, no. 6 (June 2017): 352–58. http://dx.doi.org/10.1089/omi.2017.0048.
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Crowley, P. J., and L. J. Brady. "Evaluation of the effects ofStreptococcus mutanschaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production." Molecular Oral Microbiology 31, no. 1 (October 7, 2015): 59–77. http://dx.doi.org/10.1111/omi.12130.
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Cuthbertson, Leslie, Iain L. Mainprize, James H. Naismith, and Chris Whitfield. "Pivotal Roles of the Outer Membrane Polysaccharide Export and Polysaccharide Copolymerase Protein Families in Export of Extracellular Polysaccharides in Gram-Negative Bacteria." Microbiology and Molecular Biology Reviews 73, no. 1 (March 2009): 155–77. http://dx.doi.org/10.1128/mmbr.00024-08.
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SUMMARY Many bacteria export extracellular polysaccharides (EPS) and capsular polysaccharides (CPS). These polymers exhibit remarkably diverse structures and play important roles in the biology of free-living, commensal, and pathogenic bacteria. EPS and CPS production represents a major challenge because these high-molecular-weight hydrophilic polymers must be assembled and exported in a process spanning the envelope, without compromising the essential barrier properties of the envelope. Emerging evidence points to the existence of molecular scaffolds that perform these critical polymer-trafficking functions. Two major pathways with different polymer biosynthesis strategies are involved in the assembly of most EPS/CPS: the Wzy-dependent and ATP-binding cassette (ABC) transporter-dependent pathways. They converge in an outer membrane export step mediated by a member of the outer membrane auxiliary (OMA) protein family. OMA proteins form outer membrane efflux channels for the polymers, and here we propose the revised name outer membrane polysaccharide export (OPX) proteins. Proteins in the polysaccharide copolymerase (PCP) family have been implicated in several aspects of polymer biogenesis, but there is unequivocal evidence for some systems that PCP and OPX proteins interact to form a trans-envelope scaffold for polymer export. Understanding of the precise functions of the OPX and PCP proteins has been advanced by recent findings from biochemistry and structural biology approaches and by parallel studies of other macromolecular trafficking events. Phylogenetic analyses reported here also contribute important new insight into the distribution, structural relationships, and function of the OPX and PCP proteins. This review is intended as an update on progress in this important area of microbial cell biology.
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Hao, Jiejie, Cui Hao, Lijuan Zhang, Xin Liu, Xiaolin Zhou, Yunlou Dun, Haihua Li, et al. "OM2, a Novel Oligomannuronate-Chromium(III) Complex, Promotes Mitochondrial Biogenesis and Lipid Metabolism in 3T3-L1 Adipocytes via the AMPK-PGC1α Pathway." PLOS ONE 10, no. 7 (July 15, 2015): e0131930. http://dx.doi.org/10.1371/journal.pone.0131930.
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Johnston, Joanne L., Stephen J. Billington, Volker Haring, and Julian I. Rood. "Complementation Analysis of the Dichelobacter nodosus fimN, fimO, and fimP Genes inPseudomonas aeruginosa and Transcriptional Analysis of thefimNOP Gene Region." Infection and Immunity 66, no. 1 (January 1, 1998): 297–304. http://dx.doi.org/10.1128/iai.66.1.297-304.1998.
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ABSTRACT The causative agent of ovine footrot, the gram-negative anaerobeDichelobacter nodosus, produces polar type IV fimbriae, which are the major protective antigens. The D. nodosusgenes fimN, fimO, and fimP are homologs of the Pseudomonas aeruginosa fimbrial assembly genes, pilB, pilC, and pilD, respectively. Both the pilD and fimP genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the D. nodosus genes were introduced intoP. aeruginosa strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that thefimP gene complemented a pilD mutant ofP. aeruginosa for both fimbrial assembly and protein secretion. However, the fimN and fimO genes did not complement pilB or pilC mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the P. aeruginosafimbrial assembly system. The transcriptional organization and regulation of the fimNOP gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two ς70-type promoters located upstream of ORFM, an open reading frame proximal to fimN. Transcription of theD. nodosus fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.
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Aragon, Virginia, Sherry Kurtz, Antje Flieger, Birgid Neumeister, and Nicholas P. Cianciotto. "Secreted Enzymatic Activities of Wild-Type andpilD-Deficient Legionella pneumophila." Infection and Immunity 68, no. 4 (April 1, 2000): 1855–63. http://dx.doi.org/10.1128/iai.68.4.1855-1863.2000.
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ABSTRACT Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by apilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release ofp-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.
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Ranava, David, Yiying Yang, Luis Orenday-Tapia, François Rousset, Catherine Turlan, Violette Morales, Lun Cui, et al. "Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope stress." eLife 10 (April 13, 2021). http://dx.doi.org/10.7554/elife.67817.
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In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.
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Mamou, Gideon, Federico Corona, Ruth Cohen-Khait, Nicholas G. Housden, Vivian Yeung, Dawei Sun, Pooja Sridhar, et al. "Peptidoglycan maturation controls outer membrane protein assembly." Nature, June 15, 2022. http://dx.doi.org/10.1038/s41586-022-04834-7.
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AbstractLinkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1–5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the β-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.
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Schiffrin, Bob, Jonathan M. Machin, Theodoros K. Karamanos, Anastasia Zhuravleva, David J. Brockwell, Sheena E. Radford, and Antonio N. Calabrese. "Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding." Communications Biology 5, no. 1 (June 8, 2022). http://dx.doi.org/10.1038/s42003-022-03502-w.
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AbstractCorrect folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the β-barrel assembly machinery (BAM). How unfolded substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA β1-β16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.
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Hart, Elizabeth M., Meera Gupta, Martin Wühr, and Thomas J. Silhavy. "The Synthetic Phenotype of ΔbamBΔbamEDouble Mutants Results from a Lethal Jamming of the Bam Complex by the Lipoprotein RcsF." mBio 10, no. 3 (May 21, 2019). http://dx.doi.org/10.1128/mbio.00662-19.
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ABSTRACTThe selective permeability of the Gram-negative outer membrane (OM) is maintained by integral β-barrel outer membrane proteins (OMPs). The heteropentomeric β-barrel assembly machine (Bam) folds and inserts OMPs into the OM. Coordination of the essential proteins BamA and BamD is critical for OMP assembly and therefore the viability of the cell. The role of the nonessential lipoproteins BamBCE has yet to be characterized; however, genetic evidence suggests that they have nonoverlapping roles in OMP assembly. In this work, we quantify changes of the proteome in the conditional lethal ΔbamBΔbamEdouble mutant. We show that cells lacking BamB and BamE have a global OMP defect that is a result of a lethal obstruction of an assembly-competent Bam complex by the lipoprotein RcsF. RcsF is a stress-sensing lipoprotein that is threaded through the lumen of abundant β-barrel OMPs by the Bam complex to expose the amino terminus on the cell surface. We demonstrate that simply removing this lipoprotein corrects the severe OMP assembly defect of the double mutant nearly as efficiently as a previously isolated suppressor mutation inbamA. We propose that BamB and BamE play crucial, nonoverlapping roles to coordinate the activities of BamA and BamD during OMP biogenesis.IMPORTANCEProtein assembly into lipid bilayers is an essential process that ensures the viability of diverse organisms. In Gram-negative bacteria, the heteropentomeric β-barrel assembly machine (Bam) folds and inserts proteins into the outer membrane. Due to its essentiality, outer membrane protein (OMP) assembly by the Bam complex is an attractive target for antibiotic development. Here, we show that the conditional lethal phenotype of a mutant lacking two of the three nonessential lipoproteins, BamB and BamE, is caused by lethal jamming of the stripped-down Bam complex by a normally surface-exposed lipoprotein, RcsF. The heterotrimeric Bam complex (BamA, BamD, BamC) is nearly as efficient as the wild-type complex in OMP assembly if RcsF is removed. Our study highlights the importance of BamB and BamE in regulating the interaction between BamA and BamD and expands our understanding of the role of the Bam complex in outer membrane biogenesis.
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Soltes, Garner R., Nicholas R. Martin, Eunhae Park, Holly A. Sutterlin, and Thomas J. Silhavy. "Distinctive Roles for Periplasmic Proteases in the Maintenance of Essential Outer Membrane Protein Assembly." Journal of Bacteriology 199, no. 20 (August 7, 2017). http://dx.doi.org/10.1128/jb.00418-17.
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ABSTRACT Outer membrane protein (OMP) biogenesis in Escherichia coli is a robust process essential to the life of the organism. It is catalyzed by the β-barrel assembly machine (Bam) complex, and a number of quality control factors, including periplasmic chaperones and proteases, maintain the integrity of this trafficking pathway. Little is known, however, about how periplasmic proteases recognize and degrade OMP substrates when assembly is compromised or whether different proteases recognize the same substrate at distinct points in the assembly pathway. In this work, we use well-defined assembly-defective mutants of LptD, the essential lipopolysaccharide assembly translocon, to show that the periplasmic protease DegP degrades substrates with assembly defects that prevent or impair initial contact with Bam, causing the mutant protein to accumulate in the periplasm. In contrast, another periplasmic protease, BepA, degrades a LptD mutant substrate that has engaged the Bam complex and formed a nearly complete barrel. Furthermore, we describe the role of the outer membrane lipoprotein YcaL, a protease of heretofore unknown function, in the degradation of a LptD substrate that has engaged the Bam complex but is stalled at an earlier step in the assembly process that is not accessible to BepA. Our results demonstrate that multiple periplasmic proteases monitor OMPs at distinct points in the assembly process. IMPORTANCE OMP assembly is catalyzed by the essential Bam complex and occurs in a cellular environment devoid of energy sources. Assembly intermediates that misfold can compromise this essential molecular machine. Here we demonstrate distinctive roles for three different periplasmic proteases that can clear OMP substrates with folding defects that compromise assembly at three different stages. These quality control factors help ensure the integrity of the permeability barrier that contributes to the intrinsic resistance of Gram-negative organisms to many antibiotics.
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Tata, Muralidhar, and Anna Konovalova. "Improper Coordination of BamA and BamD Results in Bam Complex Jamming by a Lipoprotein Substrate." mBio 10, no. 3 (May 21, 2019). http://dx.doi.org/10.1128/mbio.00660-19.
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ABSTRACT The β-barrel assembly machinery, the Bam complex, is central to the biogenesis of integral outer membrane proteins (OMPs) as well as OMP-dependent surface-exposed lipoproteins, such as regulator of capsule synthesis protein F (RcsF). Previous genetic analysis established the model that nonessential components BamE and BamB have overlapping, redundant functions to enhance the kinetics of the highly conserved BamA/BamD core. Here we report that BamE plays a specialized nonredundant role in the Bam complex required for surface exposure of RcsF. We show that the lack of bamE, but not bamB, completely abolishes assembly of RcsF/OMP complexes and establish that the inability to assemble RcsF/OMP complexes is a molecular reason underlying all synthetic lethal interactions of ΔbamE. Our genetic analysis and biochemical cross-linking suggest that RcsF accumulates on BamA when BamA cannot engage with BamD because of its limited availability or the incompatible conformation. The role of BamE is to promote proper coordination of RcsF-bound BamA with BamD to complete OMP assembly around RcsF. We show that in the absence of BamE, RcsF is stalled on BamA, thus blocking its function, and we identify the lipoprotein RcsF as a bona fide jamming substrate of the Bam complex. IMPORTANCE The β-barrel assembly machinery, the Bam complex, consists of five components, BamA to -E, among which BamA and BamD are highly conserved and essential. The nonessential components are believed to play redundant roles simply by improving the rate of β-barrel folding. Here we show that BamE contributes a specific and nonoverlapping function to the Bam complex. BamE coordinates BamA and BamD to form a complex between the lipoprotein RcsF and its partner outer membrane β-barrel protein, allowing RcsF to reach the cell surface. In the absence of BamE, RcsF accumulates on BamA, thus blocking the activity of the Bam complex. As the Bam complex is a major antibiotic target in Gram-negative bacteria, the discovery that a lipoprotein can act as a jamming substrate may open the door for development of novel Bam complex inhibitors.
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Alvira, Sara, Daniel W. Watkins, Lucy Troman, William J. Allen, James S. Lorriman, Gianluca Degliesposti, Eli J. Cohen, et al. "Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis." eLife 9 (November 4, 2020). http://dx.doi.org/10.7554/elife.60669.
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The outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrel Outer-Membrane Proteins (OMPs) – are first secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones, for example SurA, which prevent aggregation. OMPs are then offloaded to the β-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL) – an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC – contacts BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Furthermore, the proton-motive force (PMF) across the inner-membrane acts at distinct stages of protein secretion: (1) SecA-driven translocation through SecYEG and (2) communication of conformational changes via SecDF across the periplasm to BAM. The latter presumably drives efficient passage of OMPs. These interactions provide insights of inter-membrane organisation and communication, the importance of which is becoming increasingly apparent.
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Ricci, Dante P., Jaclyn Schwalm, Michelle Gonzales-Cope, and Thomas J. Silhavy. "The Activity and Specificity of the Outer Membrane Protein Chaperone SurA Are Modulated by a Proline Isomerase Domain." mBio 4, no. 4 (August 13, 2013). http://dx.doi.org/10.1128/mbio.00540-13.
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ABSTRACTSurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) inEscherichia 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 activityin vivodespite destabilization of the proteinin 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.IMPORTANCEThe dominantsurAmutations described here alter amino acid residues that are highly conserved in eukaryotic homologs of SurA, including Pin1, 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 asSalmonella,Shigella, andEscherichia coliO157: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.
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"Escherichia coli BepA has proteolytic and chaperone-like functions and acts in the degradation and biogenesis of β-barrel outer membrane proteins (OMP). The tetratricopeptide repeat (TPR) domain of BepA (orange) interacts with proteins of the β-barrel ass." Molecular Microbiology 106, no. 5 (November 20, 2017): i. http://dx.doi.org/10.1111/mmi.13522.
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Alaei, Sarah R., Jin Ho Park, Stephen G. Walker, and David G. Thanassi. "Peptide-Based Inhibitors of Fimbrial Biogenesis inPorphyromonas gingivalis." Infection and Immunity 87, no. 3 (January 14, 2019). http://dx.doi.org/10.1128/iai.00750-18.
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ABSTRACTPeriodontitis is a progressive inflammatory disease that affects roughly half of American adults. Colonization of the oral cavity by the Gram-negative bacterial pathogenPorphyromonas gingivalisis a key event in the initiation and development of periodontal disease. Adhesive surface structures termed fimbriae (pili) mediate interactions ofP. gingivaliswith other bacteria and with host cells throughout the course of disease. TheP. gingivalisfimbriae are assembled via a novel mechanism that involves proteolytic processing of lipidated precursor subunits and their subsequent polymerization on the bacterial surface. Given their extracellular assembly mechanism and central roles in pathogenesis, theP. gingivalisfimbriae are attractive targets for anti-infective therapeutics to prevent or treat periodontal disease. Here we confirm that conserved sequences in the N and C termini of the Mfa1 fimbrial subunit protein perform critical roles in subunit polymerization. We show that treatment ofP. gingivaliswith peptides corresponding to the conserved C-terminal region inhibits the extracellular assembly of Mfa fimbriae on the bacterial surface. We also show that peptide treatment interferes with the function of Mfa fimbriae by reducingP. gingivalisadhesion toStreptococcus gordoniiin a dual-species biofilm model. Finally, we show that treatment of bacteria with similar peptides inhibits extracellular polymerization of the Fim fimbriae, which are also expressed byP. gingivalis. These results support a donor strand-based assembly mechanism for theP. gingivalisfimbriae and demonstrate the feasibility of using extracellular peptides to disrupt the biogenesis and function of these critical periodontal disease virulence factors.
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Wen, Zezhang T., Ashton N. Jorgensen, Xiaochang Huang, Kassapa Ellepola, Lynne Chapman, Hui Wu, and L. Jeannine Brady. "Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans." Molecular Oral Microbiology, December 3, 2020. http://dx.doi.org/10.1111/omi.12318.
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Filipović, Maša, Darja Flegar, Sara Aničić, Dino Šisl, Tomislav Kelava, Nataša Kovačić, Alan Šućur, and Danka Grčević. "Transcriptome profiling of osteoclast subsets associated with arthritis: A pathogenic role of CCR2hi osteoclast progenitors." Frontiers in Immunology 13 (December 15, 2022). http://dx.doi.org/10.3389/fimmu.2022.994035.
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IntroductionThe existence of different osteoclast progenitor (OCP) subsets has been confirmed by numerous studies. However, pathological inflammation-induced osteoclastogenesis remains incompletely understood. Detailed characterization of OCP subsets may elucidate the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. In our study, we rely on previously defined OCP subsets categorized by the level of CCR2 expression as circulatory-like committed CCR2hi OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2lo OCPs of immature phenotype and behavior.MethodsIn order to perform transcriptome characterization of those subsets in the context of collagen-induced arthritis (CIA), we sorted CCR2hi and CCR2lo periarticular bone marrow OCPs of control and arthritic mice, and performed next-generation RNA sequencing (n=4 for each group) to evaluate the differential gene expression profile using gene set enrichment analysis with further validation.ResultsA disparity between CCR2hi and CCR2lo subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2hi OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2lo OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2hi (92 genes) than in CCR2lo (43 genes) OCPs. Genes associated with the osteoclastogenic pathway (Fcgr1, Socs3), and several genes involved in cell adhesion and migration (F11r, Cd38, Lrg1) identified the CCR2hi subset and distinguish CIA from control group, as validated by qPCR (n=6 for control mice, n=9 for CIA mice). The latter gene set showed a significant positive correlation with arthritis clinical score and frequency of CCR2hi OCPs. Protein-level validation by flow cytometry showed increased proportion of OCPs expressing F11r/CD321, CD38 and Lrg1 in CIA, indicating that they could be used as disease markers. Moreover, osteoclast pathway-identifying genes remained similarly expressed (Fcgr1) or even induced by several fold (Socs3) in preosteoclasts differentiated in vitro from CIA mice compared to pre-cultured levels, suggesting their importance for enhanced osteoclastogenesis of the CCR2hi OCPs in arthritis.ConclusionOur approach detected differentially expressed genes that could identify distinct subset of OCPs associated with arthritis as well as indicate possible therapeutic targets aimed to modulate osteoclast activity.
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Colas, Vincent, Philippe Barre, Frederik van Parijs, Lukas Wolters, Yannick Quitté, Tom Ruttink, Isabel Roldán-Ruiz, Abraham J. Escobar Gutiérrez, and Hilde Muylle. "Seasonal Differences in Structural and Genetic Control of Digestibility in Perennial Ryegrass." Frontiers in Plant Science 12 (January 4, 2022). http://dx.doi.org/10.3389/fpls.2021.801145.
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Perennial ryegrass is an important forage crop in dairy farming, either for grazing or haying purposes. To further optimise the forage use, this study focused on understanding forage digestibility in the two most important cuts of perennial ryegrass, the spring cut at heading and the autumn cut. In a highly diverse collection of 592 Lolium perenne genotypes, the organic matter digestibility (OMD) and underlying traits such as cell wall digestibility (NDFD) and cell wall components (cellulose, hemicellulose, and lignin) were investigated for 2 years. A high genotype × season interaction was found for OMD and NDFD, indicating differences in genetic control of these forage quality traits in spring versus autumn. OMD could be explained by both the quantity of cell wall content (NDF) and the quality of the cell wall content (NDFD). The variability in NDFD in spring was mainly explained by differences in hemicellulose. A 1% increase of the hemicellulose content in the cell wall (HC.NDF) resulted in an increase of 0.81% of NDFD. In autumn, it was mainly explained by the lignin content in the cell wall (ADL.NDF). A 0.1% decrease of ADL.NDF resulted in an increase of 0.41% of NDFD. The seasonal traits were highly heritable and showed a higher variation in autumn versus spring, indicating the potential to select for forage quality in the autumn cut. In a candidate gene association mapping approach, in which 503 genes involved in cell wall biogenesis, plant architecture, and phytohormone biosynthesis and signalling, identified significant quantitative trait loci (QTLs) which could explain from 29 to 52% of the phenotypic variance in the forage quality traits OMD and NDFD, with small effects of each marker taken individually (ranging from 1 to 7%). No identical QTLs were identified between seasons, but within a season, some QTLs were in common between digestibility traits and cell wall composition traits confirming the importance of hemicellulose concentration for spring digestibility and lignin concentration in NDF for autumn digestibility.
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Jiang, Min, Zifan Liu, Junjie Shao, Jingjing Zhou, Haiming Wang, Chao Song, Xin Li, et al. "Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity." Frontiers in Physiology 13 (November 10, 2022). http://dx.doi.org/10.3389/fphys.2022.1039913.
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Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor α (ERα)-NRF1-OMI-mitophagy signaling was involved in the dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) under simulated microgravity. Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.
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Nelson, Cassandra E., Weiliang Huang, Luke K. Brewer, Angela T. Nguyen, Maureen A. Kane, Angela Wilks, and Amanda G. Oglesby-Sherrouse. "Proteomic Analysis of thePseudomonas aeruginosaIron Starvation Response Reveals PrrF Small Regulatory RNA-Dependent Iron Regulation of Twitching Motility, Amino Acid Metabolism, and Zinc Homeostasis Proteins." Journal of Bacteriology 201, no. 12 (April 8, 2019). http://dx.doi.org/10.1128/jb.00754-18.
Full textAbstract:
ABSTRACTIron is a critical nutrient for most microbial pathogens, and the immune system exploits this requirement by sequestering iron. The opportunistic pathogenPseudomonas aeruginosaexhibits a high requirement for iron yet an exquisite ability to overcome iron deprivation during infection. Upon iron starvation,P. aeruginosainduces the expression of several high-affinity iron acquisition systems, as well as the PrrF small regulatory RNAs (sRNAs) that mediate an iron-sparing response. Here, we used liquid chromatography-tandem mass spectrometry to conduct proteomics of the iron starvation response ofP. aeruginosa. Iron starvation increased levels of multiple proteins involved in amino acid catabolism, providing the capacity for iron-independent entry of carbons into the tricarboxylic acid (TCA) cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were increased, highlighting the central role of iron inP. aeruginosametabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins and increased levels of twitching motility proteins. Subsequent analyses provided evidence for the regulation of many of these proteins via posttranscriptional regulatory events, some of which are dependent upon the PrrF sRNAs. Moreover, we showed that iron-regulated twitching motility is partially dependent upon theprrFlocus, highlighting a novel link between the PrrF sRNAs and motility. These findings add to the known impacts of iron starvation inP. aeruginosaand outline potentially novel roles for the PrrF sRNAs in iron homeostasis and pathogenesis.IMPORTANCEIron is central for growth and metabolism of almost all microbial pathogens, and as such, this element is sequestered by the host innate immune system to restrict microbial growth. Here, we used label-free proteomics to investigate thePseudomonas aeruginosairon starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes, including twitching motility, are regulated through the iron-responsive PrrF small regulatory RNAs. As such, this study demonstrates the power of proteomics for defining stress responses of microbial pathogens.