Chemical Conditionality

Ruiz, Natividad; Falcone, Brian V.; Kahne, Daniel; Silhavy, Thomas J.

doi:10.1016/j.cell.2005.02.014

Cited by 276 publications

(188 citation statements)

References 27 publications

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“…However, assembly of a considerable number of outer membrane proteins into the outer membrane was delayed in the secB null mutant. Recently, an outer membrane protein complex consisting of the proteins YeaT, NlpB, YfgL, and YfiO has been shown to act as an insertion machinery for outer membrane proteins (52,53). In our outer membrane two-dimensional electrophoresis gels, we identified YeaT and lipoprotein NlpB.…”

Section: Defining the Role Of E Coli Secbmentioning

confidence: 76%

Defining the Role of the Escherichia coli Chaperone SecB Using Comparative Proteomics

Baars

Ytterberg

Drew

et al. 2006

Journal of Biological Chemistry

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To improve understanding and identify novel substrates of the cytoplasmic chaperone SecB in Escherichia coli, we analyzed a secB null mutant using comparative proteomics. The secB null mutation did not affect cell growth but caused significant differences at the proteome level. In the absence of SecB, dynamic protein aggregates containing predominantly secretory proteins accumulated in the cytoplasm. Unprocessed secretory proteins were detected in radiolabeled whole cell lysates. Furthermore, the assembly of a large fraction of the outer membrane proteome was slowed down, whereas its steady state composition was hardly affected. In response to aggregation and delayed sorting of secretory proteins, cytoplasmic chaperones DnaK, GroEL/ES, ClpB, IbpA/B, and HslU were up-regulated severalfold, most likely to stabilize secretory proteins during their delayed translocation and/or rescue aggregated secretory proteins. The SecB/A dependence of 12 secretory proteins affected by the secB null mutation (DegP, FhuA, FkpA, OmpT, OmpX, OppA, TolB, TolC, YbgF, YcgK, YgiW, and YncE) was confirmed by "classical" pulse-labeling experiments. Our study more than triples the number of known SecB-dependent secretory proteins and shows that the primary role of SecB is to facilitate the targeting of secretory proteins to the Sec-translocase.The periplasmic and outer membrane proteins in the Gram-negative bacterium Escherichia coli need to cross the cytoplasmic membrane to reach their final destination. The vast majority of these secretory proteins are translocated through the cytoplasmic membrane via the Sec-translocase (1, 2). The core of the Sec-translocase is comprised of integral membrane proteins SecY and SecE, which form a protein conducting channel (3). The peripheral subunit SecA drives polypeptide chains in an ATP-dependent manner into and through the Sec-translocase (1).It is generally assumed that secretory proteins in E. coli are targeted to the Sec-translocase by the cytoplasmic protein SecB in a mostly posttranslational fashion (4 -8). However, direct evidence for SecB dependence is only established for six secretory proteins (PhoE, LamB, MBP, OmpF, GBP, and OmpA), whereas four secretory proteins (PhoA, Lpp, RbsB, and ␤-lac) do not seem to require SecB (9 -12, 55). SecB also has the capacity to assist the chaperone DnaK in the folding of proteins, as shown in vitro with luciferase as a model substrate (12). This indicates that SecB has the potential to assist the folding of cytoplasmic proteins. The successful complementation of a DnaK/trigger factor (TF) 2 double mutant strain by overexpression of SecB, and cross-linking of SecB to nascent chains of both secretory and cytoplasmic proteins in SecBenriched lysates support this notion (13).SecB does not bind to signal sequences and peptide library screens suggested a very loosely defined SecB binding "motif " (12). This motif, which is ϳ9 residues long, is enriched in aromatic and basic residues, whereas acidic residues are disfavored. It theoretically occurs every 20 -30 re...

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Section: Defining the Role Of E Coli Secbmentioning

confidence: 76%

Defining the Role of the Escherichia coli Chaperone SecB Using Comparative Proteomics

Baars

Ytterberg

Drew

et al. 2006

Journal of Biological Chemistry

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“…To identify new proteins involved in maintaining the integrity of the cell envelope, we screened the Keio collection for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of envelope defects (9). The Keio collection is a systematic collection of E. coli deletion mutants that contain disruptions in 3,985 nonessential genes, including several hundreds of uncharacterized open reading frames (10).…”

Section: Resultsmentioning

confidence: 99%

Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

Nicolaes¹,

Hajjaji²,

Davis³

et al. 2013

Journal of Bacteriology

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The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.A ntibiotic resistance has become a worldwide problem that threatens the effectiveness of many medicines used today to treat bacterial infections. A particularly serious threat is the emergence of Gram-negative pathogens, including Escherichia coli and Pseudomonas aeruginosa, which are resistant to all of the available antibacterial agents (1). It is therefore urgent to develop new antibiotics active against Gram-negative bacteria, which requires a deep understanding of the biology of these organisms. Proteins playing a role in the assembly of the cell envelope are attractive targets for antibiotic development because this structure is essential for viability and protection against toxic compounds such as antibiotics.The envelope of Gram-negative bacteria is characterized by the presence of two concentric membranes, the inner membrane (IM) and outer membrane (OM), which are separated by the periplasm, a compartment that represents between 10 and 20% of the total cell volume and contains the peptidoglycan layer, or sacculus (2-4). The peptidoglycan sacculus is a heteropolymeric macromolecule composed of relatively short glycan strands and peptide cross-links, which serve to maintain cell morphology and give protection from turgor pressure. Although several proteins that play a key role in the biogenesis of the cell envelope have been identified recently (5-7), we have a poor understanding of how this complex architecture is assembled and of how envelope biogenesis is coordinated with cell growth and division. For instance, we do not know how the phospholipids that ...

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“…We identified LptD as a protein whose correct folding was affected by the absence of DsbC. The synthetic phenotype of the surAdsbC mutant fits well with the identification of LptD as a DsbC substrate, because strains expressing low levels or mutated versions of LptD also are sensitive to antibiotics (28,31). Regarding the growth defect exhibited by the surAdsbC mutant at low temperatures, we propose that it results from perturbations in the lipid content of the OM caused by problems in the LPS insertion process.…”

Section: Discussionmentioning

confidence: 54%

“…LptD is also a true SurA substrate (8). Moreover, LptD, which inserts LPS in the outer leaflet of the OM, is an essential player in envelope biogenesis, and cells expressing LptD mutants have a decreased OM integrity (28). LptD was therefore a prime candidate.…”

Section: Resultsmentioning

confidence: 99%

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

Denoncin

Vertommen

Paek

et al. 2010

Journal of Biological Chemistry

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The assembly of the ␤-barrel proteins present in the outer membrane (OM) of Gram-negative bacteria is poorly characterized. After translocation across the inner membrane, unfolded ␤-barrel proteins are escorted across the periplasm by chaperones that reside within this compartment. Two partially redundant chaperones, SurA and Skp, are considered to transport the bulk mass of ␤-barrel proteins. We found that the periplasmic disulfide isomerase DsbC cooperates with SurA and the thiol oxidase DsbA in the folding of the essential ␤-barrel protein LptD. LptD inserts lipopolysaccharides in the OM. It is also the only ␤-barrel protein with more than two cysteine residues. We found that surAdsbC mutants, but not skpdsbC mutants, exhibit a synthetic phenotype. They have a decreased OM integrity, which is due to the lack of the isomerase activity of DsbC. We also isolated DsbC in a mixed disulfide complex with LptD. As such, LptD is identified as the first substrate of DsbC that is localized in the OM. Thus, electrons flowing from the cytoplasmic thioredoxin system maintain the integrity of the OM by assisting the folding of one of the most important ␤-barrel proteins. The outer membrane (OM)4 is a complex macromolecular structure that is essential for the viability of Gram-negative bacteria and serves as a permeability barrier against hydrophobic antibiotics. It is a unique asymmetric lipid bilayer with phospholipids forming the inner leaflet and lipopolysaccharides (LPS) forming the outer leaflet. The proteins that are present in the OM are either lipoproteins that are anchored to the inner leaflet via a lipid moiety or ␤-barrel proteins that are integral membrane proteins. The mechanisms that govern the assembly of the OM are poorly understood (1-3). In particular, we have a poor understanding of how the ␤-barrel proteins, which are synthesized in the cytoplasm and translocated unfolded across the inner membrane by the Sec translocon, are then transported across the periplasm and inserted in the OM. According to the most widely accepted model, unfolded ␤-barrel proteins are escorted across the periplasm by soluble periplasmic chaperones that deliver them to an OM multiprotein complex involving the ␤-barrel protein BamA (3, 4). Two partially redundant periplasmic chaperone pathways have been described in the Escherichia coli periplasm (5, 6). The first one involves SurA, a periplasmic prolyl cistrans-isomerase, which is considered as the primary chaperone in the periplasm. SurA has been shown to be involved in the biogenesis of the most abundant ␤-barrel proteins, such as OmpA, OmpF, and OmpC (6, 7), and is the preferred chaperone for the essential protein LptD (previously Imp) (8). The second pathway involves the chaperone Skp and the protease/ chaperone protein DegP. This pathway has been proposed to serve as a backup for SurA, assisting the biogenesis of the proteins that fell off the SurA pathway (6). The fact that surAskp and surAdegP mutants are not viable (5) indicates that cells cannot survive without at least o...

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Chemical Conditionality

Cited by 276 publications

References 27 publications

Defining the Role of the Escherichia coli Chaperone SecB Using Comparative Proteomics

Defining the Role of the Escherichia coli Chaperone SecB Using Comparative Proteomics

Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

The Protein-disulfide Isomerase DsbC Cooperates with SurA and DsbA in the Assembly of the Essential β-Barrel Protein LptD

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