Biogenesis of Outer Membranes in Gram-Negative Bacteria

Tokuda, Harukuni

doi:10.1271/bbb.80778

Cited by 117 publications

(116 citation statements)

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“…LptE is anchored to the inner leaflet of the OM via an N-terminal lipid moiety. To understand the role of this lipid anchor in bringing the LptD/E complex together in vivo, we engineered a plasmid in which the coding sequencing of LptE, excluding its signal sequence and the N-terminal lipidated cysteine (a.a. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], is placed after the sequence encoding the pelB leader peptide. This plasmid constitutively expresses a soluble version of LptE in the periplasm that is no longer lipidated at the N terminus.…”

Section: Resultsmentioning

confidence: 99%

“…Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3,4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.…”

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confidence: 99%

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Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

191

247

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Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. T he OM of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed almost entirely of LPS and the inner leaflet of phospholipids (PL) (Fig. 1) (1, 2). Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3, 4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

191

247

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“…Subsequently, a lipoprotein-specific signal peptidase (Lsp; also called signal peptidase II) cleaves the signal peptide at the N-terminal side of lipobox Cys. Finally, a phospholipid:apolipoprotein transacylase (Lnt) covalently attaches a fatty acid to the amide group of the N-terminal Cys to create the mature lipoprotein (Tokuda, 2009) (see Fig. 2).…”

Section: Discussionmentioning

confidence: 99%

“…the Ala of LimB; Fig. 2) are translocated to the outer membrane with the assistance of Lol sorting-system proteins in the periplasm (Tokuda, 2009;Yamaguchi et al, 1988). Based upon characteristics of the predicted LimB protein (Fig.…”

Section: Discussionmentioning

confidence: 99%

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

2011

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Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, 7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-offlight mass spectrometry. BLAST analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by .95 % on FW blots done in the presence of EDTA, imidazole, Ni 2+ or Zn 2+ , and roughly halved in the presence of Co 2+ or Fe 3+ . The limB gene was maximally expressed at 3-7 days post-infection in Coxiellainfected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (~7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (~8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.

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“…These lipoproteins from F. tularensis localize to the outer bacterial membrane and activate the mammalian inflammatory response through toll-like receptor 2 (TLR2) signaling (19 -22). The central importance of bacterial lipoproteins in the virulence of F. tularensis parallels other Gram-negative pathogens, including Neisseria meningitidis, Borrelia burgdorferi, and Escherichia coli, where lipoproteins are essential to their infectivity and their recognition by the host immune system (23,24).…”

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confidence: 99%

Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

Filippova

Weston

Kuhn

et al. 2013

Journal of Biological Chemistry

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Background: Acyl protein thioesterases control protein S-acylation at cellular membranes. Results: FTT258 is a serine hydrolase with broad substrate specificity that binds to bacterial membranes and exists in two distinct conformations. Conclusion: Conformational changes in FTT258 are correlated with catalytic activity. Significance: Structural rearrangement dually regulates the membrane binding and catalytic activity of acyl protein thioesterases.

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Biogenesis of Outer Membranes in Gram-Negative Bacteria

Cited by 117 publications

References 60 publications

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

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