Dataset Information

The gain-of-function allele bamA _E470K bypasses the essential requirement for BamD in ?-barrel outer membrane protein assembly.

ABSTRACT: The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral ?-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the ?-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamA _E470K , that bypasses the requirement for BamD. Although bamD::kan bamA _E470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

SUBMITTER: Hart EM

PROVIDER: S-EPMC7414184 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

The gain-of-function allele bamAE470K bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly.

Hart Elizabeth M EM Gupta Meera M Wühr Martin M Silhavy Thomas J TJ

Proceedings of the National Academy of Sciences of the United States of America 20200716 31

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP-BamA-and four lipoproteins-BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD ...[more]

PMID: 32675245

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Project description:Most integral outer membrane proteins (OMPs) of Gram-negative bacteria, such as Escherichia coli, assume a ?-barrel structure. The ?-barrel assembly machine (Bam), a five-member complex composed of ?-barrel OMP BamA and four associated lipoproteins, BamB, BamC, BamD, and BamE, folds and inserts OMPs into the outer membrane. The two essential proteins BamA and BamD interact to stabilize two subcomplexes, BamAB and BamCDE, and genetic and structural evidence suggests that interactions between BamA and BamD occur via an electrostatic interaction between a conserved aspartate residue in a periplasmic domain of BamA and a conserved arginine in BamD. In this work, we characterize charge-change mutations at these key BamA and BamD residues and nearby charged residues in BamA with respect to OMP assembly and Bam complex stability. We show that Bam complex stability does not correlate with function, that BamA and BamD must adopt at least two active conformational states during OMP assembly, and that these charged residues are not required for function. Rather, these charged residues are important for coordinating the activities of BamA and BamD to allow efficient OMP assembly. We present a model of OMP assembly wherein recognition and binding of unfolded OMP substrate by BamA and BamD induce a signaling interaction between the two proteins, causing conformational changes necessary for the assembly reaction to proceed. By analogy to signal sequence recognition by SecYEG, we believe these BamA-BamD interactions ensure that both substrate and complex are competent for OMP assembly before the assembly reaction commences.IMPORTANCE Conformational changes in the proteins of the ?-barrel assembly machine (Bam complex) are associated with the folding and assembly of outer membrane proteins (OMPs) in Gram-negative bacteria. We show that electrostatic interactions between the two essential proteins BamA and BamD coordinate conformational changes upon binding of unfolded substrate that allow the assembly reaction to proceed. Mutations affecting this interaction are lethal not because they destabilize the Bam complex but rather because they disrupt this coordination. Our model of BamA-BamD interactions regulating conformation in response to proper substrate interaction is reminiscent of conformational changes the secretory (Sec) machinery undergoes after signal sequence recognition that ensure protein quality control.

Dataset Information

The gain-of-function allele bamA _E470K bypasses the essential requirement for BamD in ?-barrel outer membrane protein assembly.

Publications

The gain-of-function allele <i>bamA</i><sub><i>E470K</i></sub> bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

The gain-of-function allele bamA E470K bypasses the essential requirement for BamD in ?-barrel outer membrane protein assembly.

Publications

The gain-of-function allele <i>bamA</i><sub><i>E470K</i></sub> bypasses the essential requirement for BamD in β-barrel outer membrane protein assembly.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

The gain-of-function allele bamA _E470K bypasses the essential requirement for BamD in ?-barrel outer membrane protein assembly.