Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability.

Sampson, Barbara A.; Misra, Rajeev; Benson, Spencer A.

doi:10.1093/genetics/122.3.491

Cited by 230 publications

(87 citation statements)

References 26 publications

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“…Given the enormous stability of paired β-strands within a membrane environment, it seemed unlikely that the displacement activity of Skp would also extend to late-stage folding intermediates. To address this possibility, we first examined the effects of skp deletion on the late-stalled substrates LptD4213 (45,54) The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine effect on LptD4213 assembly (SI Appendix, Fig. S1A), in agreement with previous observations that LptD4213 is not a substrate of DegP (46,55).…”

Section: Combs and Silhavysupporting

confidence: 76%

“…Given the enormous stability of paired β-strands within a membrane environment, it seemed unlikely that the displacement activity of Skp would also extend to late-stage folding intermediates. To address this possibility, we first examined the effects of skp deletion on the late-stalled substrates LptD4213 ( 45 , 54 ) and LamB ΔL5 , both of which would be predicted to have templated a substantial number of β-strands prior to the point of stalling. Deletion of skp and/or degP had no discernible effect on LptD4213 assembly ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

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The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

Combs

Silhavy

2021

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

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The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

show abstract

Section: Combs and Silhavysupporting

confidence: 76%

“…Given the enormous stability of paired β-strands within a membrane environment, it seemed unlikely that the displacement activity of Skp would also extend to late-stage folding intermediates. To address this possibility, we first examined the effects of skp deletion on the late-stalled substrates LptD4213 ( 45 , 54 ) and LamB ΔL5 , both of which would be predicted to have templated a substantial number of β-strands prior to the point of stalling. Deletion of skp and/or degP had no discernible effect on LptD4213 assembly ( SI Appendix , Fig.…”

Section: Resultsmentioning

confidence: 99%

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

Combs

Silhavy

2021

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

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“…2C ) by adding MgrB peri to an E. coli culture. To enable the MgrB peri peptide to access the periplasm, we used an imp-4213 strain, which has increased outer membrane permeability ( 35 ). We monitored PhoQ activity with a GFP reporter fused to the promoter region of a PhoQ/PhoP-regulated gene (pUA66 P mgtLA -gfp ) in the presence or absence of chemically synthesized MgrB peri .…”

Section: Resultsmentioning

confidence: 99%

Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase

et al. 2020

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The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella and related bacteria. Previous studies have shown that MgrB, a 47 amino acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine scanning mutagenesis of the periplasmic region of MgrB further indicates that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

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“…1). Unlike the yfgL mutations, the intragenic suppressor strains carrying imp4213 cannot grow on this medium (Sampson et al, 1989). However, we discovered that mutation imp61 increases resistance to all the other antibiotics tested (Table 1).…”

Section: Introduction Mycin But Not Vancomycin or Erythromycin (Figurementioning

confidence: 88%

Chemical Conditionality

Ruiz

Falcone

Kahne

et al. 2005

Cell

275

169

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The assembly of the Escherichia coli outer membrane (OM) is poorly understood. Although insight into fundamental cellular processes is often obtained from studying mutants, OM-defective mutants have not been very informative because they generally have nonspecific permeability defects. Here we show that toxic small molecules can be used in selections employing strains with permeability defects to create particular chemical conditions that demand specific suppressor mutations. Suppressor phenotypes are correlated with the physical properties of the small molecules, but the mutations are not in their target genes. Instead, mutations allow survival by partially restoring membrane impermeability. Using "chemical conditionality," we identified mutations in yfgL, and, here and in the accompanying paper by Wu et al. published in this issue of Cell (Wu et al., 2005), we show that YfgL is part of a multiprotein complex involved in the assembly of OM beta barrel proteins. We posit that panels of toxic small molecules will be useful for generating chemical conditionalities that enable identification of genes required for organelle assembly in other organisms.

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Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability.

Cited by 230 publications

References 26 publications

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase

Chemical Conditionality

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