Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of  -Barrel Outer Membrane Proteins, Including That of BamA Itself

Leonard-Rivera, Margaret; Misra, Rajeev

doi:10.1128/jb.00825-12

Cited by 55 publications

(59 citation statements)

References 37 publications

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“…Our results also show that the function of BamB becomes critical when this communication network is compromised. How structural information is transduced from P5 to L6 remains to be determined, although it is conceivable that L6 directly contacts the periplasmic domain of BamA as its length is more than sufficient to traverse the barrel lumen and extend into the periplasmic space (28,29). Notably, suppressor mutations in L6 restore viability to strains lacking BamB, -C, and -E, corroborating our assertion that a functional relationship exists between the BamA β-barrel and the Bam lipoproteins (9).…”

Section: Discussionsupporting

confidence: 55%

Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli

Rigel

Ricci

Silhavy

2013

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

120

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In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMPs) are assembled by the beta-barrel assembly machine (Bam) complex. The essential components of this complex are the OMP BamA [which contains a carboxyl-terminal β-barrel and an amino-terminal periplasmic module composed of five polypeptide transport associated (POTRA) domains] and the lipoprotein BamD. In Escherichia coli, the Bam complex also contains three nonessential lipoproteins (BamBCE), all of which require the barrel-proximal POTRA domain (P5) for stable interactions with BamA. We have previously reported that the BamA β-barrel assumes two different conformations. A method for conformation-specific labeling of BamA described here reveals that these conformers reflect the degree of surface exposure of the conserved sixth extracellular loop (L6). L6 is surface accessible in one conformation but not in the other, likely because it occupies the lumen of the BamA β-barrel in the latter case. A gain-of-function mutation that promotes Bam activity (bamDR197L) and a loss-of-function mutation that decreases the activity of Bam (ΔbamE) both favor surface exposure of BamA L6, suggesting that BamD and BamE normally act to control L6 exposure through opposing functions. These results, along with the synthetic lethality of the bamDR197L ΔbamE double mutant, imply a cyclic mechanism in which the Bam lipoproteins regulate the conformation of BamA during the OMP assembly reaction. Our results further suggest that BamDE controls L6 exposure via conformational signals transmitted through P5 to L6.genetics | OM biogenesis | Omp85 | membrane protein folding |

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Section: Discussionsupporting

confidence: 55%

Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli

Rigel

Ricci

Silhavy

2013

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

120

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“…It has been proposed that the passenger domain of its substrate passes through an enlarged barrel combining those of TamA and the autotransporter, after which the composite barrel splits to release the autotransporter's barrel into the membrane 12 . The fact that two features of the barrel, L6 and a specific sequence motif in the b-barrel strand B13 are conserved and essential for function 10,11,25,26 corroborates the essential role of the b barrel in the transport process for the entire superfamily.…”

Section: Discussionmentioning

confidence: 76%

“…We have recently shown that H1 is mobile within the pore and can move to the periplasm 23 . The extracellular loop L6, which folds back into the pore, harbours a conserved sequence motif characteristic of the Omp85 superfamily and is essential for function 9,[24][25][26] . According to the current secretion model, FhaB, the FHA precursor, is exported across the cytoplasmic membrane by the Sec machinery and chaperoned by DegP in the periplasm [27][28][29] .…”

mentioning

confidence: 99%

Translocation path of a substrate protein through its Omp85 transporter

et al. 2014

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TpsB proteins are Omp85 superfamily members that mediate protein translocation across the outer membrane of Gram-negative bacteria. Omp85 transporters are composed of N-terminal POTRA domains and a C-terminal transmembrane b-barrel. In this work, we track the in vivo secretion path of the Bordetella pertussis filamentous haemagglutinin (FHA), the substrate of the model TpsB transporter FhaC, using site-specific crosslinking. The conserved secretion domain of FHA interacts with the POTRA domains, specific extracellular loops and strands of FhaC and the inner b-barrel surface. The interaction map indicates a funnel-like pathway, with conformationally flexible FHA entering the channel in a non-exclusive manner and exiting along a four-stranded b-sheet at the surface of the FhaC barrel. This sheet of FhaC guides the secretion domain of FHA along discrete steps of translocation and folding. This work demonstrates that the Omp85 barrel serves as a channel for translocation of substrate proteins.

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“…Note that vancomycin, due to its large size (molecular weight, 1,485.71), is normally excluded from entering Gram-negative cells. However, E. coli mutants defective in the biogenesis of the outer membrane display elevated susceptibility to vancomycin (55,56,57,58). Therefore, PA␤N clearly has the potential to breach the outer membrane permeability barrier if not expelled from the cells.…”

Section: Discussionmentioning

confidence: 99%

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

et al. 2015

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The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine ␤-naphthylamide (PA␤N) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PA␤N and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PA␤N, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PA␤N. Strong action of PA␤N in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PA␤N acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PA␤N acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PA␤N in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PA␤N on membrane integrity are compounded in cells unable to extrude PA␤N. IMPORTANCEThe increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds. Multidrug resistance among human bacterial pathogens remains a grave social concern. Numerous strategies have been proposed to curtail the rampant increase in multidrug resistance among human pathogens, ranging from the effective integration of pharmacokinetic and pharmacodynamic parameters and implementation of antimicrobial stewardship (1) to the development of novel antibiotics based on either an existing or a novel chemical scaffold, exploitation of new cellular targets, and directly tackling the cellular mechanisms that confer multidrug resistance (2). Efflux of antibiotics from the cell is one of the common mechanisms of antibiotic resistance in bacteria, with resistance developing when the rate of drug efflux across the membrane exceeds that of drug influx (3).Bacterial genomes encode several membrane-bound multidrug efflux systems (4, 5). These systems are usually under t...

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Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of -Barrel Outer Membrane Proteins, Including That of BamA Itself

Cited by 55 publications

References 37 publications

Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli

Conformation-specific labeling of BamA and suppressor analysis suggest a cyclic mechanism for β-barrel assembly in Escherichia coli

Translocation path of a substrate protein through its Omp85 transporter

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

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