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The gain-of-function allele bamAE470K bypasses the essential requirement for BamD in β-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) are assembled into the OM by the beta-barrel assembly machine (BAM), which is composed of one OMP, BamA, and four lipoproteins, BamBCDE. BamBCE can be removed individually with only minor effects on barrier function. However, depletion of BamA or BamD, the only BAM components conserved in all diderm bacteria, causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamAE470K, that bypasses the requirement for BamD. Although bamD::kan bamAE470K cells exhibit growth defects, they assemble OMPs with surprising robustness. Our results demonstrate that the reactions required for beta-barrel folding and membrane integration are catalyzed solely by BamA.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Escherichia Coli

SUBMITTER: Meera Gupta

LAB HEAD: Martin Wuhr

PROVIDER: PXD017962 | Pride | 2020-07-03

REPOSITORIES: Pride

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The synthetic phenotype of bamBbamE double mutants results from a lethal jamming of the Bam complex by the lipoprotein RcsF

Project description:In Gram-negative bacteria, such as Escherichia coli, the heteropentomeric -barrel assembly machine (Bam) folds and inserts proteins into the outer membrane of the cell envelope. Here, we show that the conditional lethal phenotype of a mutant lacking two of the three nonessential lipoproteins, BamB and BamE, is caused by the lethal jamming of the stripped down Bam complex by a normally surface-exposed lipoprotein, RcsF. Our study highlights the importance of the nonessential Bam complex lipoproteins, BamB and BamE, in regulating the interaction between the essential Bam proteins, BamA and BamD and expands our understanding of the role of the Bam complex in outer membrane biogenesis.

2019-04-24 | PXD012335 | Pride

The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins

Project description:In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β‐barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling and in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. The functional implications of these interactions were investigated by two alternative and complementary approaches. The number of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation was evaluated by both western blot analysis and quantitative tandem mass spectrometry in MRM scan mode. In both experiments, a gradual decrease in outer membrane protein production with time was observed because of Mag-2 treatment.

2023-03-11 | PXD037418 | Pride

The role of the periplasmic chaperones SurA, Skp and DegP for fitness, outer membrane integrity, antibiotic susceptibility and virulence of Acinetobacter baumannii AB5075: same-same, but different?

Project description:The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants or dryness. Main constituents of the OM are integral OM β-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli (Ec), Yersinia enterocolitica (Ye) and Pseudomonas aeruginosa (Pa), the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage, the chaperones SurA, Skp and DegP, which facilitate the passage of β-barrel OMPs through the periplasm, and the β-barrel assembly machinery (BAM) which facilitates insertion into the OM. In Ec, Ye and Pa the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotics, and hypovirulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii (Ab), we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to Ec, Ye and Pa, AB5075 tolerates the lack of SurA, Skp or DegP with only weak mutant phenotypes. While the double knockout strains surAskp and surAdegP are conditionally lethal in Ec, all double deletions were well tolerated by AB5075. Strikingly, even a triple knockout strain of AB5075, lacking surA, skp and degP, was viable. Our findings underline the extreme adaptibility of Ab and suggest the existence of mechanisms bypassing or acting redundantly to the known OMP biogenesis pathway comprising SurA, Skp and DegP.

2022-08-29 | PXD022004 | Pride

Role of proteins belonging to the OMP biogenesis machinery for virulence of Yersinia enterocolitica

Project description:The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier protecting Gram-negative species from harmful environmental influences. At the same time it facilitates the import of nutrients, the export of proteins, the passage of signaling molecules and also harbors proteins associated with virulence. Transmembrane traffic particularly is facilitated by membrane-integral proteins. In order to insert these into the OM, an essential oligomeric membrane-associated protein complex, the beta-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones (termed the OMP biogenesis machinery as an entity herein) may serve as attractive targets to develop novel antimicrobial or antiinfective agents. We aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica (Ye) as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, both BamB and SurA are promising targets for the development of new antimicrobials in the future.

2018-10-17 | PXD004429 | Pride

An Outer Membrane Protein-Targeting Antibiotic Against Resistant Gram-Negative Bacterial Pathogens

Project description:There is an urgent need for novel antibiotics against carbapenem and 3rd generation cephalosporin-resistant Gram-negative pathogens, for which the last-resort antibiotics have lost most of their efficacy. We describe here a novel class of synthetic antibiotics that was inspired from natural product-derived scaffolds. The antibiotics have an unprecedented mechanism of action, which targets the main component (BamA) of the Bam folding machinery required for folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. This OMPTA (outer membrane protein-targeting antibiotic) class shows potent activity against multidrug-resistant Gram-negative ESKAPE pathogens and overcomes colistin-resistance both in vitro and in vivo. A clinical candidate has the potential to address life threatening Gram-negative infections with high unmet medical need.

2019-10-22 | PXD010174 | Pride

Effect of OMP on gene expression in the olfactory epithelium

Project description:OMP is expressed only in mature olfactory sensory neurons, arguing that its function is specially suited to the needs of OSNs. However none of the properties of OMP suggest any direct role in regulating gene expression in OSNs. Our data confirms that gene expression in the olfactory epithelium of mice lacking OMP is indistinguishable from mice expressing OMP. We used affymerix M430v2.0 gene chips to cover as much of genome as possible and observed no statistically significant differences in mRNA abundance between the two genotypes.These results imply that OMPs ability to regulate signal transduction with in OSNs has little effect on gene expression and that its ability to promote mitosis in neighbouring cells (under culture conditions) is insufficiently active under normal laboratory housing conditions to generate detectable differences in gene expression patterns. Keywords: genetic modification

2007-04-30 | GSE4927 | GEO

Transcription profiling of mouse olfactory epithelium from heterozygous and homozygouse OMP-GFP animals

2008-06-13 | E-GEOD-4927 | biostudies-arrayexpress

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Project description:Insertion of lipopolysaccharide (LPS) into the outer membrane (OM) of Gram-negative bacteria is mediated by a druggable OM translocon consisting of a beta-barrel membrane protein, LptD, and a lipoprotein, LptE. The beta-barrel assembly machinery (BAM) assembles LptD together with LptE to form a plug-and-barrel structure. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls LPS translocon activation. Here we report the discovery of LptM (formerly YifL), a conserved lipoprotein that assembles together with LptD and LptE at the BAM complex. We demonstrate that LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds and be released as mature LPS translocon by the BAM complex. Inactivation of LptM causes the accumulation of non-natively oxidized LptD, making disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon.

2023-10-11 | PXD041774 | Pride

Evidence for widespread cytoplasmic structuring into mesoscopic condensates

Project description:Eukaryotic cytoplasm organizes itself via membrane-bound organelles and membrane-less biomolecular condensates (BMCs). Known BMCs exhibit liquid-like properties and are typically visualized on the scale of ~1 µm. They have been studied mostly by microscopy, examining select individual proteins. Here, we investigate the global organization of native cytoplasm with quantitative proteomics, using differential pressure filtration and dilution experiments. These assays revealed that BMCs form predominantly at the mesoscale of ~100 nm, which we validated via microscopy. Our data indicate that at least 18% of the proteome is organized via such mesoscale BMCs, suggesting that cells widely employ dynamic liquid-like clustering to organize their cytoplasm, at surprisingly small length scales.

2023-11-02 | PXD029879 | Pride

Localisation of outer membrane proteins in Treponema denticola by quantitative proteome analyses of outer membrane vesicles and cellular fractions

Project description:The identification and localisation of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging largely due to the structural uniqueness of their outer membranes (OM), the high abundance of inner membrane (IM) lipoproteins and the low abundance of Omps. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localised to the OM comprising 59 lipoproteins, 25 β-barrel proteins and six other putative OM-associated proteins. 28 lipoproteins were localised to the IM and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signal for their differential localisation to the OM and IM. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant β-barrel Omps TDE2508 and TDE1717 formed large complexes. Several Omps were identified to have homologs in T. pallidum that have not yet been identified.

2019-02-20 | PXD011495 | Pride

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