Colicin-Mediated Transport of DNA through the Iron Transporter FepA

Cohen-Khait, Ruth; Harmalkar, Ameya; Pham, Phuong; Webby, Melissa N.; Housden, Nicholas G.; Elliston, Emma; Hopper, Jonathan T. S.; Mohammed, Shabaz; Robinson, Carol V.; Gray, Jeffrey J.; Kleanthous, Colin

doi:10.1128/mbio.01787-21

Cited by 11 publications

(13 citation statements)

References 47 publications

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“…The labelling efficiency (typically 0.8 fluorophores per protein) was estimated spectrophotometrically (V550 spectrophotometer, Jasco). Colicin B–GFP or colicin B–mCherry expression and purification: the expression and purification of these proteins has been previously described 27 . Pyocin S5–AF488 expression and purification: the expression and purification of this protein has previously been described 55 .…”

Section: Methodsmentioning

confidence: 99%

“…We focused on two TonB-dependent transporters (TBDTs), the siderophore transporter FepA and the vitamin B 12 transporter BtuB. Both TBDTs were labelled with high-affinity, fluorescently labelled colicins that exploit these OMPs as receptors 8 , 27 . ColB was fused to mCherry or GFP to locate FepA and ColE9 was labelled with Alexa Fluor 488 (AF488) to locate BtuB.…”

Section: Bam Activity Is Linked To the Cell Cyclementioning

confidence: 99%

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Peptidoglycan maturation controls outer membrane protein assembly

Mamou

Corona

Cohen-Khait

et al. 2022

Nature

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Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1–5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the β-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.

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Section: Methodsmentioning

confidence: 99%

Section: Bam Activity Is Linked To the Cell Cyclementioning

confidence: 99%

Peptidoglycan maturation controls outer membrane protein assembly

Mamou

Corona

Cohen-Khait

et al. 2022

Nature

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“…Cells (5 to 8 ml) were added to a 10 cm by 10 cm petri dish on an ice pack in a CL-1000 Ultraviolet Crosslinker and exposed to UV (365 nm) for 90 min, with mixing at 45 min. OM fractions containing OmpF, BtuB ( 42 ), or FepA ( 66 ) were extracted following lysis by sonication (no lysozyme was added; thus, PG-interacting proteins remained insoluble) and through a series of centrifugation steps, with solubilization in 2% β-OG ( 42 , 67 ).…”

Section: Methodsmentioning

confidence: 99%

Lipids mediate supramolecular outer membrane protein assembly in bacteria

et al. 2022

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β Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric β barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

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“…The labelling efficiency (typically 0.8 fluorophores/protein) was estimated spectrophotometrically (V550 spectrophotometer, Jasco). Colicin B-GFP/mCherry expression and purification: The expression and purification of these proteins has been previously described at Cohen-Khait et al ., 2021 26 .…”

Section: Methodsmentioning

confidence: 99%

“…We focused on two TonB-dependent transporters (TBDTs), the siderophore transporter FepA and the vitamin B 12 transporter BtuB. Both TBDTs were labelled with high-affinity, fluorescently-labelled colicins which exploit these OMPs as receptors 8, 26 . ColB, specific for FepA, was fused to mCherry or GFP, and ColE9, specific for BtuB, was labelled with AF488.…”

Section: Mainmentioning

confidence: 99%

Peptidoglycan maturation controls spatiotemporal organisation of outer membrane proteins in Escherichia coli

Mamou

Corona

Cohen-Khait

et al. 2022

Preprint

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Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial to the maintenance of cellular integrity and enable survival in challenging environments. The functionality of the outer membrane relies on outer membrane proteins (OMPs), which are inserted by the β-barrel assembly machine, BAM. Previous work has shown that growing Escherichia coli cells segregate old OMPs towards the poles by an unknown mechanism. Here, we demonstrate that peptidoglycan underpins the spatiotemporal organisation of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa, associates with BAM poorly and has little impact on its activity, leading to preferential insertion of OMPs at division sites. Synchronising OMP biogenesis to cell wall growth enables bacteria to replenish their OMPs by binary partitioning. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation. This coordination offers new possibilities for the design of antibiotics that disrupt cell envelope integrity.

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Colicin-Mediated Transport of DNA through the Iron Transporter FepA

Cited by 11 publications

References 47 publications

Peptidoglycan maturation controls outer membrane protein assembly

Peptidoglycan maturation controls outer membrane protein assembly

Lipids mediate supramolecular outer membrane protein assembly in bacteria

Peptidoglycan maturation controls spatiotemporal organisation of outer membrane proteins in Escherichia coli

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