Bacterial iron acquisition mediated by outer membrane translocation and cleavage of a host protein

Mosbahi, Khédidja; Wojnowska, Marta; Albalat, Amaya; Walker, Daniel

doi:10.1073/pnas.1800672115

Cited by 33 publications

(48 citation statements)

References 37 publications

(53 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Levels of FerPot obtained from cell extracts increased at the same rate in whole cell extracts and rates of removal of ferredoxin from the media were similar ( Figure 1c). These data show that FusC does not play a role in protein import and the role of FusC in iron acquisition is likely restricted to proteolytic processing of ferredoxin as we previously reported 11 .…”

Section: Ferredoxin Import Is Independent Of Fusc But Requires Protonsupporting

confidence: 72%

“…Our recent discovery that ferredoxin is imported into the periplasm of P. carotovorum revealed an unprecedented example of protein uptake for nutrient acquisition in Gramnegative bacteria 11 . In this work, we define key aspects of the mechanism of ferredoxin transport across the outer membrane.…”

Section: Discussionmentioning

confidence: 99%

“…The Fus operon, TonB and ExbBD sequences were determined from the genome sequence of P. carotovorum LMG2410 27 . Genes in of PcLMG2410 were deleted using the lambda red method as described previously 11,28 . The primers used for amplifying the kanamycin cassette from pKD4 template plasmid, gene sequences from genomic DNA and plasmid site-directed mutagenesis are listed in Table S1.…”

Section: Generation Of Gene Knockout Strains and Plasmidsmentioning

confidence: 99%

“…FusC, FusBCTD, TonBCTD, FusANTR-GFP and all ferredoxin proteins were overproduced in E. coli and purified as described previously 11,12 , except spinach ferredoxin (FerSp) which was purchased from Sigma. GFP alone used as a negative control in ITC was produced by cleavage of FusANTR-GFP with TEV protease for 2 hours at RT, at 50:1 ratio.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…Growth enhancement in the presence of ferredoxin was performed on solid media as previously described 11 . Briefly, 10 ml of 0.8% pre-cooled agar was supplemented with 50 µl of mid-log culture in LB media and poured onto an LB agar base containing 400 µM 2,2'-bipyridine (and 0.2 mM IPTG where specified).…”

Section: Growth Enhancement Assaysmentioning

confidence: 99%

See 4 more Smart Citations

FusB energises import across the outer membrane through direct interaction with its ferredoxin substrate

Wojnowska

Walker

2019

Preprint

Self Cite

View full text Add to dashboard Cite

We recently described an example of protein import into Gram-negative bacteria for nutrient acquisition whereby phytopathogenic Pectobacterium spp. are able to import ferredoxin into the periplasm for proteolytic processing and iron release via the ferredoxin uptake system. Although the outer membrane ferredoxin receptor FusA and the periplasmic ferredoxin processing protease, FusC, have been identified, the mechanistic basis of ferredoxin import is poorly understood. Based on structural analysis of the FusC-ferredoxin complex, an additional role for FusC in direct mediation of protein import has been suggested.In this model, FusC acts as a periplasmic anchor that enables ferredoxin to traverse the outer membrane via FusA using a Brownian ratchet mechanism, akin to that proposed for the transport of mitochondrial proteins across the mitochondrial outer membrane. In this work we demonstrate that FusC does not play a role in protein import and show that, instead, protein translocation across the outer membrane is dependent on the TonB-like protein FusB. In contrast to the loss of FusC, loss of FusB or FusA abolishes ferredoxin transport to the periplasm, demonstrating that FusA and FusB work in concert to transport ferredoxin across the outer membrane. Interestingly, in addition to interaction with the TonB-box region of FusA, FusB also forms a complex with the ferredoxin substrate, with complex formation required for substrate transport. These data suggest that ferredoxin transport requires energy transduction from the cytoplasmic membrane via FusB for both removal of the FusA plug domain and for substrate translocation through the lumen of the FusA barrel.Protein translocation occurs by direct interaction with the an N-terminal intrinsically unstructured region of the toxin that, similar to the TBDRs, carries a TonB-binding motif 10 .We recently demonstrated that TBDR-mediated iron acquisition from the iron-sulphur cluster containing protein ferredoxin represents an unprecedented example of protein translocation into the bacterial cell for nutrient acquision 11 . Ferredoxin binding at the cell surface is mediated by the TBDR FusA and, following transport of intact ferredoxin into the periplasm, the substrate is subjected to proteolytic processing by the M16 protease FusC 11,12 .Cleavage by FusC results in release of the iron-sulphur cluster and is required for effective iron acquisition from ferredoxin by Pectobacterium. Together with the genes encoding FusA and FusC, the Fus operon contains two additional genes, with fusB encoding a TonBhomologue and fusD a putative ABC transporter 12 . Interestingly, the M-type pectocins M1 and M2, which we have previously described, parasitise the ferredoxin uptake system through an N-terminal ferredoxin domain that is highly homologous to plant ferredoxins 13,14 .

show abstract

Section: Ferredoxin Import Is Independent Of Fusc But Requires Protonsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%