TonB and uptake of the siderophore enterobactin

Iron

Found in iron sulphur clusters and heme groups Catalyses redox reactions Comes in 2 forms

Fe(II) – soluble; can diffuse freely through outer membrane; uptake by ABC transporter; easily oxidised to Fe(III)

Fe(III) – forms insoluble hydroxides via Fenton reaction; leads to accumulation of toxic hydroxyl radicals

Iron

Important in pathogenicity

Excess iron increases virulence of many human pathogens including Escherichia, Klebsiella, Salmonella

Direct correlation between LD50 of Vibrio vulnificans and iron availability

Iron

Iron therefore kept bound to proteins in cellular environments

In mammals, bound to transferrin, lactoferrin, and ferritin among others

Free concentration in humans 10^-24M How do bacteria obtain iron?

Siderophores

Siderophores are small iron chelating molecules produced by bacteria

Have specific uptake systems

Include enterobactin

Enterobactin uptake

TonB

TonB provides energy for FepA

Energy generated by protomotive force on cytoplasmic membrane by ExbB:ExbD

TonB stores energy as conformational change

Energises FepATonB C terminal in

complex with FhuA

TonB

26kDa membrane protein, 239 AA in E. Coli 3 domains: N terminal, linker and C terminal N terminal (residues 1-32)

Interacts with ExbB and ExbD Transmembrane helix and short

cytoplasmic tail Contains Sec signal sequence for export

into the CM Replacement of this region results in loss

of interaction with ExbB and loss of activity

TonB

Linker region (residues 66-102) Proline rich; contains repeats of Glu-Pro and

Lys-Pro Highly structured conformation Can extend 10nm, allows complex to span the

periplasm Full length only necessary in high salinity

TonB C terminal

TonB C terminal interacts with outer membrane receptors

Forms a complex with receptor through B strand exchange

B strand from receptor displaces TonB B strand forming mixed B sheet

TonB C terminal (yellow) in complex with FhuA showing B sheet structure

Controversy

Multiple lines of evidence have shown TonB has the ability to dimerise with itself

Unlikely to represent in vivo interaction

Project

Aim To create a website combining literature

review with bioinformatic analysis of the enterobactin uptake system

Tools Microsoft expression web BLAST search ClustalW Jalview

Website

Can show multiple forms of media Easy to navigate to particular information Large diagrams do not obscure text Interactability

Demonstration of website

Findings of bioinformatic analysis

C terminal of TonB highly conserved Aliphatic residue at Ile193 (Leu, Ile, Val) Tyrosine-proline pairing Tyr163-Pro164 Large insertions in Synechococcus spp.