The unstructured domain of colicin <scp>N</scp> kills <i><scp>E</scp>scherichia coli</i>

Johnson, Christopher L.; Ridley, Helen; Pengelly, Robert J.; Salleh, Mohd Zulkifli; Lakey, Jeremy H.

doi:10.1111/mmi.12260

Cited by 18 publications

(22 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This arrangement would position the unstructured N‐terminal T domain to search for a nearby opening in one of the ∼ 10 5 copies of OmpF. As has been shown to occur for the T domains of colicin E3 and E9 and suggested by the colicin N resistance of OmpF mutant G119N (Fourel et al ., ; Housden et al ., 2005; 2010; 2013; Yamashita et al ., ), the T domain could then thread into one of the three pores of the trimeric OmpF, using the recently identified OBS of colicin N to bind within the pore (Johnson et al ., ). Once the unfolded T domain has threaded through a pore of OmpF, presumably via some sort of pushing/pulling mechanism (Housden et al ., ; Thoren and Krantz, ), the TolA binding site on the T domain (Raggett et al ., ) would be exposed in the periplasm.…”

mentioning

confidence: 97%

Daring to be different: colicin N finds another way

Jakes

2014

Molecular Microbiology

View full text Add to dashboard Cite

Summary The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. In this issue of Molecular Microbiology, Johnson et al. show that the receptor-binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor-binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.

show abstract

mentioning

confidence: 97%

Daring to be different: colicin N finds another way

Jakes

2014

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…To define the in vivo effects more precisely we studied the kinetics of ColN activity which can be accurately followed in real time by using an ion‐selective electrode to measure the release of intracellular potassium from affected cells. We used this extremely sensitive assay (Johnson et al ., ) to measure the in vivo activity of ColN on various Keio (Baba et al ., ) LPS synthetic pathway mutants. First, the K + efflux measurements confirmed that Δ waaF cells, which fail to add the either HepII or HepIII, which we now have shown by STD‐NMR to be involved in ColN‐R binding, were resistant to ColN (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…ColN was shown to bind directly to OmpF by ITC but a chymotryptic fragment missing the N‐terminal 67 residues of the T‐domain or the isolated R‐domain alone showed much lower affinity (Evans et al ., 1996a). After the OBS in ColE9 was identified at the tip of the disordered T‐domain (Housden et al ., ), the site of the strong OmpF interaction in ColN was also pinpointed as a linear OBS epitope at the extreme N‐terminus (Johnson et al ., ). However, whereas a point mutation in OBS (F14G) was enough to abolish in vitro OmpF binding it hardly affected ColN activity (Johnson et al ., ).…”

Section: Discussionmentioning

confidence: 97%

“…After the OBS in ColE9 was identified at the tip of the disordered T‐domain (Housden et al ., ), the site of the strong OmpF interaction in ColN was also pinpointed as a linear OBS epitope at the extreme N‐terminus (Johnson et al ., ). However, whereas a point mutation in OBS (F14G) was enough to abolish in vitro OmpF binding it hardly affected ColN activity (Johnson et al ., ). Thus OmpF binding by the T‐domain OBS of ColN is not a critical receptor binding step.…”

Section: Discussionmentioning

confidence: 97%

“…ColN, like ColIa, utilizes and binds to a single type of OMP, OmpF, as its receptor (Evans et al ., 1996a) but its R‐domain (ColN‐R) has a very weak affinity for OmpF (Evans et al ., 1996b). This situation was recently clarified as it has been shown that ColN, like ColE9, contains an OBS in its flexible T‐domain (ColN‐T) (Johnson et al ., ). The Kd measured for ColN‐T binding to OmpF was found to be ∼ 3 μM, similar to the 2 μM and 24 μM measured for OBS1 and OBS2 respectively of ColE9 (Housden et al ., ).…”

Section: Introductionmentioning

confidence: 97%

See 2 more Smart Citations

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Johnson

Ridley

Marchetti

et al. 2014

Molecular Microbiology

Self Cite

View full text Add to dashboard Cite

SummaryColicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPSΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

show abstract

Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals

2014

Self Cite

View full text Add to dashboard Cite

Amphipols (APol) are polymers which can solubilise and stabilise membrane proteins (MP) in aqueous solutions. In contrast to conventional detergents, APol are able to keep MP soluble even when the free APol concentration is very low. Outer membrane protein F (OmpF) is the most abundant MP commonly found in the outer membrane (OM) of Escherichia coli. It plays a vital role in the transport of hydrophilic nutrients, as well as antibiotics, across the OM. In the present study, APol was used to solubilise OmpF to characterize its interactions with molecules such as lipopolysaccharides (LPS) or colicins. OmpF was reconstituted into APol by the removal of detergents using Bio-Beads followed by size-exclusion chromatography (SEC) to remove excess APol. OmpF/APol complexes were then analysed by SEC, dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM showed that in the absence of free APol–OmpF associated as long filaments with a thickness of ~6 nm. This indicates that the OmpF trimers lie on their sides on the carbon EM grid and that they also favour side by side association. The formation of filaments requires APol and occurs very rapidly. Addition of LPS to OmpF/APol complexes impeded filament formation and the trimers form 2D sheets which mimic the OM. Consequently, free APol is undoubtedly required to maintain the homogeneity of OmpF in solutions, but ‘minimum APol’ provides a new phase, which can allow weaker protein–protein and protein–lipid interactions characteristic of native membranes to take place and thus control 1D–2D crystallisation.

show abstract

The unstructured domain of colicin N kills Escherichia coli

Cited by 18 publications

References 54 publications

Daring to be different: colicin N finds another way

Daring to be different: colicin N finds another way

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals

Contact Info

Product

Resources

About