Colicin Pore-Forming Domains Bind toEscherichia ColiTrimeric Porins
23
Citation
15
Reference
10
Related Paper
Citation Trend
Abstract:
Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin−P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin−porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.Keywords:
Porin
Colicin
Colicin
Porin
Inner membrane
Cite
Citations (0)
A strain of Escherichia coli, selected on the basis of its resistance to colicin N, reveals distinct structural and functional alterations in unspecific OmpF porin. A single mutation [Gly-119-->Asp (G119D)] was identified in the internal loop L3 that contributes critically to the formation of the construction inside the lumen of the pore. X-ray structure analysis to a resolution of 3.0 A reveals a locally altered peptide backbone, with the side chain of residue Asp-119 protruding into the channel, causing the area of the constriction (7 x 11 A in the wild type) to be subdivided into two intercommunicating subcompartments of 3-4 A in diameter. The functional consequences of this structural modification consist of a reduction of the channel conductance by about one-third, of altered ion selectivity and voltage gating, and of a decrease of permeation rates of various sugars by factors of 2-12. The structural modification of the mutant protein affects neither the beta-barrel structure nor those regions of the molecule that are exposed at the cell surface. Considering the colicin resistance of the mutant, it is inferred that in vivo, colicin N traverses the outer membrane through the porin channel or that the dynamics of the exposed loops are affected in the mutant such that these may impede the binding of the toxin.
Colicin
Porin
Cite
Citations (126)
To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle.Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment.Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains.The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.
Cite
Citations (15)
Porins are pore-forming outer-membrane proteins which serve as a non-specific pathway for the entry of hydrophilic molecules into Gram-negative bacteria. We studied four strains of Haemophilus influenzae that had decreased permeability to chloramphenicol associated with diminished quantities of a 40 kDa major outer-membrane protein. Isogenic pairs of organisms containing and lacking this protein were compared. The latter strains grew more slowly and were less permeable to sucrose and raffinose. They were also more resistant to multiple hydrophilic antibiotics than an isogenic strain containing the 40 kDa protein and were less permeable to penicillin G and chloramphenicol. We conclude that the 40 kDa outer-membrane protein functions as a porin in H. influenzae.
Porin
Strain (injury)
Cite
Citations (32)
Colicin N kills sensitive Escherichia coli cells by first binding to its trimeric receptor (OmpF) via its receptor binding domain. It then uses OmpF to translocate across the outer membrane and in the process it also needs domains II and III of the protein TolA. Recent studies have demonstrated sodium dodecyl sulfate- (SDS) dependent complex formation between trimeric porins and TolA-II. Here we demonstrate that colicin N forms similar complexes with the same trimeric porins and that this association is unexpectedly solely dependent upon the pore-forming domain (P-domain). No binding was seen with the monomeric porin OmpA. In mixtures of P-domain and TolA with OmpF porin, only binary and no ternary complexes were observed, suggesting that binding of these proteins to the porin is mutually exclusive. Pull-down assays in solution show that porin−P-domain complexes also form in the presence of outer membrane lipopolysaccharide. This indicates that an additional colicin−porin interaction may occur within the outer membrane, one that involves the colicin pore domain rather than the receptor-binding domain. This may help to explain the role of porins and TolA-II in the later stages of colicin translocation.
Porin
Colicin
Cite
Citations (23)
The OmpF porin plays a central role during the colicin uptake by sensitive Escherichia coli cells. Lipopolysaccharide‐OmpF complexes ( ‐1b LPS‐OmpF), which contain one tightly bound and no loosely bound LPS molecules for each porin trimer, is able to recognize and bind to immobilized colicins. This association is specific to colicins A and N, which both use the OmpF porin as receptor, and depends on the presence of the porin‐receptor domain on the bacteriocin molecule. The ‐1b LPS‐OmpF complex protects sensitive cells against colicin A and N activity. The protection level depends on the native conformation, as demonstrated by heat denaturation of the trimeric porin which abolishes the protection. This indicates that the purified OmpF trimer presents an affinity site for the colicin which efficiently mimics the native cellular receptor site. These results are discussed with regard to the conformation of the receptor site and to the early steps of colicin uptake.
Porin
Colicin
Trimer
Cite
Citations (11)
Summary: Cells of Escherichia coli, newly infected with the colicin I factor (colI), showed an enhanced efficiency of transfer of this factor (HFC), and were also more likely to undergo lethal colicin synthesis, than were stably colicinogenic cells. Up to 20% of the cells of stably colI + strains were induced to produce colicin by ultraviolet irradiation, and from such irradiated cultures transfer of the colI factor occurred more efficiently. To account for these results, it is proposed that the colI factor exists as an autonomous non-chromosomal genetic element which sets up its own system of self-regulation within cells of stably colicinogenic strains.
Colicin
Cite
Citations (32)
Porin
Colicin
Immunogold labelling
C-terminus
Translocase
Inner membrane
Cite
Citations (8)
Colicin
Porin
Cell envelope
Internalization
Inner membrane
Cell membrane
Cite
Citations (85)
The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules > 600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (> 6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B 12 receptor in Escherichia coli , the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpF-binding sites, OBS1 (residues 2–18) and OBS2 (residues 54–63), which flank the TBE and bind with K d s of 2 and 24 μM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow cross-sectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein.
Colicin
Porin
Cite
Citations (89)