High level expression of His-tagged colicin pore-forming domains and reflections on the sites for pore formation in the inner membrane

Fridd, Susan L.; Gökçe, İ̇sa; Lakey, Jeremy H.

doi:10.1016/s0300-9084(02)01418-9

Cited by 16 publications

(18 citation statements)

References 32 publications

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“…1A). E. coli ’s colicin N immunity protein (Cni) (35, 36), although slightly smaller (174 aa), is equally constituted of 4 TMHs and shares ~20% amino acid identity with ImnH proteins.…”

Section: Resultsmentioning

confidence: 99%

A Natural ChimericPseudomonasBacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Ghequire

Kemland

Anoz-Carbonell

et al. 2017

mBio

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Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin’s activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module. We demonstrate strain-specific inhibitory activity for PmnH, a Pseudomonas bacteriocin with an unprecedented dual-toxin architecture, hosting both a colicin M domain, potentially interfering with peptidoglycan synthesis, and a novel colicin N-type domain, a pore-forming module distinct from the colicin Ia-type domain in Pseudomonas aeruginosa pyocin S5. A downstream-linked gene product confers PmnH immunity upon susceptible strains. This protein, ImnH, has a transmembrane topology similar to that of Pseudomonas colicin M-like and pore-forming immunity proteins, although homology with either of these is essentially absent. The enhanced killing activity of PmnH under iron-limited growth conditions reflects parasitism of the ferrichrome-type transporter for entry into target cells, a strategy shown here to be used as well by monodomain colicin M-like bacteriocins from pseudomonads. The integration of a second type of toxin module in a bacteriocin gene could offer a competitive advantage against bacteria displaying immunity against only one of both toxic activities.

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“…1A). E. coli ’s colicin N immunity protein (Cni) (35, 36), although slightly smaller (174 aa), is equally constituted of 4 TMHs and shares ~20% amino acid identity with ImnH proteins.…”

Section: Resultsmentioning

confidence: 99%

A Natural ChimericPseudomonasBacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Ghequire

Kemland

Anoz-Carbonell

et al. 2017

mBio

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“…Characterization of WT and Mutant p190H 6 -The N-terminal hexahistidine tag has been incorporated previously in the channel domain of colicin A and N, for ease of purification (30). Structural and functional characterizations of His-tagged colicin A and N channel domains have shown that the incorporation of the hexahistidine tag did not significantly alter the channel properties of these two proteins (30).…”

Section: Resultsmentioning

confidence: 99%

“…Structural and functional characterizations of His-tagged colicin A and N channel domains have shown that the incorporation of the hexahistidine tag did not significantly alter the channel properties of these two proteins (30). In the present study, we used intrinsic Trp fluorescence as a probe of protein conformation as well as in vitro channel activity analysis to assess the impact of how the engineered hexahistidine tag, the introduction of cysteine residues, and the bimane labeling affected the structural and functional integrity of the protein.…”

Section: Resultsmentioning

confidence: 99%

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

Musse

Wang

deLeon

et al. 2006

Journal of Biological Chemistry

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Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membranebound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic ␣-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic ␣-helix (3.7 ؎ 0.1 residues per turn and 97 ؎ 3.0°, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, because of nutrient depletion or overcrowding, and these proteins often target sensitive bacterial strains (1). The lethal actions of colicins against their target cells are manifested in a number of different modes that include the following: (i) formation of depolarizing ion channels in the cytoplasmic membrane, (ii) inhibition of protein and peptidoglycan synthesis, and (iii) degradation of cellular nucleic acids (1-7). In this context, the bacterial machinery responsible for colicin biological activity feature important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that harbors the naturally occurring colE1 plasmid; the whole colicin consists of three functional segments, the translocation, receptor-binding domains, and channel-forming domains. Initially, the receptor-binding domain (8) interacts with the vitamin B 12 receptor of target cells (9). Following receptor recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm (10). In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state and then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The cha...

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“…This construct, named pKSJ373, gave a poor yield of badly degraded or impure E1ΔR, so the genes for ImmE1 and E1ΔR were subsequently cloned into pET52-b (Novagen/EMD Millipore) so that the colicin deletion protein would be overexpressed with a cleavable C-terminal His 10 tag for subsequent purification by metal chelation chromatography. Since cells bearing a gene for colicin must also carry the ability to synthesize the cognate immunity protein for that colicin (45), the gene for ImmE1 was first cloned with its own promoter sequence by PCR from pKSJ373, with AvrII sites at either end, and inserted into the unique AvrII site in pET52-b, creating pKSJ375. The resulting plasmid confers immunity to E1 on cells bearing it.…”

Section: Methodsmentioning

confidence: 99%

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Jakes

2017

J Bacteriol

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Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCEThe Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.KEYWORDS TolC, colicins, drug efflux, membrane translocation, toxins E scherichia coli competes for scarce resources by making plasmid-encoded protein toxins called colicins, which efficiently kill closely related bacteria. All of the few dozen or so colicins that have been identified kill their targets by one of a few basic mechanisms: (i) making an ion-permeable channel in the inner membrane of the target cell, which depolarizes and kills the cell (1); (ii) enzymatically cleaving its rRNA, tRNA, or DNA in the cytoplasm (2, 3); or (iii) degrading peptidoglycan precursors in the periplasm (4, 5). Regardless of their ultimate killing mechanism, all colicins must cross at least the outer membrane in order to reach their targets. The way one particular colicin, E1, makes that transit is the subject of this study.Colicins have a well-defined domain structure, with the killing (catalytic or channelforming) domain at the ...

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High level expression of His-tagged colicin pore-forming domains and reflections on the sites for pore formation in the inner membrane

Cited by 16 publications

References 32 publications

A Natural ChimericPseudomonasBacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

A Natural ChimericPseudomonasBacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

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