The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Karen S. Jakes - One of the best experts on this subject based on the ideXlab platform.
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The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding.
Journal of bacteriology, 2016Co-Authors: Karen S. JakesAbstract: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. Importance The 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.
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Translocation of TonB Dependent Colicins
Biophysical Journal, 2013Co-Authors: Karen S. JakesAbstract:Colicins are protein toxins produced by E. coli to kill closely related competitor E. coli. Colicins initially attach to target bacteria by binding to outer membrane receptors, most of which are TonB-dependent nutrient transporters—22-stranded -barrels plugged by their amino-terminal domains. One group of Colicins, which use the Tol proteins, TolA,B,Q,R, for uptake, have been shown to use OmpF or TolC as their translocators after the initial step when the Colicin binds to its receptor. The N-terminal translocation domain of the Colicin actually threads into the translocator (Housden et al. (2010) PNAS: 107: 20412).Until recently, no translocator or “second receptor” had been identified for the other family of Colicins, those that use TonB and ExbB and D for uptake. Recently, Colicin Ia was shown to use a second copy of its primary receptor, Cir, as its translocator (Jakes, K.S. and Finkelstein, A. (2010) Mol. Micro.: 75: 567). In an attempt to determine whether other TonB-dependent Colicins also use a second copy of their primary receptor as a translocator, I have made chimera constructs with Colicin M, substituting the receptor-binding domain of Colicin E3 for that of Colicin M, and also deleting the entire receptor-binding domain. These constructs were insoluble and went into inclusion bodies. Dissolving the inclusion bodies in 8M urea, renaturing by dilution and dialysis, and purifying by nickel chelation yielded a small amount of pure protein that had no in vivo killing activity. However, the chimera blocks killing by Colicin E3, demonstrating that it binds the E3 receptor, BtuB. Osmotic shock to bypass receptor binding and translocation showed that the enzymatic moiety of the chimera is also active.These results suggest that Colicin M translocates differently than Colicin Ia and may not normally use a translocator remote from its primary binding site.
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Translocation trumps receptor binding in Colicin entry into Escherichia coli.
Biochemical Society transactions, 2012Co-Authors: Karen S. JakesAbstract:Of the steps involved in the killing of Escherichia coli by Colicins, binding to a specific outer-membrane receptor was the best understood and earliest characterized. Receptor binding was believed to be an indispensable step in Colicin intoxication, coming before the less well-understood step of translocation across the outer membrane to present the killing domain to its target. In the process of identifying the translocator for Colicin Ia, I created chimaeric Colicins, as well as a deletion missing the entire receptor-binding domain of Colicin Ia. The normal pathway for Colicin Ia killing was shown to require two copies of Cir: one that serves as the primary receptor and a second copy that serves as translocator. The novel Ia Colicins retain the ability to kill E. coli, even in the absence of receptor binding, as long as they can translocate via their Cir translocator. Experiments to determine whether Colicin M uses a second copy of its receptor, FhuA, as its translocator were hampered by precipitation of Colicin M chimaeras in inclusion bodies. Nevertheless, I show that receptor binding can be bypassed for killing, as long as a translocation pathway is maintained for Colicin M. These experiments suggest that Colicin M, unlike Colicin Ia, may normally use a single copy of FhuA as both its receptor and its translocator. Colicin E1 can kill in the absence of receptor binding, using translocation through TolC.
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Dissecting the Pathway for Colicin Ia Translocation across the E. coli Outer Membrane
Biophysical Journal, 2011Co-Authors: Karen S. Jakes, Alan FinkelsteinAbstract:The bacterial protein toxins called Colicins all share the common challenges both of binding to and crossing the E. coli outer membrane, regardless of their ultimate target in susceptible cells. All Colicins have co-opted, as their primary binding receptors, one of a family of outer membrane proteins normally involved in the uptake of essential nutrients, such as siderophore-bound iron or cobalamin. Many Colicins then use a porin, such as OmpF, as their translocator across the outer membrane. For another family of Colicins, no translocator had ever been genetically or functionally identified. We recently showed that one of those Colicins, Colicin Ia, uses a second copy of its receptor, Cir, in an entirely different way_as its translocator. Here, we begin to dissect the steps by which the translocation domain of the Colicin traverses the outer membrane through the Cir protein. Genetically attaching a folded protein at the C-terminus of the isolated T domain yields a protein that protects sensitive cells from killing by Colicin Ia significantly more efficiently than does the purified T domain alone. Therefore, the chimeric T domain protein appears to stop or slow down translocation better than T domain. The effects of the interaction of T domain with the periplasmic protein, TonB, will be reported.
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Colicin ia uses cir protein both as its primary receptor and as its translocon
Biophysical Journal, 2009Co-Authors: Karen S. Jakes, Susan K Buchanan, Rodolfo Ghirlando, Alan FinkelsteinAbstract:Colicin Ia is a bactericidal protein that kills E. coli by making a voltage-dependent ion channel in their inner membrane and de-energizing them. To do this, the Colicin or its pore-forming domain must cross the outer bacterial membrane. Like all Colicins, Ia first binds to an outer membrane receptor. The crystal structure of Colicin Ia bound to its outer membrane receptor, the 22-strand plugged β-barrel protein, Cir, suggests the plug doesn't move upon Ia binding. Therefore, another pathway is needed for the Colicin to cross the outer membrane. Group A Colicins, like E3, use the periplasmic and inner membrane proteins TolA,B,Q,R,Pal in translocation; a second outer membrane protein, such as the porin OmpF or TolC, has been shown to serve as a “second receptor” and be involved in passage of the Colicin across the outer membrane.Unlike the Tol-dependent Colicins, no “second receptor” has ever been identified for the group B, or TonB-dependent Colicins, such as Colicin Ia. We show here that Colicin Ia uses one copy of Cir as its receptor, for the initial binding step, and then searches for a second copy of Cir for translocation across the outer membrane. We constructed a chimeric Colicin with the C-terminal channel-forming domain and N-terminal translocation domain (T-domain) of Colicin Ia and the receptor-binding domain of Colicin E3. Although this Colicin now requires the E3 receptor protein, BtuB, presumably for binding, it still requires both the Cir and TonB proteins for killing. Furthermore, we show that the purified 225-residue Colicin Ia T-domain can compete with this hybrid Colicin and protect E. coli from killing, in vivo. These results imply that the T-domain binds to and opens a channel in Cir through which the Colicin reaches the periplasm.
Colin Kleanthous - One of the best experts on this subject based on the ideXlab platform.
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o antigen dependent Colicin insensitivity of uropathogenic escherichia coli
Journal of Bacteriology, 2018Co-Authors: Connor Sharp, Julian Parkhill, Christine J Boinett, Amy K Cain, Nicholas G Housden, Sandip Kumar, Keith Turner, Colin KleanthousAbstract:ABSTRACT The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, Colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward Colicins that is independent of Colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have Colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the Colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that Colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward Colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing Colicin insensitivity. We also show, by microscopy of fluorescently labelled Colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward Colicins and that O-antigen in LPS is central to this role. IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although Colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring Colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent Colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that Colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, Colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).
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Swimming against the tide: progress and challenges in our understanding of Colicin translocation
Nature Reviews Microbiology, 2010Co-Authors: Colin KleanthousAbstract:Colicins are folded protein toxins that must translocate across one or both of the Escherichia coli cell membranes to induce cell death. In this Progress article, Colin Kleanthous discusses recent advances in our understanding of the molecular determinants of Colicin translocation into E. coli and the novel insights that this has provided into host protein function. Colicins are folded protein toxins that face the formidable task of translocating across one or both of the Escherichia coli cell membranes in order to induce cell death. This translocation is achieved by parasitizing host proteins. There has been much recent progress in our understanding of the early stages of Colicin entry, including the binding of outer-membrane nutrient transporters and porins and the subsequent recruitment of periplasmic and inner-membrane proteins that, together, trigger translocation. As well as providing insights into how these toxins enter cells, these studies have highlighted some surprising similarities in the modes of action of the systems that Colicins subvert.
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the role of electrostatics in Colicin nuclease domain translocation into bacterial cells
Journal of Biological Chemistry, 2007Co-Authors: Daniel Walker, Richard James, Khedidja Mosbahi, Mireille Vankemmelbeke, Colin KleanthousAbstract:Abstract The mechanism(s) by which nuclease Colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated Colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin Colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl β-d-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming Colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a Colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between Colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated Colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease Colicin uptake but that nuclease Colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA+ ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction.
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Colicin biology.
Microbiology and Molecular Biology Reviews, 2007Co-Authors: Eric Cascales, Colin Kleanthous, Roland Lloubès, Denis Duché, Kathleen Postle, Susan Buchanan, Margaret Riley, Stephen Slatin, Danièle CavardAbstract:Colicins are proteins produced by and toxic for some strains of Escherichia coli. They are produced by strains of E. coli carrying a Colicinogenic plasmid that bears the genetic determinants for Colicin synthesis, immunity, and release. Insights gained into each fundamental aspect of their biology are presented: their synthesis, which is under SOS regulation; their release into the extracellular medium, which involves the Colicin lysis protein; and their uptake mechanisms and modes of action. Colicins are organized into three domains, each one involved in a different step of the process of killing sensitive bacteria. The structures of some Colicins are known at the atomic level and are discussed. Colicins exert their lethal action by first binding to specific receptors, which are outer membrane proteins used for the entry of specific nutrients. They are then translocated through the outer membrane and transit through the periplasm by either the Tol or the TonB system. The components of each system are known, and their implication in the functioning of the system is described. Colicins then reach their lethal target and act either by forming a voltage-dependent channel into the inner membrane or by using their endonuclease activity on DNA, rRNA, or tRNA. The mechanisms of inhibition by specific and cognate immunity proteins are presented. Finally, the use of Colicins as laboratory or biotechnological tools and their mode of evolution are discussed.
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immunity proteins enzyme inhibitors that avoid the active site
Trends in Biochemical Sciences, 2001Co-Authors: Colin Kleanthous, Daniel WalkerAbstract:Abstract Immunity proteins are high affinity inhibitors of Colicins – SOS-induced toxins released by bacteria during times of stress. Recent work has shown that nuclease-specific immunity proteins are exosite inhibitors, binding adjacent to the enzyme active site and inhibiting Colicin activity indirectly. Unusually, their binding sites comprise a near contiguous sequence that lies N-terminal to active site sequences, raising the possibility that immunity proteins bind Colicins co-translationally. Exosite binding accounts for the extensive sequence diversity seen at the interfaces of Colicin–immunity protein complexes, which is not only a selective advantage to Colicin-producing bacteria, but also represents a powerful model system for studying specificity in protein–protein recognition.
Volkmar Braun - One of the best experts on this subject based on the ideXlab platform.
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periplasmic chaperone fkpa is essential for imported Colicin m toxicity
Molecular Microbiology, 2008Co-Authors: Julia Hullmann, Volkmar Braun, Silke I Patzer, Christin Romer, Klaus HantkeAbstract:Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by Colicin M entering cells from the outside. Highly active Colicin M preparations were inactive against fkpA mutant cells; 10(4)-fold dilutions killed fkpA(+) cells. Three previously isolated spontaneous mutants tolerant to Colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to Colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were Colicin M-sensitive. Isolated FkpA-His reduced Colicin M-His cleavage by proteinase K and renatured denatured Colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate Colicin M. Among the tested Colicins, only Colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound Colicin M retained activity. We propose that Colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding Colicin M into an active toxin in the periplasm and PPIase is essential for Colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.
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characterization of Colicin s4 and its receptor ompw a minor protein of the escherichia coli outer membrane
Journal of Bacteriology, 1999Co-Authors: Holger Pilsl, David Smajs, Volkmar BraunAbstract:Analysis of the nucleotide sequence of an Escherichia coli Colicin S4 determinant revealed 76% identity to the pore-forming domain of the Colicin A protein, 77% identity to the Colicin A immunity protein, and 82% identity to the Colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of Colicin S4, has 94% identity to the N-terminal region of Colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other Colicins. Mutants that lacked the OmpW protein were resistant to Colicin S4.
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The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein
Journal of bacteriology, 1998Co-Authors: Holger Pilsl, David Smajs, Volkmar BraunAbstract:Pore-forming Colicins form voltage-dependent ion channels in the cytoplasmic membrane of sensitive bacteria. Colicin U belongs to the family of channel-forming Colicins (22), which consist of three domains responsible for translocation through the outer membrane (N-terminal domain), binding to the receptor (central domain), and channel formation (C-terminal domain). Crystal structures of the pore-forming domains of Colicins A, E1, and Ia have been determined at atomic resolution (4, 16, 27). In the water-soluble state, the pore-forming domains are arranged similarly and consist of a central hydrophobic hairpin (helices 8 and 9) surrounded by eight amphipathic helices. The structure of the membrane pore is less clear. Upon contact with the cytoplasmic membrane, the Colicins unfold and the hydrophobic hairpin inserts into the lipid bilayer. It is debated whether the hydrophobic hairpin is oriented parallel to the bilayer or whether it assumes a transmembrane arrangement, and how its arrangement and that of the other helices change upon voltage-dependent pore formation (1, 3, 11–13, 15). In Colicin Ia, at least helices 5 and 6 are translocated across the membrane in response to a transmembrane voltage (21), whereas helices 8 and 9 are inserted voltage-independent into the membrane (9). For the purpose of this paper, the general agreement that helices 8 and 9 are embedded in the membrane is of relevance. Sequence similarities separate the pore-forming Colicins into the A-type (Colicins A, B, N, and U) and the E1-type (Colicins E1, 5, K, 10, Ia, and Ib) Colicins (Fig. (Fig.1).1). The corresponding immunity proteins have been classified into the same two groups (17, 20, 23). The Colicin A immunity protein (Cai) has four transmembrane segments, and its N and C termini are located in the cytoplasm (8), whereas the immunity proteins of Colicin E1 (23) and Colicin 5 (17) cross the cytoplasmic membrane three times, with the N terminus in the cytoplasm and the C terminus in the periplasm. FIG. 1 Hydrophobic hairpin sequences (helices 8 and 9) of the channel-forming domains of the E1-type (Colicins E1, 5, K, 10, Ia, and Ib) and A-type (Colicins B, N, U, and A) Colicins and of bacteriocin 28b. The hydrophobic amino acids are indicated in boldface, ... In this study, we show that deletion of residues 575 to 583, which we propose to form the tip of the hydrophobic helical hairpin, did not alter the cytotoxic activity of Colicin U. We further demonstrate that the tip sequence is a main determinant for the specific recognition of Colicin U by the cognate immunity protein. In addition, we determined the transmembrane topology of the Colicin U immunity protein and show that it corresponds with the immunity protein of Colicin A.
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The tip of the hydrophobic hairpin of Colicin U is dispensablefor Colicin U
Journal of Bacteriology, 1998Co-Authors: Holger Pilsl, David Smajs, Volkmar BraunAbstract:The hydrophobic C terminus of pore-forming Colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of Colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of Colicin U by the Colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of Colicin U resulted in a fully active Colicin that was no longer inactivated by the Colicin U immunity protein. Replacement of eight amino acids at the tip of the Colicin U hairpin by the corresponding amino acids of the related Colicin B resulted in Colicin U(575-582ColB), which was inactivated by the Colicin U immunity protein to 10% of the level of inactivation of the wild-type Colicin U. The Colicin B immunity protein inactivated Colicin U(575-582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of Colicin U and of Colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for Colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming Colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type Colicins. The Colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.
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Import and Export of Colicin M
Bacteriocins Microcins and Lantibiotics, 1992Co-Authors: Volkmar Braun, S. Gaisser, C. Glaser, R. Harkness, T. Ölschäger, J MendeAbstract:Colicin M is unique among the Colicins in that it does not belong to the Colicins which form pores in the cytoplasmic membrane oftarget cells, or display nuclease activities (Braun et a.., 1974; Harkness & Olschlager, 1991). Rather, Colicin M inhibits murein biosynthesis (Schaller et a.., 1982) by interfering with the dephosphorylation of C55-polyisoprenyl pyrophosphate, which leads to cell lysis (Harkness & Braun, 1989a). Colicin M also inhibits lipopolysaccharide O-antigensynthesis since this involves the same step ofbactoprenyl phosphate regeneration (Harkness & Braun 1989b). However, prevention of O-antigen synthesis does not kill cells, as viable rough mutants are isolated.
David Smajs - One of the best experts on this subject based on the ideXlab platform.
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Colicin Z, a structurally and functionally novel Colicin type that selectively kills enteroinvasive Escherichia coli and Shigella strains.
Scientific reports, 2019Co-Authors: Lenka Micenková, Dirk Linke, Juraj Bosák, Jiri Kucera, Matěj Hrala, Tereza Dolejšová, Ondrej Šedo, Radovan Fišer, David SmajsAbstract:Colicin production in Escherichia coli (E. coli) strains represents an important trait with regard to microbial survival and competition in the complex intestinal environment. A novel Colicin type, Colicin Z (26.3 kDa), was described as a product of an original producer, extraintestinal E. coli B1356 strain, isolated from the anorectal abscess of a 17 years-old man. The 4,007 bp plasmid (pColZ) was completely sequenced and Colicin Z activity (cza) and Colicin Z immunity (czi) genes were identified. The cza and czi genes are transcribed in opposite directions and encode for 237 and 151 amino acid-long proteins, respectively. Colicin Z shows a narrow inhibitory spectrum, being active only against enteroinvasive E. coli (EIEC) and Shigella strains via CjrC receptor recognition and CjrB- and ExbB-, ExbD-mediated Colicin translocation. All tested EIEC and Shigella strains isolated between the years 1958-2010 were sensitive to Colicin Z. The lethal effect of Colicin Z was found to be directed against cell wall peptidoglycan (PG) resulting in PG degradation, as revealed by experiments with Remazol Brilliant Blue-stained purified peptidoglycans and with MALDI-TOF MS analyses of treated PG. Colicin Z represents a new class of Colicins that is structurally and functionally distinct from previously studied Colicin types.
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Colicins produced by the Escherichia fergusonii strains closely resemble Colicins encoded by Escherichia coli
FEMS microbiology letters, 2002Co-Authors: David Smajs, Jan Šmarda, Sandor E. Karpathy, George M WeinstockAbstract:Plasmid DNA of six Escherichia fergusonii Colicinogenic strains (three producers of Colicin E1, two of Ib and one of Ia) was isolated and the Colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of Colicin E1, one of Colicin Ib, and one of Colicin Ia were identified as well as new variants of the Colicin E1 and Colicin Ib immunity proteins and the Colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 Colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.
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Colicins produced by the Escherichia fergusonii strains closelyresemble Colicins coded by E. coli
Fems Microbiology Letters, 2002Co-Authors: David Smajs, Jan Šmarda, Sandor E. Karpathy, George M WeinstockAbstract:Plasmid DNA of six Escherichia fergusonii Colicinogenic strains (three producers of Colicin E1, two of Ib and one of Ia) was isolated and the Colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of Colicin E1, one of Colicin Ib, and one of Colicin Ia were identified as well as new variants of the Colicin E1 and Colicin Ib immunity proteins and the Colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 Colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.
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genetic organization of plasmid coljs encoding Colicin js activity immunity and release genes
Journal of Bacteriology, 2001Co-Authors: David Smajs, George M WeinstockAbstract:Colicins are plasmid-encoded toxic exoproteins that are produced by Colicinogenic strains of Escherichia coli and some related species of the family Enterobacteriaceae (28, 29). To date, at least 23 Colicin types have been described in detail (19, 27, 30, 34). They exert an inhibitory effect on sensitive bacteria of the same family and preferably on strains of the same species. The molecular masses of Colicins range between 29,000 and 75,000 Da (7). Colicin polypeptide chains can be divided into separate functional domains, each of which is responsible for one step in the interaction between the Colicin and a sensitive bacterium. The central domain of Colicins is involved in the attachment of the Colicin molecule to a specific outer membrane receptor protein, the N-terminal domain mediates translocation through the cell envelope, and the C-terminal domain exerts the lethal effect (4, 6, 7). At least 12 different outer membrane proteins have been shown to be Colicin receptors, two different translocation systems (Ton and Tol) used by Colicins have been identified, and six different modes of molecular lethal action of Colicins have been described (7, 10, 22, 34). Some molecules initially described as Colicins were later reclassified as microcins, e.g., Colicin V as microcin V and Colicin X as microcin B19. In contrast to these oligopeptide microcins (3), Colicins are larger proteins. Moreover, Colicins are not posttranslationally modified, are usually inducible by the SOS response, and also differ from microcins by the mode of export from the producer bacteria. Colicin Js was originally described as a bacteriocin of Shigella sonnei Colicinotype 7 (1, 2). In 1987, Colicin type 7 was reclassified in accordance with Fredericq's original classification scheme (14) and designated Colicin Js. Its particular physicochemical and biological characteristics were published (33). For a number of reasons, Colicin Js appeared to be a rather exceptional Colicin type: producer bacteria, as well as the indicator strain S. sonnei 17 (Colicin type 6), were involved in outbreaks of epidemic diarrhea (12). Colicin Js showed a unique antimicrobial spectrum, being inactive against standard E. coli Colicin indicators. Indirect fluorimetry measurements indicated that the mode of action of Colicin Js was not analogous to that of either pore-forming or nuclease-type Colicins (33). Colicin Js was shown to be active against enteroinvasive E. coli (EIEC) serotypes (17). The sensitivity to Js was 90% associated with the ability of EIEC strains to produce experimental keratoconjunctivitis in rabbits. Strains belonging to EIEC serotypes that were not sensitive to Colicin Js were, as a rule, negative in the enteroinvasiveness test (17). This communication presents a number of new details of the Colicin Js system. These include the structure of the Colicin Js coding region on plasmid ColJs; identification of genes for Colicin activity, immunity, and release; and molecular characterization of the Js polypeptide.
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characterization of Colicin s4 and its receptor ompw a minor protein of the escherichia coli outer membrane
Journal of Bacteriology, 1999Co-Authors: Holger Pilsl, David Smajs, Volkmar BraunAbstract:Analysis of the nucleotide sequence of an Escherichia coli Colicin S4 determinant revealed 76% identity to the pore-forming domain of the Colicin A protein, 77% identity to the Colicin A immunity protein, and 82% identity to the Colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of Colicin S4, has 94% identity to the N-terminal region of Colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other Colicins. Mutants that lacked the OmpW protein were resistant to Colicin S4.
H Bénédetti - One of the best experts on this subject based on the ideXlab platform.
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Import of Colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm.
Molecular Microbiology, 2001Co-Authors: L. Journet, E Bouveret, A Rigal, C Lazdunski, R Lloubès, H BénédettiAbstract:Several proteins of the Tol/Pal system are required for group A Colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain. Both interactions are required for Colicin translocation. Using in vivo and in vitro approaches, we show in this study that Colicin A also interacts with a third component of the Tol/Pal system required for Colicin import, TolR. This interaction is specific to Colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the Colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the Colicin A N-terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the Colicin A N-terminal domain and appear to be arranged linearly in the Colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A Colicin translocation.
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Distinct regions of the Colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli.
Molecular Microbiology, 1998Co-Authors: E Bouveret, A Rigal, C Lazdunski, H BénédettiAbstract:Group A Colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of Colicins is involved in the import process. The N-terminal domains of Colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of Colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A Colicins, the 'TolA box', was important for Colicin A import but was not involved in the Colicin A-TolA interaction. It was, however, involved in the Colicin A-TolB interaction. The interactions of Colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the Colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire Colicin A for import into the cells. Our results demonstrate that different regions of the Colicin A N-terminal domain interact with TolA and TolB. The Colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB.
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The N-terminal domain of Colicin E3 interacts with TolB which is involved in the Colicin translocation step.
Molecular Microbiology, 1997Co-Authors: E Bouveret, A Rigal, C Lazdunski, H BénédettiAbstract:Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A Colicins and the TonB system for group B Colicins. The N-terminal domain of Colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of Colicin E3 (group A) and Colicin B (group B). Producing cells became specifically tolerant to entire extracellular Colicins of the same group. The periplasmic N-terminal domains therefore compete with entire Colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the Colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the Colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the Colicin E3 domain. The N-terminal domain of a translocation-defective Colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in Colicin E3 translocation.
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Colicin transport, channel formation and inhibition
1996Co-Authors: H Bénédetti, V. GeliAbstract:This chapter discusses the molecular mechanisms involved in Colicin translocation across the outer membrane, the insertion of pore-forming Colicins into the inner membrane and the inhibition of their lethal activities by the corresponding specific immunity proteins. The chapter focuses on Colicin A, that the immunity protein interacts with the pore-forming domain of the Colicin as it inserts into the inner membrane and that it prevents it from opening its channel as normally in the presence of membrane potential. The transmembrane helices of the immunity protein might somehow interact with membrane inserted portions of the Colicin A channel in order to block any further conformational changes necessary for the channel opening. The chapter discusses the mode of action of Colicin that is divided into three steps. They first bind to a specific receptor at the cell surface. For that purpose, some Colicins have parasitized proteins of the outer membrane whose function is dedicated to the transport of iron siderophores (FepA, FhuA, FhuE, and Cir), of vitamin B12 (BtuB), or nucleotides (Tsx). Others have parasitized the major porin OmpF through which small hydrophilic solutes with MW of up to 650 Daltons.
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Quantification of group A Colicin import sites.
Journal of Bacteriology, 1995Co-Authors: D. Duche, H Bénédetti, V. Geli, L. Letellier, D. BatyAbstract:Pore-forming Colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these Colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A Colicins use the Tol proteins; group B Colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine Colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of Colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of Colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 Colicin A receptors and translocation sites, respectively.