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Colin Kleanthous - One of the best experts on this subject based on the ideXlab platform.
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bifurcated binding of the ompf receptor underpins import of the bacteriocin colicin n into escherichia coli
Journal of Biological Chemistry, 2020Co-Authors: Katarina Bartos Jansen, Nicholas G Housden, Jonathan T S Hopper, Carol V Robinson, Patrick George Inns, Renata Kaminska, Hagan Bayley, Sejeong Lee, Colin KleanthousAbstract:Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A Colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A Colicins.
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bifurcated binding of the ompf receptor underpins import of the bacteriocin colicin n into escherichia coli
Journal of Biological Chemistry, 2020Co-Authors: Katarina Bartos Jansen, Nicholas G Housden, Jonathan T S Hopper, Carol V Robinson, Patrick George Inns, Renata Kaminska, Hagan Bayley, Colin KleanthousAbstract:: Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A Colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer-membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide (LPS) as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that LPS enhances this binding. Approx. 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A Colicins.
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Colicins exploit native disorder to gain cell entry: a hitchhiker's guide to translocation
2020Co-Authors: Daniel A Bonsor, Nicola A Meenan, Colin KleanthousAbstract:Abstract The translocation of protein toxins into a cell relies on a myriad of protein-protein interactions. One such group of toxins are enzymatic E Colicins, protein antibiotics produced by Escherichia coli in times of stress. These proteins subvert ordinary nutrient uptake mechanisms to enter the cell and unleash nuclease activity. We, and others, have previously shown that uptake of ColE9 (colicin E9) is dependent on engagement of the OM (outer membrane) receptors BtuB and OmpF as well as recruitment of the periplasmic protein TolB, forming a large supramolecular complex. Intriguingly, Colicins bind TolB using a natively disordered region to mimic the interaction of TolB with Pal (peptidoglycan-associated lipoprotein). This is thought to trigger OM instability and prime the system for translocation. Here, we review key interactions in the assembly of this 'colicin translocon' and discuss the key role disorder plays in achieving uptake
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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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Microbiology (1 996), 142,1569-1 580 Printed in Great Britain The biology of E Colicins: paradigms and paradoxes ARTICLE
2015Co-Authors: Richard I James, Colin Kleanthous, Geoffrey R. MooreAbstract:Colicins are plasmid-encoded antibacterial proteins that are classified into groups on the basis of the cell surface receptor to which they bind. The E Colicins, for example, all bind to the product of the chromosomal bttrB gene, which is an essential component of the high-affinity transport system for vitamin B,, (cobalamin) in Escbericbia coli (Di Masi e t al., 1973). Each E colicin plasmid encodes the production of a specific immunity protein which protects the producing cell against the cytotoxic activity of its colicin, located at the C-terminus of the protein, by binding to this domain (Jakes e t al., 1974; Sidikaro & Nomura, 1974; Wallis e t al., 1992a). Based on immunity tests (Watson e t al., 1981; Cooper & James, 1984) the E group Colicins have been subdivided into nine types (ColEl-E9), and these fall into one of three cytotoxi
William A. Cramer - One of the best experts on this subject based on the ideXlab platform.
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On mechanisms of colicin import: the outer membrane quandary
The Biochemical journal, 2018Co-Authors: William A. Cramer, Onkar Sharma, Stanislav D. ZakharovAbstract:Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (K d ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease Colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming Colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.
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pathways of colicin import utilization of btub ompf porin and the tolc drug export protein
Biochemical Society Transactions, 2012Co-Authors: Stanislav D. Zakharov, Onkar Sharma, Mariya V. Zhalnina, Eiki Yamashita, William A. CramerAbstract:Pathway I. Group A nuclease Colicins parasitize and bind tightly ( K d ≤ 10 −9 M) to the vitamin B 12 receptor on which they diffuse laterally in the OM (outer membrane) and use their long (≥100 A; 1 A=0.1 nm) receptor-binding domain as a ‘fishing pole’ to locate the OmpF porin channel for translocation. Crystal structures of OmpF imply that a disordered N-terminal segment of the colicin T-domain initiates insertion. Pathway II. Colicin N does not possess a ‘fishing pole’ receptor-binding domain. Instead, it uses OmpF as the Omp (outer membrane protein) for reception and translocation, processes in which LPS (lipopolysaccharide) may also serve. Keio collection experiments defined the LPS core that is used. Pathway III. Colicin E1 utilizes the drug-export protein TolC for import. CD spectra and thermal-melting analysis predict: (i) N-terminal translocation (T) and central receptor (BtuB) -binding (R) domains are predominantly α-helical; and (ii) helical coiled-coil conformation of the R-domain is similar to that of Colicins E3 and Ia. Recombinant colicin peptides spanning the N-terminal translocation domain defined TolC-binding site(s). The N-terminal 40-residue segment lacks the ordered secondary structure. Peptide 41–190 is helical (78%), co-elutes with TolC and occluded TolC channels. Driven by a trans-negative potential, peptides 82–140 and 141–190 occluded TolC channels. The use of TolC for colicin E1 import implies that the interaction of this colicin with the other Tol proteins does not occur in the periplasmic space, but rather through Tol domains in the cytoplasmic membrane, thus explaining colicin E1 cytotoxicity towards a strain in which a 234 residue periplasmic TolA segment is deleted.
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genome wide screens novel mechanisms in colicin import and cytotoxicity
Molecular Microbiology, 2009Co-Authors: Onkar Sharma, Mariya V. Zhalnina, Kirill A Datsenko, Sara Ess, Barry L Wanner, William A. CramerAbstract:Protein import and export across cellular membranes involves an assembly of integral membrane polypeptides that provide a conduit for protein transfer across the hydrophobic membrane. Colicin import into bacteria is a model for the study of protein import across bacterial membranes. Colicins are produced by E. coli in response to stresses such as nutrient depletion and overcrowding, and kill closely related sibling cells that contain the set of receptors and trans-envelope import proteins, but do not contain the respective immunity protein. Based on known components of the translocation network utilized for cell entry, Colicins have been divided into two groups. Group A utilizes the Tol network consisting of genes tolA, tolB, tolQ, tolR and pal (Nagel de Zwaig and Luria, 1967; Davies and Reeves, 1975a; Lazzaroni et al., 2002). Group B employs the Ton network comprised of tonB, exbB and exbD (Davies and Reeves, 1975b; Braun et al., 2002). Loss of the colicin receptors in the outer membrane, proteins in the translocation pathway, or of the immunity protein, are the only known mechanisms of colicin resistance or tolerance. The first step in colicin import is binding to its outer membrane primary receptor. The protein component of this primary receptor is known to be BtuB for colicin A, E1, E2, E3 and E7, OmpF for colicin N, and FepA for Colicins B and D. Subsequent to binding to the primary binding steps, nuclease E Colicins, for which the import process has been studied extensively (Di Masi et al., 1973; Mock and Pugsley, 1982; Benedetti et al., 1989; Bouveret et al., 1997; Garinot-Schneider et al., 1997; Kurisu et al., 2003; Housden et al., 2005; Duche et al., 2006; Loftus et al., 2006; Duche, 2007; Sharma et al., 2007; Yamashita et al., 2008), utilize a second receptor/translocator to accomplish translocation across the outer membrane. This secondary receptor/translocator is OmpF for colicin A, TolC for colicin E1 and OmpF/OmpC for Colicins E2, E3 and E7. No secondary receptor/translocator has yet been identified for colicin N and the group B Colicins. In addition to the primary receptor and secondary receptor/translocator several other proteins have been identified that are responsible for colicin import across the double membrane of the target cell. A total of ten genes, btuB, iutA, ompF, ompC, tolC, tolQRAB and tsx have been identified to play a role in the uptake of group A Colicins, while the corresponding number for group B Colicins is nine ((cir, exbBD, fepA, fhuA, lepB, tonB, tsx (Cascales et al., 2007) and the recently identified fkpA (Hullmann et al., 2008)). This number is considerably smaller than the number of proteins showed to be involved in protein transport across the mitochondrial double membrane. In the mitochondrial system, virtually all the pre-proteins traverse the outer membrane through the initial entry gate, the TOM (translocase of the outer membrane) complex that consists of seven polypeptides. The full translocation of the pre-proteins across the inner membrane into the mitochondrial matrix requires 10 more polypeptides (Bohnert et al., 2007; Bolender et al., 2008). Thus, on the one hand, it might be expected that additional components, proteins or lipids as yet unidentified, could be involved in colicin uptake across the E. coli double membrane. On the other, it is possible that some gene(s) previously proposed to be required for colicin cytotoxicity are not essential. The “Keio Collection” (www.EcoliHub.org/GenoBase) can be used to clarify this situation. This collection, a single-gene knockout library of the entire E. coli genome (Datsenko and Wanner, 2000; Baba et al., 2006), consists of 3985 strains, in duplicate, inoculated in 96-well plates, each well consisting of a distinct single-gene knockout mutant. In the present study, the collection was screened for the sensitivity of each knockout strain to eight Colicins, A, B, D, E1, E2, E3, E7 and N, allowing for the identification of knockout strains that are able to grow in the presence of colicin. These colicin ‘tolerant’ or ‘resistant’ single gene knockout strains were further analyzed by complementation with the respective genes from the “ASKA” orfeome library, which restored the colicin sensitivity to the knockout strain. Since only the genes non-essential for cellular viability are represented in the Keio collection, proteins that are required by colicin but which are also essential for cell growth (e. g. lepB for colD) will not be scored in the present genomic screening. It is noted that while a ‘positive hit’ implies that the particular gene is important for the colicin cytotoxicity, a ‘negative hit’, where a gene deletion does not affect colicin activity, is equally significant. A ‘negative hit’ implies that, barring a redundancy in the gene function, the gene is not critical for colicin cytotoxicity. Through this study we have been able to define the requirement of each of the 3985 genes in the Keio collection for colicin cytotoxicity.
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crystal structures of the ompf porin function in a colicin translocon
The EMBO Journal, 2008Co-Authors: Eiki Yamashita, Stanislav D. Zakharov, Onkar Sharma, Mariya V. Zhalnina, William A. CramerAbstract:The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A Colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.
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Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon
The Journal of biological chemistry, 2007Co-Authors: Onkar Sharma, Stanislav D. Zakharov, Mariya V. Zhalnina, Kirill A Datsenko, Barry L Wanner, Eiki Yamashita, William A. CramerAbstract:The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E Colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.
Stanislav D. Zakharov - One of the best experts on this subject based on the ideXlab platform.
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On mechanisms of colicin import: the outer membrane quandary
The Biochemical journal, 2018Co-Authors: William A. Cramer, Onkar Sharma, Stanislav D. ZakharovAbstract:Current problems in the understanding of colicin import across the Escherichia coli outer membrane (OM), involving a range of cytotoxic mechanisms, are discussed: (I) Crystal structure analysis of colicin E3 (RNAase) with bound OM vitamin B12 receptor, BtuB, and of the N-terminal translocation (T) domain of E3 and E9 (DNAase) inserted into the OM OmpF porin, provide details of the initial interaction of the colicin central receptor (R)- and N-terminal T-domain with OM receptors/translocators. (II) Features of the translocon include: (a) high-affinity (K d ≈ 10-9 M) binding of the E3 receptor-binding R-domain E3 to BtuB; (b) insertion of disordered colicin N-terminal domain into the OmpF trimer; (c) binding of the N-terminus, documented for colicin E9, to the TolB protein on the periplasmic side of OmpF. Reinsertion of the colicin N-terminus into the second of the three pores in OmpF implies a colicin anchor site on the periplasmic side of OmpF. (III) Studies on the insertion of nuclease Colicins into the cytoplasmic compartment imply that translocation proceeds via the C-terminal catalytic domain, proposed here to insert through the unoccupied third pore of the OmpF trimer, consistent with in vitro occlusion of OmpF channels by the isolated E3 C-terminal domain. (IV) Discussion of channel-forming Colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC.
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pathways of colicin import utilization of btub ompf porin and the tolc drug export protein
Biochemical Society Transactions, 2012Co-Authors: Stanislav D. Zakharov, Onkar Sharma, Mariya V. Zhalnina, Eiki Yamashita, William A. CramerAbstract:Pathway I. Group A nuclease Colicins parasitize and bind tightly ( K d ≤ 10 −9 M) to the vitamin B 12 receptor on which they diffuse laterally in the OM (outer membrane) and use their long (≥100 A; 1 A=0.1 nm) receptor-binding domain as a ‘fishing pole’ to locate the OmpF porin channel for translocation. Crystal structures of OmpF imply that a disordered N-terminal segment of the colicin T-domain initiates insertion. Pathway II. Colicin N does not possess a ‘fishing pole’ receptor-binding domain. Instead, it uses OmpF as the Omp (outer membrane protein) for reception and translocation, processes in which LPS (lipopolysaccharide) may also serve. Keio collection experiments defined the LPS core that is used. Pathway III. Colicin E1 utilizes the drug-export protein TolC for import. CD spectra and thermal-melting analysis predict: (i) N-terminal translocation (T) and central receptor (BtuB) -binding (R) domains are predominantly α-helical; and (ii) helical coiled-coil conformation of the R-domain is similar to that of Colicins E3 and Ia. Recombinant colicin peptides spanning the N-terminal translocation domain defined TolC-binding site(s). The N-terminal 40-residue segment lacks the ordered secondary structure. Peptide 41–190 is helical (78%), co-elutes with TolC and occluded TolC channels. Driven by a trans-negative potential, peptides 82–140 and 141–190 occluded TolC channels. The use of TolC for colicin E1 import implies that the interaction of this colicin with the other Tol proteins does not occur in the periplasmic space, but rather through Tol domains in the cytoplasmic membrane, thus explaining colicin E1 cytotoxicity towards a strain in which a 234 residue periplasmic TolA segment is deleted.
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crystal structures of the ompf porin function in a colicin translocon
The EMBO Journal, 2008Co-Authors: Eiki Yamashita, Stanislav D. Zakharov, Onkar Sharma, Mariya V. Zhalnina, William A. CramerAbstract:The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A Colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 A OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co-crystallization of OmpF with the unfolded 83 residue glycine-rich N-terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 A structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly-glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno-extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.
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Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon
The Journal of biological chemistry, 2007Co-Authors: Onkar Sharma, Stanislav D. Zakharov, Mariya V. Zhalnina, Kirill A Datsenko, Barry L Wanner, Eiki Yamashita, William A. CramerAbstract:The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E Colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg452 at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.
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on the role of lipid in colicin pore formation
Biochimica et Biophysica Acta, 2004Co-Authors: Elena A Kotova, Stanislav D. Zakharov, Yuri N Antonenko, William A. CramerAbstract:Insights into the protein–membrane interactions by which the C-terminal pore-forming domain of Colicins inserts into membranes and forms voltage-gated channels, and the nature of the colicin channel, are provided by data on: (i) the flexible helix-elongated state of the colicin pore-forming domain in the fluid anionic membrane interfacial layer, the optimum anionic surface charge for channel formation, and voltage-gated translocation of charged regions of the colicin domain across the membrane; (ii) structure–function data on the voltage-gated K + channel showing translocation of an arginine-rich helical segment through the membrane; (iii) toroidal channels formed by small peptides that involve local participation of anionic lipids in an inverted phase. It is proposed that translocation of the colicin across the membrane occurs through minimization of the Born charging energy for translocation of positively charged basic residues across the lipid bilayer by neutralization with anionic lipid head groups. The resulting pore structure may consist of somewhat short, ca. 16 residues, trans-membrane helices, in a locally thinned membrane, together with surface elements of inverted phase lipid micelles. D 2004 Published by Elsevier B.V.
Nicholas G Housden - One of the best experts on this subject based on the ideXlab platform.
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colicin mediated transport of dna through the iron transporter fepa
Mbio, 2021Co-Authors: Ruth Cohenkhait, Nicholas G Housden, Ameya Harmalkar, Phuong Pham, Melissa N Webby, Emma Elliston, Jonathan T S Hopper, Shabaz Mohammed, Carol V Robinson, Jeffrey J GrayAbstract:Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of Colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.
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colicin mediated transport of dna through the iron transporter fepa
bioRxiv, 2021Co-Authors: Ruth Cohenkhait, Nicholas G Housden, Ameya Harmalkar, Phuong Pham, Melissa N Webby, Emma Elliston, Jonathan T S Hopper, Shabaz Mohammed, Carol V Robinson, Jeffrey J GrayAbstract:ABSTRACT Colicins are protein antibiotics used by bacteria to eliminate competing Escherichia coli. A key event in the selective colicin function is a highly specific initial recognition step of an outer membrane (OM) receptor, which consequently allows the active transport of the colicin across the otherwise impervious OM. Though the colicin-receptor interaction is exclusive, the translocation process is likely to be universal as many receptors and Colicins have surprisingly similar 3D folds. Here, using a combination of photo-activated crosslinking, mass spectrometry, and structural modeling, we reveal how colicin B (ColB) associates with its OM receptor FepA. We demonstrate that complex formation is coincident with a large-scale conformational change in the colicin. In vivo crosslinking experiments and further simulations of the translocation process indicate that part of the colicin engages active transport by disguising itself to part of the cellular receptor. Applying live-cell fluorescence imaging we were able to follow ColB into E. coli and localize it within the periplasm. Finally, we demonstrate that single-stranded DNA coupled to ColB is transported into the bacterial periplasm, emphasizing that the import routes of Colicins can be exploited to carry large cargo molecules into Gram-negative bacteria.
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bifurcated binding of the ompf receptor underpins import of the bacteriocin colicin n into escherichia coli
Journal of Biological Chemistry, 2020Co-Authors: Katarina Bartos Jansen, Nicholas G Housden, Jonathan T S Hopper, Carol V Robinson, Patrick George Inns, Renata Kaminska, Hagan Bayley, Sejeong Lee, Colin KleanthousAbstract:Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A Colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that lipopolysaccharide (LPS) enhances this binding. Approximately 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A Colicins.
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bifurcated binding of the ompf receptor underpins import of the bacteriocin colicin n into escherichia coli
Journal of Biological Chemistry, 2020Co-Authors: Katarina Bartos Jansen, Nicholas G Housden, Jonathan T S Hopper, Carol V Robinson, Patrick George Inns, Renata Kaminska, Hagan Bayley, Colin KleanthousAbstract:: Colicins are Escherichia coli-specific bacteriocins that translocate across the outer bacterial membrane by a poorly understood mechanism. Group A Colicins typically parasitize the proton-motive force-linked Tol system in the inner membrane via porins after first binding an outer-membrane protein receptor. Recent studies have suggested that the pore-forming group A colicin N (ColN) instead uses lipopolysaccharide (LPS) as a receptor. Contrary to this prevailing view, using diffusion-precipitation assays, native state MS, isothermal titration calorimetry, single-channel conductance measurements in planar lipid bilayers, and in vivo fluorescence imaging, we demonstrate here that ColN uses OmpF both as its receptor and translocator. This dual function is achieved by ColN having multiple distinct OmpF-binding sites, one located within its central globular domain and another within its disordered N terminus. We observed that the ColN globular domain associates with the extracellular surface of OmpF and that LPS enhances this binding. Approx. 90 amino acids of ColN then translocate through the porin, enabling the ColN N terminus to localize within the lumen of an OmpF subunit from the periplasmic side of the membrane, a binding mode reminiscent of that observed for the nuclease colicin E9. We conclude that bifurcated engagement of porins is intrinsic to the import mechanism of group A Colicins.
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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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immunity protein release from a cell bound nuclease colicin complex requires global conformational rearrangement
Microbiology Open, 2013Co-Authors: Mireille Vankemmelbeke, Richard James, Colin Kleanthous, Nicholas G Housden, Christopher N. PenfoldAbstract:Nuclease Colicins bind their target receptor BtuB in the outer membrane of sensitive Escherichia coli cells in the form of a high-affinity complex with their cognate immunity proteins. The release of the immunity protein from the colicin complex is a prerequisite for cell entry of the colicin and occurs via a process that is still relatively poorly understood. We have previously shown that an energy input in the form of the cytoplasmic membrane proton motive force is required to promote immunity protein (Im9) release from the colicin E9/Im9 complex and colicin cell entry. We report here that engineering rigidity in the structured part of the colicin translocation domain via the introduction of disulfide bonds prevents immunity protein release from the colicin complex. Reduction of the disulfide bond by the addition of DTT leads to immunity protein release and resumption of activity. Similarly, the introduction of a disulfide bond in the DNase domain previously shown to abolish channel formation in planar bilayers also prevented immunity protein release. Importantly, all disulfide bonds, in the translocation as well as the DNase domain, also abolished the biological activity of the Im9-free colicin E9, the reduction of which led to a resumption of activity. Our results show, for the first time, that conformational flexibility in the structured translocation and DNase domains of a nuclease colicin is essential for immunity protein release, providing further evidence for the hypothesis that global structural rearrangement of the colicin molecule is required for disassembly of this high-affinity toxin-immunity protein complex prior to outer membrane translocation.
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energy dependent immunity protein release during tol dependent nuclease colicin translocation
Journal of Biological Chemistry, 2009Co-Authors: Mireille Vankemmelbeke, Christopher N. Penfold, Colin Kleanthous, Geoffrey R. Moore, Ying Zhang, Richard JamesAbstract:Nuclease Colicins bind their target receptor in the outer membrane of sensitive cells in the form of a high affinity complex with their cognate immunity proteins. Upon cell entry the immunity protein is lost from the complex by means that are poorly understood. We have developed a sensitive fluorescence assay that has enabled us to study the molecular requirements for immunity protein release. Nuclease Colicins use members of the tol operon for their translocation across the outer membrane. We have demonstrated that the amino-terminal 80 residues of the colicin E9 molecule, which is the region that interacts with TolB, are essential for immunity protein release. Using tol deletion strains we analyzed the cellular components necessary for immunity protein release and found that in addition to a requirement for tolB, the tolA deletion strain was most affected. Complementation studies showed that the mutation H22A, within the transmembrane segment of TolA, abolishes immunity protein release. Investigation of the energy requirements demonstrated that the proton motive force of the cytoplasmic membrane is critical. Taken together these results demonstrate for the first time a clear energy requirement for the uptake of a nuclease colicin complex and suggest that energy transduced from the cytoplasmic membrane to the outer membrane by TolA could be the driving force for immunity protein release and concomitant translocation of the nuclease domain.
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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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the cytotoxic domain of colicin e9 is a channel forming endonuclease
Nature Structural & Molecular Biology, 2002Co-Authors: Khedidja Mosbahi, Richard James, Geoffrey R. Moore, Christelle Lemaitre, Anthony H Keeble, Hamid Mobasheri, Bertrand Morel, Edward J A LeaAbstract:Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of Colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.
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Inhibition of a ribosome-inactivating ribonuclease: the crystal structure of the cytotoxic domain of colicin E3 in complex with its immunity protein
Structure, 2000Co-Authors: Stephen B. Carr, Colin Kleanthous, Daniel Walker, Richard James, Andrew M. HemmingsAbstract:Abstract Background: The cytotoxicity of most ribonuclease E Colicins towards Escherichia coli arises from their ability to specifically cleave between bases 1493 and 1494 of 16S ribosomal RNA. This activity is carried by the C-terminal domain of the colicin, an activity which if left unneutralised would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms a complex with the ribonuclease domain effectively suppressing its activity. Results: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central β-sheet elaborated with a short N-terminal helix, the residues of which form a well-packed interface with the immunity protein. Conclusions: The structure of the ribonuclease domain of colicin E3 is novel and forms an interface with its inhibitor which is significantly different in character to that reported for the DNase colicin complexes with their immunity proteins. The structure also gives insight into the mode of action of this class of enzymatic Colicins by allowing the identification of potentially catalytic residues. This in turn reveals that the inhibitor does not bind at the active site but rather at an adjacent site, leaving the catalytic centre exposed in a fashion similar to that observed for the DNase Colicins. Thus, E. coli appears to have evolved similar methods for ensuring efficient inhibition of the potentially destructive effects of the two classes of enzymatic Colicins.