Dataset Information

Interaction of the colicin K bactericidal toxin with components of its import machinery in the periplasm of Escherichia coli.

ABSTRACT: Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. We first produced KT fused to a signal sequence for periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.

SUBMITTER: Barneoud-Arnoulet A

PROVIDER: S-EPMC2976459 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Interaction of the colicin K bactericidal toxin with components of its import machinery in the periplasm of Escherichia coli.

Barnéoud-Arnoulet Aurélie A Gavioli Marthe M Lloubès Roland R Cascales Eric E

Journal of bacteriology 20100924 22

Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for bin ...[more]

PMID: 20870776

Similar Datasets

Project description: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.IMPORTANCE Escherichia 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).

Project description:The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery.The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Interaction of the colicin K bactericidal toxin with components of its import machinery in the periplasm of Escherichia coli.

Publications

Interaction of the colicin K bactericidal toxin with components of its import machinery in the periplasm of Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets