Rapid Detection of Colicin E9-Induced DNA Damage Using
            <i>Escherichia coli</i>
            Cells Carrying SOS Promoter-
            <i>lux</i>
            Fusions

Vankemmelbeke, Mireille; Healy, Bryan; Gr, Moore; Kleanthous, Colin; Penfold, Christopher N.; James, Richard

doi:10.1128/jb.187.18.6601.2005

Cited by 3 publications

(7 citation statements)

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“…A “gamma value” was calculated to quantify colicin activity ( Materials and Methods ), with higher gamma values corresponding to a greater colicin activity within the cell. 50 These experiments also indicated that L18A–L19A–L37A provides protection against ColE7, to a level similar to that provided by wild-type Im7 ( Fig. 5 b).…”

Section: Resultssupporting

confidence: 56%

“…This system utilises E. coli DPD1718, which has been engineered to contain a heterologous luxCDABE gene complex under the control of the cellular stress-induced E. coli recA promoter. 50 The luxA , luxB , luxC , luxD and luxE genes express the structural proteins responsible for bacterial bioluminescence. Hence, luminescence can be detected when the luxCDABE genes are expressed under the control of a stress-induced promoter activated by colicin-induced DNA damage.…”

Section: Resultsmentioning

confidence: 99%

“…Hence, luminescence can be detected when the luxCDABE genes are expressed under the control of a stress-induced promoter activated by colicin-induced DNA damage. 50 To exploit this assay, we transformed E. coli DPD1718 cells with the pTrc99a vector containing the gene for L18A–L19A–L37A. Cells were grown to an OD 600 (optical density at 600 nm) of 0.3, and protein expression was then induced by the addition of 1 mM IPTG for 1 h. The cells were then challenged by the addition of 4 nM ColE7, and luminescence, indicative of the induction of the stress response, was monitored ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This assay makes use of an SOS-inducible chromosomal lux operon to detect DNA damage induced by ColE7 in reporter cells. 50 E. coli DPD1718 contain a fusion of the E. coli recA promoter region with the Photorhabdus luminescens luxCDABE reporter integrated into the lacZ locus of E. coli DPD1692. 50 E. coli DPD1718 were transformed with a pTrc99a plasmid encoding the Im7 gene of interest, and overnight cultures were grown in LB at 37 °C in 10-ml culture volumes.…”

Section: Methodsmentioning

confidence: 99%

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Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Pashley

Morgan

Kalverda

et al. 2012

Journal of Molecular Biology

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The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, 15N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (Rh = 25 Å) relative to the urea-denatured state (Rh ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.

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Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Pashley

Morgan

Kalverda

et al. 2012

Journal of Molecular Biology

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“…Using an SOS–lux reporter system, James and co-workers demonstrated that translocation of the ColE9 DNase to the cytoplasm (detected by DNA damage-induced luminescence) takes the same amount of time whether the Im protein is bound or not (Vankemmelbeke et al 2005), suggesting Im protein dissociation is not rate-limiting for cell-killing. Using an EGFP–Im2 fusion as well as Western blotting, the Lloubes lab showed that the Im protein from the ColE2–Im2 complex dissociates on the same timescale as the kinetics of cell-killing and that the full translocon machinery is needed for the separation of Col from Im protein (Duche et al 2006).…”

Section: Col Translocation Across the Om And The Role Of The Pmfmentioning

confidence: 99%

Nuclease colicins and their immunity proteins

Papadakos

Wojdyla

Kleanthous

2011

Quart. Rev. Biophys.

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It is more than 80 years since Gratia first described 'a remarkable antagonism between two strains of Escherichia coli'. Shown subsequently to be due to the action of proteins (or peptides) produced by one bacterium to kill closely related species with which it might be cohabiting, such bacteriocins have since been shown to be commonplace in the internecine warfare between bacteria. Bacteriocins have been studied primarily from the twin perspectives of how they shape microbial communities and how they penetrate bacteria to kill them. Here, we review the modes of action of a family of bacteriocins that cleave nucleic acid substrates in E. coli, known collectively as nuclease colicins, and the specific immunity (inhibitor) proteins that colicin-producing organisms make in order to avoid committing suicide. In a process akin to targeting in mitochondria, nuclease colicins engage in a variety of cellular associations in order to translocate their cytotoxic domains through the cell envelope to the cytoplasm. As well as informing on the process itself, the study of nuclease colicin import has also illuminated functional aspects of the host proteins they parasitize. We also review recent studies where nuclease colicins and their immunity proteins have been used as model systems for addressing fundamental problems in protein folding and protein-protein interactions, areas of biophysics that are intimately linked to the role of colicins in bacterial competition and to the import process itself.

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Applying multi‐omics toward tumor microbiome research

Zhang

Kandalai

Zhou

et al. 2023

iMeta

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Rather than a "short-term tenant," the tumor microbiome has been shown to play a vital role as a "permanent resident," affecting carcinogenesis, cancer development, metastasis, and cancer therapies. As the tumor microbiome has great potential to become a target for the early diagnosis and treatment of cancer, recent research on the relevance of the tumor microbiota has attracted a wide range of attention from various scientific fields, resulting in remarkable progress that benefits from the development of interdisciplinary technologies.However, there are still a great variety of challenges in this emerging area, such as the low biomass of intratumoral bacteria and unculturable character of some microbial species. Due to the complexity of tumor microbiome research (e.g., the heterogeneity of tumor microenvironment), new methods with high spatial and temporal resolution are urgently needed. Among these developing methods, multi-omics technologies (combinations of genomics, transcriptomics, proteomics, and metabolomics) are powerful approaches that can facilitate the understanding of the tumor microbiome on different levels of the central dogma. Therefore, multi-omics (especially single-cell omics) will make enormous impacts on the future studies of the interplay between microbes and tumor microenvironment. In this review, we have systematically summarized the advances in multi-omics and their existing and potential applications in tumor microbiome research, thus providing an omics toolbox for investigators to reference in the future.

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Rapid Detection of Colicin E9-Induced DNA Damage Using Escherichia coli Cells Carrying SOS Promoter- lux Fusions

Cited by 3 publications

References 0 publications

Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Nuclease colicins and their immunity proteins

Applying multi‐omics toward tumor microbiome research

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