Improved bacterial SOS promoter∷lux fusions for genotoxicity detection

Davidov, Yaakov; Rozen, Ramona; Smulski, Dana R.; Dyk, Tina K. Van; Vollmer, Amy Cheng; Elsemore, David A.; LaRossa, Robert A.; Belkin, Shimshon

doi:10.1016/s1383-5718(99)00233-8

Cited by 111 publications

(72 citation statements)

References 28 publications

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“…E. coli BL21(DE3) or ER2566 (Novagen) was used as the host strain for the expression vector pET21a (Novagen), which has a strong IPTG-inducible T7 polymerase promoter and a C-terminal polyhistidine tag (His tag) to facilitate the purification of overexpressed proteins. E. coli DPD1718 contains a fusion of the E. coli recA promoter region to the Photorhabdus luminescens luxCDABE reporter integrated into the lacZ locus of E. coli DPD1692 (8). E. coli DPD2377 is E. coli DM800(pDEW634) that contains a plasmid-borne yigN fusion to the P. luminescens luxCDABE reporter (27).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2005

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ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.Colicins are plasmid-encoded antibacterial proteins that are secreted as part of the stress response system of Escherichia coli to kill other bacteria. They are classified into groups on the basis of the cell surface receptor on the target cells to which they bind. The E colicins all bind to the product of the chromosomal btuB gene, an outer membrane protein that is an essential component of the high-affinity transport system for vitamin B 12 in E. coli, and they require the outer membrane protein OmpF as a coreceptor (13). Based on immunity tests, the E group colicins have been subdivided into nine types, ColE1 to ColE9. These fall into one of three cytotoxic classes: the membrane-depolarizing, or pore-forming, agent ColE1 (7); the DNases, colicins E2, E7, E8, and E9 (6); and the RNases, colicins E3, E4, E5, and E6 (17, 20). Cells producing enzymatic colicins protect themselves against the action of their own toxin by coproducing a tightly binding, inactivating immunity protein. The resulting colicin-immunity complex is released from the cells, and the immunity protein is lost from the complex upon entry into susceptible cells (13).In common with most colicins, the enzymatic E-type colicins consist of three functional domains. The killing activity is contained in the C-terminal domain, which can be isolated as a stable and active protein (11,14,21,31). The central section contains the receptor-binding (R) domain, while the N-terminal T domain is responsible for translocation of the cytotoxic domain into the cytoplasm of the target cell (1, 10). After binding to their outer membrane receptors, group A colicins, such as colicin E9, are translocated across the membrane in a process that is mediated by the tol system (1, 10). Translocation requires a specific pentapeptide sequence in the T domain, known initially as the TolA box, which is now known to interact with TolB. ColE9 contains a TolB box from residues 35 to 39, DGSGW, which has been shown by mutagenesis to be important for its killing action and for the interaction of the T domain with the translocation protein TolB. The mechanism by which TolB recognizes and s...

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Section: Methodsmentioning

confidence: 99%

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

et al. 2005

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“…Using a concentration of 0.4 nM, which is within the concentration range that produces a linear response in the reporter assay, it is possible to accurately compare the DNA damage-inducing activity of the ColE9 mutant protein/Im9 complexes, expressed as a gamma value (8), at a fixed time after addition to E. coli DPD1718 cells (41) ( Table 1). The results support the largeplate assay data as the ColE9 S34A, S40A, and E42A mutant protein/Im9 complexes showed no significant DNA damageinducing activity.…”

Section: Resultsmentioning

confidence: 99%

“…This assay makes use of an SOS-inducible chromosomal lux operon to detect DNA damage induced by ColE9 in reporter cells (8,41). All assays were performed in a microtiter plate luminometer (Lucy 1; Anthos Labtech, Salzburg, Austria) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

et al. 2005

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The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.

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“…MMC is a natural occurring compound consisting of a pyrrolo (1,2-a) indole ring system with an aziridine ring.MMC is a potent DNA cross-linker, which has a strong ability to crosslink DNA with high efficiency and specificity for the sequence CpG [18]. To interact with DNA, MMC requires enzymatic activation by a one-electron pathway to a semiquinone, or by a two-electron reduction pathway to a hydroquinone [19]. MMC has several biological effects in mammalian cells, such as mutagenesis, stimulation of genetic recombination, selective inhibition of DNA synthesis, chromosome breakage and sister chromatid exchange and induction of the DNA repair system (SOS-response) [18].…”

Section: Discussionmentioning

confidence: 99%

Validation of SOS-lux Microbial Biosensors for Mutagenicity Assessment: Mitomycin-C as a Model Compound

Paton¹

2013

J Biosens Bioelectron

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Validation of SOS-lux Microbial Biosensors for Mutagenicity Assessment: Mitomycin-C as a Model Compound AbstractHuman health protection requires relating the bioaccessible concentration of a mutagen with the corresponding likely harm that could be caused through exposure. This requires designating the target receptor in need of protection, and a quantitative understanding of the likely pathways for mutagen availability. In this study, young children were selected as target receptors because of their tendency to directly ingest soils. Most data used to characterise a chemical mutagenicity has been extrapolated from rat-based assays using chemical ingestion or direct injection procedures. Mitomycin C was selected as a relevant model compound and extracted using an established in vitro digestion technique. A range of mutagenic bioassays (i.e. SOS-lux based microbial biosensors and Salmonella mutagenicity assay) were calibrated and optimised in aqueous samples, before being applied to soil extracts. The biosensors were consistently as sensitive and responsive as the traditional Salmonella assay, however, the use of microbial biosensors offered speed and ease of analysis. The data presented confirm that the in vitro digestion bioassay enabled a rapid and inexpensive technique for deriving critical values for the protection of humans exposed to soil borne mutagenic pollutants.

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Improved bacterial SOS promoter∷lux fusions for genotoxicity detection

Cited by 111 publications

References 28 publications

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

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

Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

Validation of SOS-lux Microbial Biosensors for Mutagenicity Assessment: Mitomycin-C as a Model Compound

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