Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids

Abstract

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair. Bacterial repair of DNA damage is mediated by at least two inducible systems, the recA-independent, ada-controlled adaptive response and the recA-dependent, lexA-controlled SOS response. The former responds specifically to the presence of methylated phosphotriesters generated by DNA alkylation (39). This signal activates the ada gene product, which in turn triggers the transcription of genes such as ada, alkA, alkB, and aid (7, 20, 28, 39). In contrast, the nature of the specific inducing signal of the SOS response is not yet fully defined (21, 23, 53). Upon SOS induction, the recA gene product is converted into an active and specific protease (21). Activated RecA protein cleaves the LexA repressor and other repressors such as the phage cI product (22, 35), resulting in the transcriptional derepression of several genes, among them uvrA, recA, those needed in the lytic pathway of phage (17), and others, such as sulA, that couple DNA damage to cell division (14, 15). Recent reviews have focused upon one or both of these repair systems (39, 53). Activation of such repair systems is a measure of the mutagenic and genotoxic effects of various chemical and physical treatments. Many of the gene products, however, are difficult to assay because of the nature of their enzymatic activities and the particular substrates upon which they act. Thus, investigators have used relatively inexpensive and rapid alternative approaches. Measuring the reversion of specific auxotrophic bacterial mutations is the strategy used in the Ames tests (1, 24), and detecting restoration of bioluminescence is used in the Mutatox assay (47). The use of various transcriptional fusions also allows detection of agents that interact with DNA. The umu test (31), the rec-lac test (30), the SOS chromotest (34), and the Pro-Tox assay (32) exploit the ease with which ␤-galactosidase specific activities can be determined (26), while the biochemical prophage induction assay (11, 37) measures additional effects of the SOS response. The use of these assays by several pharmaceutical companies in Europe, Japan, and the

Versatile biosensor vectors for detection and quantification of mercury

“…All the mercury whole cell biosensors referred to above used a plasmid borne biosensor construct consisting of the mercury inducible promoter P mer in combination with its regulatory gene merR, both obtained from the well described transposon Tn21 system [6]. However, some loss of sensitivity has been recorded when using plasmids instead of chromosomal inserts [7]. Furthermore, plasmids are sometimes lost from host organisms if selective pressure to maintain the plasmids is absent [8].…”

Bacterial genotoxicity bioreporters

“…The system was tested against 4-Nitroquinoline-N-oxid (4-NQO), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C (MMC) and UV radiation, as well as with hydrogen peroxide, formaldehyde, tert-butyl hydroperoxide, cumene hydroperoxide and streptonigrin. A different reporter system, the V. fischeri luxCDABE operon, was used by Vollmer and colleagues (1997) to generate several E. coli reporter strains, one of them (DPD2794) carrying a recA′::luxCDABE fusion on the multi-copy plasmid pUCD615 (Vollmer et al, 1997). As in other constructs carrying this 5-gene complement, these bioluminescent fusions allowed real-time visualization of the transcriptional responses induced by DNA damage, without the need for cell-free enzyme assays or the exogenous addition of luciferase substrates.…”

Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli

“…The recombinant bacterial strain DPD2794, containing a recA::luxCDABE fusion, was used in this study (Vollmer et al 1997). During the flask experiments, the cells were grown in Luria-Bertani (LB) medium (Difco-BRL) with 25 mg l )1 kanamycin monosulphate (Sigma) at 30°C and an initial pH of 7AE0; aeration was provided by agitation on a rotary shaker at 250 rev min )1 .…”

“…This adaptive response may be used to study and evaluate the effectiveness of the DNA repair system (Bartusiak et al 2002), and is a phenomenon by which cells exposed to low concentrations of a mutagen become significantly resistant to a subsequent exposure by the same mutagen (Izawa et al 1990; Bartusiak et al 2002). Therefore, in this study, a recombinant bioluminescent E. coli, DPD2794 with the recA promoter fused to luxCDABE originated from Vibrio fischeri (Vollmer et al 1997), was used to observe adaptive responses after the cellular DNA was damaged by three different mutagens, namely an intercalating agent [mitomycin C (MMC)], an alkylating agent [N-methyl-N-nitro-Nnitrosoguanidine (MNNG)] and a hydroxylating agent [hydrogen peroxide (H 2 O 2 )]. The DNA repair system of E. coli was studied using these three mutagens to assess the adaptive response of DPD2794.…”