Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA , umuDC , or sulA Promoters

Abstract

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 M, 1.1 M, and 141 M, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.As a result of modern life, we are in daily contact with an ever increasing myriad of substances. This has necessitated the ability to screen large numbers of samples for harmful properties like carcinogenicity. Short-term bacteriological mutagenicity assays have shown great consistency with traditional rodent bioassays and are popular due to the relative ease with which multiple substances can be screened, with only modest requirements for laboratory equipment and space.In addition to the classic Ames test (2), used as a standard mutagenicity assay since the 1970s, many assays based on the Escherichia coli DNA repair SOS response have been developed. This has been done in order to provide even cheaper and faster alternatives and to accommodate high-throughput screening (13,33,35,36). SOS-based genotoxicity assays function via simple SOS promoter/reporter gene fusions, introduced into strains of E. coli or Salmonella enterica serovar Typhimurium. SOS induction is thereby an indicator of a genotoxic presence (measured by a subsequent increase in reporter gene levels).SOS genes are controlled by a single repressor, LexA, and are derepressed when cells undergo DNA damage, due to the accumulation of single-stranded DNA-RecA complexes which act as coproteases in a LexA self-cleaving mechanism (22,28,29). Expression of a given SOS gene depends on the specific LexA-binding properties of its promoter. These properties are determined by the presence of LexA-binding sequences (SOS boxes), which share a great deal of homology but are distinct from gene to gene (14,27). The response of an SOS-based genotoxicity assay is therefore greatly influenced by the employed promoter. The preferred promoters have typically been the recA, umuDC, and sulA promoters, as was the case with the rec-lac test (32), the umu test, and the SOS chromotest, respectively. A relatively...

The role of SOS boxes in enteric bacteriocin regulation

“…Similarly, induced enteric bacteriocin promoters express low basal levels of expression and high response ratios in the current (Fig. 4) and comparable studies (Mrak et al, 2007;Norman et al, 2005;Prieto et al, 2004;Vankemmelbeke et al, 2005). Moreover, when the contributions of each of the LexA binding sites were compared, expression under a single LexA repressor resulted in higher basal levels and lower response ratios (Fig.…”

“…3). Promoter-reporter gene fusions using gfp downstream of colicin D (Norman et al, 2005) and K (Mrak et al, 2007) promoters have been shown to respond in a similar manner, with high expression/leakage ratios and rapid responses to environmental stimuli. One hypothesis to explain these observations is that bacteriocin toxins are lethal to the producing cells and have intrinsically strong promoters.…”

“…Experimental data further suggest that the proximal SOS box of recN does not effectively bind the LexA protein (van der Lelie et al, 1997). Nevertheless, when the promoters of these four genes are fused to a reporter gene (gfp or lux), low basal levels of expression and high response ratios have been reported upon induction (Norman et al, 2005;Van Dyk et al, 2001a, b;van der Lelie et al, 1997). Similarly, induced enteric bacteriocin promoters express low basal levels of expression and high response ratios in the current (Fig.…”

Transcription termination controls prophage maintenance in Escherichia coli genomes

“…We therefore wondered whether the SOS response was involved in HK620 virion formation in the presence of BCM. We used a well-characterized reporter of the SOS response, the pANO1::cda′ plasmid that contains a Pcda′-gfpmut3* fusion (Table S1) (39). This reporter was transformed into lysogenic TD2158(HK620), and cultures were monitored for GFP production in the presence of BCM or MMC, the classical inducer of the SOS response.…”

Application of reporter strains for new antibiotic screening

“…Антибиотики, вызывающие повреждение ДНК, как фторхинолоны и митомицин С, активируют систему SOS-ответа. Несколько промоторов, находящихся под контролем данной системы, recA, sulA, umuDC и cda, использовали при создании сенсоров ДНК-повреждающих антибиотиков [12,[20][21][22][23][24][25]. Промотор гена katG использован для создания сенсора на окислительное повреждение клеток [21].…”