Philippe Quillardet

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures

The SOS Chromotest is a quantitative bacterial colorimetric assay for genotoxins. Substantial validation is now available (Quillardet et al., 1985). We describe here in detail the tester strain as well as the effects of the variation of some parameters on the assay. We report a simple spot-test procedure as well as a new standard procedure which incorporate recent technical improvements aimed at simplifying the assay further.

The SOS chromotest: a review☆☆☆

The SOS chromotest is reviewed through over 100 publications corresponding to the testing of 751 chemicals. 404 (54%) of these chemicals present a genotoxic activity detectable in the SOS chromotest. Their SOS inducing potencies span more than 8 orders of magnitude. For 452 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 373 (82%) of these compounds give similar responses in both tests (236 positive and 137 negative responses). Thus the discrepancies between both tests concern 79 compounds (18%). A case by case analysis shows that many of these compounds are at the same time very weak SOS inducers and very weak mutagens. Thus we think that, most of the time, the discrepancies between the two tests may be accounted for by differences in the interpretation of the results rather than by the experimental results themselves. However, there are some compounds which are clearly SOS inducers but devoid of mutagenic activity in the Ames test (such as quinoline-1-oxide) and to a larger extent, clearly mutagenic compounds which do not induce the SOS response in the SOS chromotest (such as benzidine, cyclophosphamide, acridines, ethidium bromide). We also analyzed the correlation between SOS induction, mutagenesis and carcinogenesis according to the classification of Lewis. For 65 confirmed carcinogens (class 1), the sensitivity, i.e., the capacity to identify carcinogens, was 62% with the SOS chromotest and 77% with the Ames test. For 44 suspected carcinogens (class 2), the sensitivity was 66% with the SOS chromotest and 68% with the Ames test. Thus, we confirmed previous observations made on 83 compounds that there is a close correlation between the results given by both bacterial tests. The capacity of the Ames test to identify carcinogens is higher than that of the SOS chromotest. However, because the number of false positive compounds was lower in the SOS chromotest, the specificity, i.e., the capacity to discriminate between carcinogens and non-carcinogens of the SOS chromotest, appeared higher than that of the Ames test. Thus, the results of the SOS chromotest and of the Ames test can complement each other. The SOS chromotest is one of the most rapid and simple short-term test for genotoxins and is easily adaptable to various conditions, so that it could be used as an early--perhaps the earliest--test in a battery.

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: validation study with 83 compounds

The SOS Chromotest is a simple bacterial colorimetric assay for genotoxicity. It is based on the measure of the induction of sfiA, a gene controlled by the general repressor of the SOS system in E. coli. Expression of sfiA is monitored by means of a gene fusion with lacZ, the structural gene for beta-galactosidase. We have examined 83 compounds of various chemical classes with the SOS Chromotest using a standard procedure. Comparison of the results with those obtained in the Mutatest (the Ames test) showed that most (90%) of the mutagenic compounds were also SOS inducers. For these compounds a quantitative correlation was observed between the mutagenic potency and the SOS-inducing potency (SOSIP). The case of the 10% remaining compounds giving conflicting results in the two tests is discussed. Sensitivity, specificity and accuracy for carcinogenicity prediction have been evaluated for the SOS Chromotest and the Mutatest using 73 chemicals for which carcinogenicity data were available. In spite of some differences, similar results were obtained in the two tests. The present data indicate that the SOS Chromotest has many practical advantages and may be used as a primary screening tool or as part of a battery of short-term tests for carcinogens.

Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium

BackgroundThe colonial cyanobacterium Microcystis proliferates in a wide range of freshwater ecosystems and is exposed to changing environmental factors during its life cycle. Microcystis blooms are often toxic, potentially fatal to animals and humans, and may cause environmental problems. There has been little investigation of the genomics of these cyanobacteria.ResultsDeciphering the 5,172,804 bp sequence of Microcystis aeruginosa PCC 7806 has revealed the high plasticity of its genome: 11.7% DNA repeats containing more than 1,000 bases, 6.8% putative transposases and 21 putative restriction enzymes. Compared to the genomes of other cyanobacterial lineages, strain PCC 7806 contains a large number of atypical genes that may have been acquired by lateral transfers. Metabolic pathways, such as fermentation and a methionine salvage pathway, have been identified, as have genes for programmed cell death that may be related to the rapid disappearance of Microcystis blooms in nature. Analysis of the PCC 7806 genome also reveals striking novel biosynthetic features that might help to elucidate the ecological impact of secondary metabolites and lead to the discovery of novel metabolites for new biotechnological applications. M. aeruginosa and other large cyanobacterial genomes exhibit a rapid loss of synteny in contrast to other microbial genomes.ConclusionMicrocystis aeruginosa PCC 7806 appears to have adopted an evolutionary strategy relying on unusual genome plasticity to adapt to eutrophic freshwater ecosystems, a property shared by another strain of M. aeruginosa (NIES-843). Comparisons of the genomes of PCC 7806 and other cyanobacterial strains indicate that a similar strategy may have also been used by the marine strain Crocosphaera watsonii WH8501 to adapt to other ecological niches, such as oligotrophic open oceans.

Microcyclamide Biosynthesis in Two Strains of Microcystis aeruginosa: from Structure to Genes and Vice Versa

ABSTRACT Comparative analysis of related biosynthetic gene clusters can provide new insights into the versatility of these pathways and allow the discovery of new natural products. The freshwater cyanobacterium Microcystis aeruginosa NIES298 produces the cytotoxic peptide microcyclamide. Here, we provide evidence that the cyclic hexapeptide is formed by a ribosomal pathway through the activity of a set of processing enzymes closely resembling those recently shown to be involved in patellamide biosynthesis in cyanobacterial symbionts of ascidians. Besides two subtilisin-type proteases and a heterocyclization enzyme, the gene cluster discovered in strain NIES298 encodes six further open reading frames, two of them without similarity to enzymes encoded by the patellamide gene cluster. Analyses of genomic data of a second cyanobacterial strain, M. aeruginosa PCC 7806, guided the discovery and structural elucidation of two novel peptides of the microcyclamide family. The identification of the microcyclamide biosynthetic genes provided an avenue by which to study the regulation of peptide synthesis at the transcriptional level. The precursor genes were strongly and constitutively expressed throughout the growth phase, excluding the autoinduction of these peptides, as has been observed for several peptide pheromone families in bacteria.

A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism.

Results of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored a collaborative study to examine the intra- and inter-laboratory variation associated with the preparation and bioassay of complex chemical mixtures. The mixtures selected were National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs). 20 laboratories worldwide participated in the collaborative trial. The participating laboratories extracted the organic portion of two particulate samples--an air-particulate sample and a diesel-particulate sample--and bioassayed the extracts. The laboratories simultaneously bioassayed a NIST-prepared extract of coal tar and two control compounds (benzo[a]pyrene, and 1-nitropyrene). The bioassay method used was the Salmonella/mammalian microsome plate-incorporation test using strains TA98 and TA100. Study design also allowed for a comparison of sonication and Soxhlet extraction techniques. The mean extractable masses for the air particles and diesel particles were approximately 5% and 17.5%, respectively. The particulate samples were mutagenic in both strains with and without activation in all 20 laboratories. For TA100 the with and without activation slope values for the air particulate were 162 and 137 revertants per mg particles, respectively. For TA98 the respective diesel slope values were 268 and 269. The mutagenicity slope values for the diesel particles ranged from 3090 (TA98, +S9) to 6697 (TA100, +S9) revertants per mg particles. The coal tar solution was negative for both strains when exogenous activation was not used but was mutagenic in both strains with exogenous activation. The benzo[a]pyrene and 1-nitropyrene were used as positive controls and gave results consistent with the literature. This paper provides a complete summary of the data collected during the collaborative study. Companion papers provide further analysis and interpretation of the results.

DNA array analysis of gene expression in response to UV irradiation in Escherichia coli.

The capacity of DNA macroarrays that contain all 4290 predicted open reading frames of the E. coli K12 genome was evaluated by measuring changes in gene expression in response to irradiation by ultraviolet light (UV). UV and other DNA damaging agents are known to trigger the induction of the SOS response. This is a coordinated increase in the level of expression of a set of approximately 30 unlinked genes, the SOS genes, negatively regulated by the LexA repressor. The analysis was performed on a set of isogenic strains with mutations that affect expression of genes of the SOS system: (i) the lexA+ strain, in which the SOS system can be induced after DNA damage, (ii) lexAind- mutants in which the SOS system cannot be induced, and (iii) lexAdef mutants in which the SOS system is induced constitutively. We found that a large set of genes appeared to be either upregulated or downregulated following UV irradiation. Among the genes which appeared to be upregulated in a LexA-dependent manner, we correctly identified 9 out of 27 SOS genes printed on the arrays and one gene containing a LexA binding site. One gene, dnaN, encoding the beta subunit of DNA polymerase III holoenzyme, was identified as an upregulated gene in a LexA-independent manner. Our results were compared to those of similar studies previously published. Although the SOS response as a whole could not be illustrated by using DNA arrays, the data suggest that regulation of some SOS genes might be more complex than previously thought.

Genotoxicity of 2-nitro-7-methoxy-naphtho(2,1-b)furan (R7000): A case study with some considerations on nitrofurantoin and nifuroxazide

Two nitrofurans present broad-spectrum antimicrobial properties and some of them are used in human and veterinary medicine. Most of these molecules are mutagens and some of them were reported as carcinogens. Due to its extreme mutagenic potency in bacteria, the nitronaphtho derivative 2-nitro-7-methoxy-naphtho[2,1-b]furan (R7000) was used as a tool to analyze the mechanism of the genotoxic action of this family of chemicals. In the present paper, we review essential data on the genotoxicity of R7000 and briefly discuss the case of nitrofurantoin and nifuroxazide, two nitrofurans, still in use as urinary and gastrointestinal disinfectants.

Horizontal gene transfer of two cytoskeletal elements from a eukaryote to a cyanobacterium

Summary The concept of horizontal gene transfer (HGT) as a potent evolutionary force has prompted the re-evaluation of prokaryotic genome shaping and speciation. Horizontal gene transfer enables prokaryotes to rearrange their genomes dynamically, facilitating responses to changing environmental conditions and invasions of new ecological niches. Here we report that the genome of the cyanobacterium Microcystis aeruginosa contains a genomic island encoding proteins with extensive amino-acid sequence identity to two components of the eukaryotic actin cytoskeleton: actin itself; and profilin, an actin binding protein hitherto only known in eukaryotes. Our data indicate that a rare eukaryote-to-prokaryote HGT has introduced both sequences into the Microcystis lineage. We found both genes to be actively expressed and propose a unique role in Microcystis cell stabilization for actin, differing substantially from what is observed for bacterial actin homologs. Because we detected both eukaryote-like genes only in one strain in culture and in recent samples collected from its original habitat we suggest that both proteins may contribute to the adaptation of this strain to its specific ecological niche.