Arginine Auxotrophy Research Articles

Stationary phase expression of the arginine biosynthetic operon argCBH in Escherichia coli

BackgroundArginine biosynthesis in Escherichia coli is elevated in response to nutrient limitation, stress or arginine restriction. Though control of the pathway in response to arginine limitation is largely modulated by the ArgR repressor, other factors may be involved in increased stationary phase and stress expression.ResultsIn this study, we report that expression of the argCBH operon is induced in stationary phase cultures and is reduced in strains possessing a mutation in rpoS, which encodes an alternative sigma factor. Using strains carrying defined argR, and rpoS mutations, we evaluated the relative contributions of these two regulators to the expression of argH using operon-lacZ fusions. While ArgR was the main factor responsible for modulating expression of argCBH, RpoS was also required for full expression of this biosynthetic operon at low arginine concentrations (below 60 μM L-arginine), a level at which growth of an arginine auxotroph was limited by arginine. When the argCBH operon was fully de-repressed (arginine limited), levels of expression were only one third of those observed in ΔargR mutants, indicating that the argCBH operon is partially repressed by ArgR even in the absence of arginine. In addition, argCBH expression was 30-fold higher in ΔargR mutants relative to levels found in wild type, fully-repressed strains, and this expression was independent of RpoS.ConclusionThe results of this study indicate that both derepression and positive control by RpoS are required for full control of arginine biosynthesis in stationary phase cultures of E. coli.

Kinetic Analysis of the l-Ornithine Transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola That Is Resistant to the Transition State Analogue (R)-Nδ-(N‘-Sulfodiaminophosphinyl)-l-ornithine

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host ( niaD − sC −). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain ( niaD − sC − adeA −) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains ( niaD − sC −Δ argB adeA − or niaD − sC −Δ argB adeB −) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain ( niaD − sC −Δ argB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.

Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293.

Aspergillus fumigatus is a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in the A. fumigatus genome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion of argB and lysB, respectively. Transformation of these auxotrophic strains with plasmids carrying argB or lysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility.

Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation.

Argininosuccinate synthetase (ASS) was the first of two enzymes to convert citrulline to arginine. This pathway allowed cells to synthesize arginine from citrulline, making this amino acid nonessential for the growth of most mammalian cells. Previous studies demonstrated that several human tumor cell lines were auxotrophic for arginine due to an inability to express ASS. Selective elimination of arginine from the circulation of animals with these tumors is a potentially effective anticancer treatment. The purpose of these experiments was to determine the frequency of ASS deficiency and arginine auxotrophy in a variety of human malignant tumors. The authors analyzed the expression of ASS by immunohistochemistry with a monoclonal antibody in a variety of human tumor biopsies. They found that the incidence of ASS deficiency varied greatly with the tumor type and tissue of origin. Melanoma, hepatocellular carcinoma, and prostate carcinoma were most frequently deficient in ASS. Some human cancers were almost always positive for ASS (e.g., lung and colon carcinomas). However, other human cancers, including sarcomas, invasive breast carcinoma, and renal cell carcinoma, also were sometimes ASS deficient. These data indicated that immunohistochemical detection of ASS may prove an effective means for determining ASS deficiency in malignant human tumors and for identifying patients most likely to respond to arginine deprivation therapy. Based on these results, human clinical trials using arginine-degrading enzyme therapy to treat patients with advanced melanoma or hepatocellular carcinoma have been initiated.

Disruption of the Aspergillus fumigatus argB gene using a novel in vitro transposon-based mutagenesis approach.

We disrupted the Aspergillus fumigatus argB gene, encoding ornithine transcarbamylase, using a novel in vitro transposon-based mutagenesis approach. This approach utilizes a modified transposon containing the Neurospora crassa pyr4 gene, which is randomly inserted in vitro into a target sequence of interest. Clones in which the gene of interest has been disrupted are identified by PCR and used to transform a pyrG-deficient strain of A. fumigatus. Using this approach, we obtained arginine auxotrophs of A. fumigatus. Full characterization of the argB insertion was performed by Southern blot analysis. These strains can be supplemented by addition of arginine into the culture medium and can be fully rescued to arginine prototrophy by transformation with the intact A. fumigatus argB gene.

Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation

Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes. Toxoplasma gondii is novel in possessing a single carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to T. gondii infection. These findings are consistent with a model of host–parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.

In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate.

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis.

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95-1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95-1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris.

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

In Lactobacillus plantarum , Carbamoyl Phosphate Is Synthesized by Two Carbamoyl-Phosphate Synthetases (CPS): Carbon Dioxide Differentiates the Arginine-Repressed from the Pyrimidine-Regulated CPS

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO(2). Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (DeltaCPS-P, DeltaCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO(2)-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO(2) enrichment may be due to the low affinity of CPS-A for its substrate CO(2) or to regulation of the CP pool by the cellular CO(2)/bicarbonate level.

Generation of nuclear hybrids overcoming the natural barrier of incompatibility: transfer of nuclei from Lentinula edodes into protoplasts of Coriolus versicolor.

Heterokaryotic nuclear hybrids overcoming the natural barriers of incompatibility have been studied in basidiomycetes. To produce these nuclear hybrids between incompatible mushrooms, which have several potent pharmacological effects, nuclear transfer was performed between Lentinula edodes and Coriolus versicolor. Nuclei from serine auxotrophs of Lentinula edodes, LE207 (Ser-) were transferred into the protoplasts of arginine auxotrophs of Coriolus versicolor, CV17 (Arg-), using 30% polyethylene glycol 4000 in 10 mM CaCl2-glycine solution (pH 8.0). Nuclear transfer progenies were selected by nutritional complementation on minimal media supplemented with 0.6 M sucrose. The progenies were classified based on colony morphology to L. edodes-like, C. versicolor-like and non-parental type. Most of the progenies grew slower than either parent. The number of nuclei per cell was similar but the DNA content varied between progenies. The isozyme patterns of nuclear hybrids resembled either of the parent profiles or showed a mixed profile.

Pectinase production by a diploid construct from two Aspergillus niger overproducing mutants

Arginine auxotrophy (Arg −) was induced in two Aspergillus niger strains, previously reported as 2-deoxy- d-glucose (2DG) resistant and pectinase overproducing mutants in either solid state fermentation (SSF) or submerged fermentation (SmF). Only one of these two arginine auxotrophs (strain Aw99ARG) was transformed with a plasmid containing the A. nidulands argB gene, indicating that this strain was affected in this particular gene. A diploid (D4) was generated via parasexual recombination between these two Arg − strains and was characterized with regard to 2DG sensitivity and pectinase production. This diploid showed wild type sensitivity to 2DG, suggesting that recessive mutations at two different loci were involved in 2DG resistance in each parent strain. The diploid had also increased pectinase production in SmF (127% higher) when compared to the wild type strain, from which all pectinase overproducing mutants were previously isolated, and 10% over Aw99-iii2 strain, formerly characterized as an overproducing strain in SmF.

Molecular cloning and analysis of the argC gene from Corynebacterium glutamicum.

The argC gene encoding N-acetylglutamate 5-semialdehyde dehydrogenase has been cloned from Corynebacterium glutamicum by transforming Escherichia coli arginine auxotroph with the genomic DNA library. Based on the restriction map of the cloned DNA, we have subcloned and sequenced the minimal DNA fragment complementing the E. coli argC mutant. The coding region of the cloned gene is 1041 nucleotides long with a predicted molecular mass of about 38 kDa polypeptide. Enzyme activity and size of the expressed protein in the E. coli auxotroph carrying the recombinant argC gene revealed that the cloned gene indeed codes for N-acetylglutamate 5-semialdehyde dehydrogenase. Computer analysis of the amino acid sequences of the predicted protein revealed a strong similarity to the corresponding protein of other bacteria.

A Comparison of Auxotrophic and Wild Strains of Sclerotinia sclerotiorum Used as a Mycoherbicide Against Californian Thistle ( Cirsium arvense )

Two auxotrophic mutant strains of Sclerotinia sclerotiorum were tested in the greenhouse for pathogenicity on Cirsium arvense (Californian thistle) with and without amino acid amendments. An arginine auxotrophic mutant, with an amendment of the amino acid, followed an identical disease progress curve to that of the wild strain of the pathogen from which it was derived. However, when deprived of the amino acid amendment it was still highly pathogenic. A leucine auxotrophic mutant demonstrated poor pathogenicity without a leucine amendment, but improved pathogenicity with the addition of the amino acid. However, both of these treatments were inferior to the two wild strains tested and the arginine auxotroph with and without amendments. A field experiment was conducted on C. arvense stems in permanent pasture to compare the pathogenicity of amended auxotrophic strains and wild strains of S. sclerotiorum applied as a granule in a wheat-based carrier. The two wild strains gave significant reductions in thistle cover within 3 months of treatment, and subsequent reductions in thistle stem height and density during the following season. There was no evidence that the auxotrophic strains reduced thistle cover in the season the treatments were applied, but they did reduce subsequent stem density in the following spring. To determine disease carry-over associated with the wild and auxotrophic strains of the pathogen, rape was planted into subplots over the next three consecutive seasons. Despite substantial populations of sclerotia being present in the soil, especially in the first season after treatment of the thistles, no disease of rape caused by S. sclerotiorum was detected over the three seasons in any of the plots. Sclerotium populations of S. sclerotiorum in the soil declined by over 50% between 20 and 32 months after treatment, but there was no decline over the subsequent 12 months. The trial demonstrated that the auxotrophic strains were less field fit compared with the wild strains and that the presence of inoculum and a susceptible host to S. sclerotiorum were not the only prerequisites for disease development. It was concluded that use of a trap crop following treatment is not a suitable method for determining the risk of using this pathogen as a mycoherbicide in pasture.

Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans.

The unstable linear chromosome of Streptomyces lividans was circularized by homologous recombination and its terminal inverted repeats deleted. Strains with circularized chromosomes showed no obvious phenotypic disadvantages compared to the wild type. However, they segregated about 20 times more chloramphenicol-sensitive mutants than the wild type (24.3% vs. 1.4%), due to a higher incidence of large deletions. In addition, in all circularized chromosomes amplification of 30-60 kb fragments was observed at the new chromosomal junction, to levels of approximately 10 copies per chromosome. Arginine auxotrophs that arose spontaneously among the progeny of strains with a circularized chromosome showed high-copy-number amplification of the DNA element AUD1, as also seen in mutants of the wild type. These observations demonstrate that the circular form of the Streptomyces chromosome is more unstable than the linear one.

Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans.

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 delta strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6 delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.

Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes in Streptomyces coelicolor.

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis in Streptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolated S. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-type S. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A subfragment carrying the 5' end of argC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism in Bacillus subtilis. It is therefore likely that in S. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterised B. subtilis and Escherichia coli repressors.

Production of l-ornithine by arginine auxotrophic mutants of Brevibacterium ketoglutamicum in dual substrate-limited continuous culture

Production of l-ornithine under dual limitation of arginine and phosphate was investigated in a chemostat culture of an arginine auxotroph of Brevibacterium ketoglutamicum. Under an arginine-limited condition, culture instability, including the formation of revertant cells, could be a serious problem due to the inherent instability of the auxotrophic mutant strain. The difference in the growth rates between the auxotrophs and revertants was considered to be the main cause of the culture instability, and dual limitation with arginine and phosphate was found most appropriate to overcome this problem. When the ratio of the phosphate to arginine concentration in the feed medium was varied between 0.55 and 5.22 at fixed dilution rate of 0.1 h −1, three distinct growth phases were recognized: phosphate-limited (0.55 to 2.01), dual arginine- and phosphate-limited (2.01 to 2.16) and arginine-limited (above 2.16). In the dual arginine- and phosphate- limitation phase, the culture stability was markedly improved while the yield of l-ornithine was maintained at a high level. When the dilution rate was varied from 0.04 h −1 to 0.17 h −1 under the dual-limitation condition, maximum l-ornithine productivity was obtained at a value of 0.27 g/ l/h at D=0.1h −1.

MX100, a new Escherichia coli tester strain for use in genotoxicity studies.

The development of a new Escherichia coli tester strain for use in metabolic and mechanistic studies of genotoxins, strain MR2101/pKR11, has recently been reported. This strain, a derivative of the E. coli K12 laboratory strain AB1157, has sensitivity towards the detection of base-substitution mutagenesis, monitored by the reversion of arginine auxotrophy [argE3, (ochre)]. Besides arginine, MK2101/pKR11 is auxotrophic for histidine (hisG4), leucine (leuB6), proline (DeltaproA) and threonine (thr-1). MX100 was developed to overcome the auxotrophy for four amino acids of MR2101/pKR11 which are non-essential for the mutagenic responsiveness of the strain. We restored the biosynthesis for these four amino acids in MR2101/pKR11, resulting in strain MX100. This strain showed an almost 2-fold increase in mutagenic activity relative to MR2101/pKR11 with a set of diagnostic mutagens (aflatoxin B1, benzo[a]pyrene, 4-nitroquinoline-1-oxide, 2,7-dimethyl-benz[a]anthracene and others) and was further characterized with other types of mutagens in which it showed sensitivity towards the detection of oxidative (H2O2t-butyl-hydroperoxide, cumene-hydroperoxide, KO2) and carbonyl mutagens (methylglyoxal, malondialdehyde). As MX100 seems to have the right characteristics of a versatile genotoxicity tester strain and due to the extensive genetic and physiological knowledge of E. coli K12 in general and AB1157 in particular, we propose that MX100 could serve as mother strain for the development of specialized tester strains, of interest in studies of metabolism and/or mechanism of action of genotoxic carcinogens.