Correction method for estimating the number of reversions in bacteria

A correction of the results of mutation tests in bacteria during the induction of reversions from auxotrophy to prototrophy is described. The correction is based on a calibration curve of the spontaneous frequency of reversions and is suitable for testing doses of mutagens that exhibit a pronounced lethal effect.     

The induction and characterization of recessive suppressors of arg-2 in Schizophyllum commune

Summary Genetic analyses have been made to detect recessive suppressor mutations in eight prototrophic strains derived by treating an arginine dependent strain with hydroxylamine. The results indicate that one strain possesses a recessive suppressor, su-1, which maps outside the arg-2 locus and is capable of suppressing auxotrophy conferred by the arg-2 mutation. This suppressor is incapable of suppressing auxotrophy conferred by eight other loci. Prototrophy in the remaining seven strains resulted from either intragenic suppression, reversion, or from a suppressor mutation that is closely linked to the arg-2 locus. The results of heterokaryotic allelic tests with the seven strains indicate that the mutation to prototrophy is recessive.     

Population changes induced in Candida albicans by nalidixic acid

Cells of Candida albicans plated on media containing nalidixic acid (Nal) either die, adapt physiologically to Nal-tolerance or mutate to Nal-resistance. The fraction of a population exhibiting each response depends on the growth phase of cells when plated and their nitrogen and carbon nutrition and growth temperatures before and after plating. Nal induces Nal-resistant mutants in very high frequency but only at 37 C on plates containing i) glucose as primary carbon source and ii) adenine, a sulfur amino acid or a representative of the glutamic acid family of amino acids. Nal does not affect either forward mutation to caffeine-resistance or reverse mutation from histidine auxotrophy to prototrophy. Nal-resistant mutants produce minute colonies on Nal-free medium, respire oxidatively and are unusually sensitive to inhibitors of oxidative phosphorylation. They revert spontaneously to wild type at very high rates but can be propagated indefinitely in the absence of Nal by serial selection and replating of minute colonies. Cellular inactivation and induction of Nal-resistant mutants are greatly affected by specific inhibitors of mitochondrial macromolecular syntheses. The presence of chloramphenicol or erythromycin during exposure to Nal prevents cell death and mutation but has no effect on adaptation to Nal-tolerance. Growth on acriflavin or ethidium bromide enhances resistance of cells to inactivation when subsequently plated on Nal containing media. It is concluded that Nal-induced cellular inactivation and mutation to Nal-resistance, but not adaptation to Nal-tolerance, result from damages to the mitochondrion which are fixed or promoted by macromolecular syntheses within the mitochondrion. Implications of these findings for the therapeutic use of Nal are discussed.     

Simple method for precise determination of chemical lethality in the L-arabinose resistance test of Salmonella typhimurium

A simple method is described for the determination of the lethal effects of chemicals assayed with the L-arabinose resistance test of Salmonella typhimurium. The method uses a mixed culture of 2 isogenic bacterial strains which are distinguished on the basis of their different nutritional requirements: sensitivity or resistance to L-arabinose, auxotrophy or prototrophy to histidine and leucine. BA13 (the mutation indicator strain) is the strain for routine screening of mutagens and allows the selection of forward mutation from L-arabinose sensitivity to L-arabinose resistance. BAL13 (the survival indicator strain) is a derivative of BA13. Both bacterial strains are found to be equally sensitive to the lethal effects of mutagens. The method described permits the measurement of cell survival at the same high cell concentration as used in the measurement of the mutant yield and in the same type of minimal medium with L-arabinose and glycerol, except for the histidine supplement in the mutant plates or the leucine in the survival plates.     

Carboxymethyl Cellulose Decomposition by Intestinal Bacteria of Cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Carboxymethyl cellulose decomposition by intestinal bacteria of cockroaches

The effect of freeze-drying on phenotypic reversion of amino acid auxotrophy to prototrophy was studied in Escherichia coli. In a radioresistant strain, E. coli H/r 30 (uvr+ exr+), which can repair the deoxyribonucleic acid damaged due to freeze-drying, an increased mutation frequency from auxotrophy to prototrophy was observed with increased time of freeze-drying of the cells. On the other hand, in a radiosensitive strain, E. coli NG 30 (recA), which cannot repair the damaged deoxyribonucleic acid due to a lack of repair enzyme system, no significant reversion occurred, although the survival rate was very low. The rate of phenotypic reversion dut to freeze-drying in both E. coli RIMD 0509109 (uvr+ exr+) and RIMD 0509115 (uvr exr+) was almost the same, indicating that the phenomenon is independent of the uvr character. From these results it is concluded that mutation was induced in E. coli cells during the rehydration when the damaged deoxyribonucleic acid was repaired by exr character of the cells. Thus, we propose that a serious consideration should be paid to the freeze-drying technique to preserve bacterial cells.     

Genetic variability among archival cultures of Salmonella typhimurium

The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies. In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in these populations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.     

Mutation induction in Escherichia coli WP2 uvrA by Cerenkov emission associated with 137Cs gamma irradiation

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs gamma irradiation. Furthermore, the data strongly suggest a more than additive interaction between the gamma-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/10(7) survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by gamma irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.     

Comparison of a forward and a reverse mutation assay in Salmonella typhimurium measuring L-arabinose resistance and histidine prototrophy

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.     

Complementation of Methionine Auxotrophs of Pseudomonas aeruginosa from Cystic Fibrosis

Auxotrophic Pseudomonas aeruginosa are exclusive to respiratory infections in cystic fibrosis (CF) and bronchiectatic patients, and isolates require specific amino acids for growth on minimal media, particularly methionine. Since auxotrophic and prototrophic P. aeruginosa from CF are identical by genotyping, we investigated the genetic events leading to methionine auxotrophy (Met⁻). Most (10/13) Met⁻ strains had the same pattern of growth on methionine precursors and required methionine exclusively for growth. Back mutation to prototrophy was very low (frequencies 10⁻⁸ to <10⁻¹⁰). Complementation of the mutations leading to auxotrophy was achieved for five strains with a genomic library of P. aeruginosa PAO1. Strains with different patterns of growth on methionine precursors were complemented by clones with different restriction patterns, while identical clones complemented strains with the same pattern of growth on methionine precursors. Methionine auxotrophy in P. aeruginosa from CF results from stable chromosomal mutations, and the commonest defect is probably in gene(s) encoding enzymes that convert homocysteine to methionine. Received: 2 August 1997 / Accepted: 23 September 1997     

Molecular cloning and characterization of supQ/newD, a gene substitution system for the leuD gene of Salmonella typhimurium.

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.     

Ethyl methanesulfonate: an effective mutagen in Chlamydomonas reinhardi

Summary The effectiveness of ethyl methanesulfonate (EMS) as a mutagen inChlamydomonas reinhardi has been demonstrated in two different systems: reversion to prototrophy of an argine-requiring strain and forward mutation to auxotrophy of the wild-type strain. In these experiments, the toxic effect of EMS was very low. With certain batches of EMS, an unusually high toxicity was observed. In this case, the lethal effects seem to be attributable to the presence of minor impurities in EMS.Chargé de Recherches au Fonds National de la Recherche Scientifique.     

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells

Background

A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy.

Results

Extensive and variable losses and a few gains of catabolic functions were observed by this standardized method. Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates.

Conclusion

While the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment. Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog? system.     

Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells

This article describes the selection of auxotrophic mutants of Chinese Hamster Ovary (CHO) cells and the genetic and biochemical characterization of two mutant lines. AUXB1 is auxotrophic for glycine, adenosine, and thymidine (GAT-), whereas AUXB3 requires only glycine and adenosine (GA-). These mutants do not complement since hybrid cells formed between them are also auxotrophic. Experiments concerned with the reversion of AUXB1 to prototrophy suggest that a single genetic lesion is responsible for the multiple auxotrophy. Biochemical analysis indicates that the multiple auxotrophy of both AUXB1 and AUXB3 is a result of low levels of intracellular folates in mutant cells. Phenotypic reversion to complete or partial prototrophy can be accomplished by growing these cells in high concentrations of folic or folinic acids. However, neither the folate transport nor the dihydrofolate reductase are defective in mutant cells. Chromatographic analysis of intracellular folate derivatives indicates that while folates extracted from wild type cells exist almost exclusively as polyglutamyl derivates (primarily pentaglutamates), AUXB1 cells contain primarily folate derivates in monoglutamyl form and AUXB3 cells contain mono-, di-, and perhaps some triglutamates. This observation suggests that the enzyme responsible for linking glutamate residues onto intracellular folate derivates is the site of the biochemical lesion in the mutant cells. Our results also suggest that a possible function of polyglutamyl residues is to aid cellular retention of folates.     

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae

The hypothesis of an alternative pathway of sulphur amino acid synthesis as the basis of the prototrophy of sulphite reductase negative (Sr^-) strains of Saccharomyces cerevisiae has been rejected. Met^- mutants obtained after phenylmercuric nitrate treatment of Sr^- strains accumulate H₂S as the consequence of a metabolic block which leads to methionine auxotrophy. This mutation has been shown to be independent of the Sr locus. We assume that the molecular basis of the prototrophy of Sr^- strains resides in a leaky missense induced in the Sr gene.     

Integration of mammalian, microbial and drosophila procedures for evaluating chemical mutagens.

cis-Platinum(II)diamminodichloride (PDD), an anti-tumor agent, induced auxotrophic mutations in Escherichia coli, some of which were reverted to prototrophy by exposure to PDD, 2-aminopurine (2-AP), and N-methyl-N′-nitro-N-nitroguanidine (NTG), but not ICR derivatives. Similarly, various 2-AP-, NTG-, and ultraviolet light-induced auxotrophs were reverted to prototrophy by PDD. Some PDD-induced auxotrophs carried nonsense mutations and others could be phenotypically suppressed by growth with streptomycin. Although these findings suggest that PDD promotes base substitutions, this mutagen may also cause base subtractions because (like NTG)it induced, at reduced frequency, reversion to prototrophy of certain ICR-induced auxotrophs. Isomeric trans-platinum(II)diamminodichloride, which lacks anti-tumor activity, was an ineffective mutagen. Near-optimal conditions for PDD-induced mutagenesis entailed prolonged cultivation with low levels of mutagen where the frequency of forward mutation to auxotrophy was 10⁻³ and that of a selected trp isolate to prototrophy was 10⁻².     

Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus

A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.     

Yeast vectors for integration at the HO locus

We have constructed new yeast vectors for targeted integration of desired sequences at the Saccharomyces cerevisiae HO locus. Insertion at HO has been shown to have no effect on yeast growth, and thus these integrations should be neutral. One vector contains the KanMX selectable marker, and integrants can be selected by resistance to G418. The other vector contains the hisG-URA3-hisG cassette, and integrants can be selected by uracil prototrophy. Subsequent growth on 5-FOA permits identification of colonies where recombination between the hisG tandem repeats has led to loss of the URA3 marker and return to uracil auxotrophy. We also describe several new bacterial polylinker vectors derived from pUC21 (ampicillin resistance) and pUK21 (kanamycin resistance).     

INTERACTION OF IONIZING AND VISIBLE RADIATION IN MUTATION INDUCTION IN NEUROSPORA CRASSA

Klein , Richard M. (New York Bot. Gdn., New York, N.Y.), and Deana T. Klein. Interaction of ionizing and visible radiation in mutation induction in Neurospora crassa. Amer. Jour. Bot. 49(8): 870–874. 1962.—Conidia of the purple adenineless strain of N. crassa were irradiated with 25 kr of X rays and then exposed to far-red or red radiations or to far-red followed by red radiation. Far-red light, without effect on un-irradiated conidia, augmented the genetic damage caused by X rays as measured by survival (colony count), back mutation to adenine prototrophy, and the induction of mutants affecting colony morphology. Post-X-irradiation with red light ameliorated the severity of X-radiation as measured by survival and back mutation. The potentiation of X-ray-induced genetic damage by far-red light could be completely negated by subsequent exposure to red light.     

Stringent mating-type-regulated auxotrophy increases the accuracy of systematic genetic interaction screens with Saccharomyces cerevisiae mutant arrays

A genomic collection of haploid Saccharomyces cerevisiae deletion strains provides a unique resource for systematic analysis of gene interactions. Double-mutant haploid strains can be constructed by the synthetic genetic array (SGA) method, wherein a query mutation is introduced by mating to mutant arrays, selection of diploid double mutants, induction of meiosis, and selection of recombinant haploid double-mutant progeny. The mechanism of haploid selection is mating-type-regulated auxotrophy (MRA), by which prototrophy is restricted to a particular haploid genotype generated only as a result of meiosis. MRA escape leads to false-negative genetic interaction results because postmeiotic haploids that are supposed to be under negative selection instead proliferate and mate, forming diploids that are heterozygous at interacting loci, masking phenotypes that would be observed in a pure haploid double-mutant culture. This work identified factors that reduce MRA escape, including insertion of terminator and repressor sequences upstream of the MRA cassette, deletion of silent mating-type loci, and utilization of α-type instead of a-type MRA. Modifications engineered to reduce haploid MRA escape reduced false negative results in SGA-type analysis, resulting in >95% sensitivity for detecting gene–gene interactions.