A new experimental system for study on adaptive mutations

A super-repressed mutant of purR (purR^S), which encodes a repressor protein controlling expression of purine biosynthetic genes inSalmonella typhimurium, grew very slowly on NCE medium with 10 μg/mL Ade and lactose as sole carbon source (cannot form colonies). However, a phenomenon of late-arising mutations was observed when purR^S mutants were spread on NCE+lactose plates and subjected to a prolonged non-lethal selection. The reconstruction experiments of revertants showed that the late-arising “lac⁺” mutants are not slow growing mutants. Statistical analysis indicated that the distribution of late-arising mutants is Poisson distribution, showing that reversion occurred after plating. The result of co-transductional analysis preliminarily showed that late-arising mutation occurred at selected genepurR or 16 bp PUR box,cis element of structural genepurD. The above results suggest that the phenomenon of late-arising mutation observed by our system is a result of adaptive mutations which are different from random mutations. This is the first time to extend target genes at which adaptive mutations could occur from structural genes involved in carbon metabolism and amino acid biosynthesis totrans regulatory gene coding repressor protein. Our results have provided not only a new proof for generality of adaptive mutations but also a new system for study on adaptive mutations.     

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac⁻) but that reverts to lactose utilization (Lac⁺) when lactose is its sole energy and carbon source. The adaptive Lac⁺ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac⁻ allele, and an unknown process that affects the whole genome. Most of the Lac⁺ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac⁺ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.     

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Regulation of Newly Evolved Enzymes. III Evolution of the ebg Repressor during Selection for Enhanced Lactase Activity

The evolution of lactose utilization by lacZ deletion strains of E. coli occurs via mutations in the ebg genes. We show that one kind of mutation in the regulatory gene ebgR results in a repressor which retains the ability to repress synthesis of ebg enzymes, but which permits 4.5-fold more ebg enzyme synthesis during lactose induction than does the wild-type repressor. A comparison between the growth rate of various ebg+ strains on lactose and the amount of ebg enzyme synthesized by these strains shows that the rate of enzyme synthesis permitted by the wild-type repressor is insufficient for growth on lactose as a sole carbon source by a cell with the most active ebg lactase yet isolated. We conclude, therefore, that the evolution of lactose utilization requires both a structural and a regulatory mutation.     

Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants

From a pool of Medicago truncatula mutants—obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis—impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis. Phenotypic data relate to nodulation and mycorrhizal phenotypes. Among the five new mutants, three were classified as [Nod⁺ Fix^– Myc⁺] and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49). For the two other new mutants, one was classified as [Nod^–/+ Myc⁺] with a mutation ascribed to gene Mtsym15 (TRV48), and the other as [Nod^– Myc^-/+] with a mutation ascribed to gene Mtsym16 (TRV58). Genetic analysis of three previously described mutants has shown that [Nod^–/+ Myc⁺] TR74 mutant can be ascribed to gene Mtsym14, and that [Nod^–/+ Myc^–/+] TR89 and TRV9 mutants are ascribed to gene Mtsym2 (dmi2). Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria. This phenotype was called [Myc^–/+] and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as [Myc^–/+]. Mutant P1 was reclassified as [Nod^–/+] because of a late nodulation observed on roots of this mutant.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Activation of the bgl operon by adaptive mutation

In growing Escherichia coli K12 cells, the cryptic bgl operon is activated 98% of the time by insertions of IS1 or IS5 into the control region, designated bglR. The activated bgl operon permits utilization of the beta-glucoside sugar arbutin as a sole carbon and energy source. The bgl operon is also activated by late-occurring mutations during prolonged selection on arbutin. The late-occurring mutations that occurred during prolonged carbon starvation in the presence of arbutin were "adaptive mutations" because they were specific to the presence of arbutin, and they did not occur during prolonged starvation in the absence of arbutin. The spectrum of late-arising mutations differed from that of early-arising, growth-dependent mutations in that 20% of the late-arising mutants resulted from mutations at the hns locus. This provides the first direct evidence for adaptive mutagenesis mediated by the insertion of IS elements. Because no special genetic background is required to select Bgl+ mutants, this affords the opportunity to study IS-element-mediated adaptive mutagenesis in a variety of genetic backgrounds, including the backgrounds of natural isolates of E. coli.      

Thermal inactivation of maltase and its application to temperature-sensitive mutants of yeast

Summary Phage P22 mutationc27 defines a site required for establishment, but not maintenance of repressor synthesis. This study confirms that P22c27 is able to synthesize repressor if active repressor is present. An interaction involving gene products ofc1 andc3 and the sitec27 retards expression of the lytic genes of P22. Mutations in genec1 eliminate the retardation of lytic gene expression, butc27 does not alleviate the retardation. These results are used to construct a model that postulates that binding ofc1 andc3 products to DNA at or nearc27 is sufficient to cause retardation of lytic gene expression. The functioning ofc27 is contrasted to that of the analogouscy mutants of λ. The effect of thec27 mutation upon alleviation of “c1 repression” was studied in a partial revertant ofSalmonella typhimurium Pox-1 in whichc1 repression is exaggerated. The higher frequency of lysogenization seen in the mutant host is related to enhancedc1 repression.     

Pseudoreversion of lactose operator-constitutive mutants.

A set of pseudorevertants of lactose operator-constitutive (lacOc) mutant has been obtained. Analysis of a subset of these pseudorevertants indicates that, in some cases, the secondary mutation alters the lactose repressor (lacl gene product), whereas in others it seems to have occurred in the lactose operator (lacO) itself. Of the lacl gene mutations, the lacl8 mutation, already known to suppress all lacOc mutations nonspecifically, was recovered by a selection technique developed for this study. However, two additional lacl gene mutants were selected which appear to suppress lacOc sequences in a more-or-less specific fashion; repressor interaction with some operator sequences is facilitated, whereas the binding with lacO+ and others is attenuated concomitantly.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Net carbon exchange and evapotranspiration in postfire and intact sagebrush communities in the Great Basin

Invasion of non-native annuals across the Intermountain West is causing a widespread transition from perennial sagebrush communities to fire-prone annual herbaceous communities and grasslands. To determine how this invasion affects ecosystem function, carbon and water fluxes were quantified in three, paired sagebrush and adjacent postfire communities in the northern Great Basin using a 1-m³ gas exchange chamber. Most of the plant cover in the postfire communities was invasive species including Bromus tectorum L., Agropyron cristatum (L.) Gaertn and Sisymbrium altissimum L. Instantaneous morning net carbon exchange (NCE) and evapotranspiration (ET) in native shrub plots were greater than either intershrub or postfire plots. Native sagebrush communities were net carbon sinks (mean NCE 0.2–4.3 μmol m⁻² s⁻¹) throughout the growing season. The magnitude and seasonal variation of NCE in the postfire communities were controlled by the dominant species and availability of soil moisture. Net C exchange in postfire communities dominated by perennial bunchgrasses was similar to sagebrush. However, communities dominated by annuals (cheatgrass and mustard) had significantly lower NCE than sagebrush and became net sources of carbon to the atmosphere (NCE declined to −0.5 μmol m⁻² s⁻¹) with increased severity of the summer drought. Differences in the patterns of ET led to lower surface soil moisture content and increased soil temperatures during summer in the cheatgrass-dominated community compared to the adjacent sagebrush community. Intensive measurements at one site revealed that temporal and spatial patterns of NCE and ET were correlated most closely with changes in leaf area in each community. By altering the patterns of carbon and water exchange, conversion of native sagebrush to postfire invasive communities may disrupt surface-atmosphere exchange and degrade the carbon storage capacity of these systems.     

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 × 10⁷  viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole^–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole^– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim^– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 ×g supernatant, but produced wild-type levels of penicillin. Received: 16 April 1997 / Accepted: 26 July 1997     

D-amino acid dehydrogenase: the enzyme of the first step of D-histidine and D-methionine racemization in Salmonella typhimurium

Summary Mutants ofSalmonella typhimurium deficient in D-amino acid dehydrogenase were isolated in histidine auxotrophs able to utilize D-histidine(his-dhuA) 1. The mutants have lost the ability to utilize D-histidine and D-methionine due to mutations in the locusdadA mapped in co-transducible vicinity of the genehemA. ThedadA mutants were unable to deaminate D-histidine, D-methionine, D-alanine and several other D-amino acids to the respective keto products. Indad + strains the enzyme activity was the highest in toluenized cells. In crude sonieates it was 5 to 10 times less. Reduction of artificial electron accepters in the presence of D-amino acids behaved similarly. Keto product formation was strongly inhi-bited by cyanide. It has been concluded thereof that the deaminating enzyme is a D-amino acid dehydrogenase, the activity of which depends on structural integrity of a cell component or on a structure-bound electron accepter. The enzyme activity was inducible by adding L-or D-alanine to growth media. The induction was the highest in media with poor carbon sources. A temperature-sensitivedadA mutant was isolated. I t mapped indadA and had thermolabile D-amino acid dehydrogenase. This has indicated thatdadA is structural gene for the D-amino acid dehydrogenase. This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1. The nomenclature rules for describing genotypes and phenotypes of Demerecet al. (1966) were followed throughout this paper. E.g.dhuA ⁻ hisP⁺ mutants have Dhu⁺ phenotype, those with dhuA^{s - hisP s - mutations are phenotypically Dhu}- All strains with wild-type dhuA⁺ lOCUS are Dhu⁻.     

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement

Summary A large number of motility mutants of the gliding bacterium Myxococcus xanthus have been isolated and analyzed by transduction. Almost all nonmotile mutants are found to be double mutants. This is explained by the existence of two parallel and almost independent multi-gene systems controlling motility, in which case at least one mutation in each system would be required eliminate motility. Only one locus, called mgl, has been found to be shared by both systems. Wild type cells move singly and in groups. Single cells move if they carry a complete gene system A, the genes of which are described in the preceding paper. Groups of cells can move if they carry a complete gene system S, but single A^–S⁺ cells do not move. Linkage analysis reveals at least 9 different loci in system S. One class of S^– mutants, those mutated in the locus tgl, are conditional mutants which, after contact with tgl ⁺ cells, become temporarily motile as cell groups. Most system A mutations have little effect on fruiting but many system S mutations block it, suggesting that system S plays a role in the fruiting process.     

Mutations in the Bacillus subtilis Purine Repressor That Perturb PRPP Effector Function In Vitro and In Vivo

The Bacillus subtilis pur operon repressor (PurR) has a PRPP (5-phosphoribosyl 1-pyrophosphate) binding motif at residues 199–211. Two PurR PRPP binding region mutations (D203A and D204A) were constructed, and the effects on binding of repressor to the pur operon control site in vitro and on regulation of pur operon expression in vivo were investigated. PRPP significantly inhibited the binding of wild-type but not mutant PurR to pur operon control site DNA. In strains with the D203A and D204A mutations, pur operon expression in vivo was super-repressed by addition of adenine to the growth medium. These results support the role of PRPP in modulating the regulatory function of PurR in vivo. YabJ, the product of the distal gene in the bicistronic purR operon, is also required for PurR function in vivo. Received: 5 January 2000 / Accepted: 9 February 2000     

Genetic mapping of the G101 bacteriophage (Pseudomonas aeuruginosa by temperature-sensitive mutations

Temperature-sensitive (ts) mutations of the G101 phage were isolated after mutagenesis with hydroxylamine. A complementation analysis of 61ts mutants showed that these mutants may be divided into at least 12 complementation groups. Twots mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12ts mutations tested are localized on that side of the “c” region where the probablecI repressor gene is positioned. Sevents mutations is closely linked to thecI ₂₆ clear marker, three exhibit a closer linkage and two do not exhibit any linkage withcI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle. Received: 7 April 1998 / Accepted: 10 June 1998