The induction of pigmentation change in a non-acid-fast strain ofMycobacterium phlei by ultraviolet radiation

Five scotochromogenic mutants and 11 achromogenic mutants were induced by UV irradiation of the non-acid-fast photochromogenic PN strain ofMycobacterium phlei. Spontaneous scotochromogenic and achromogenic mutants were not obtained. Colonies of the scotochromogenic mutants are orange, except for one mutant which is ochre. Three mutants are resistant to STM. Out of 11 achromogenic mutants 3 were induced by UV treatment of the original photochromogenic strain, 8 were prepared from the scotochromogenic mutant. No significant differences in the sensitivity to UV rays were found among the scotochromogenic mutant, achromogenic mutant and the photochromogenic PN strain ofMycobacterium phlei under the given experimental conditions. Scotochromogenic mutants and most achromogenic mutants are stable and suitable for further genetic investigation. Pigmentation changes can be used as genetic marker in mutation studies.     

The induction of mutants in a non-acid-fast strain ofMycobacterium phlei with nitrous acid

The inactivation and mutagenic effets of nitrous acid on a non-acid-fast strain ofMycobacterium phlei were studied. It was found that 0.017m NaNO₂ at pH 4.4 may be used for the induction of auxotrophic mutants, scotochromogenic and achromogenic mutants and STM-resistant mutants. Three doubly auxotrophic mutants, three mutants requiring amino acids and three mutants depending on vitamins were obtained. One mutant was not classified. Eighteen scotochromogenic mutants were isolated, seventeen of them were orange. Only ten achromogenic mutants were isolated. Twelve scotochromogenic and eight achromogenic mutants could be used in further genetic studies as they did not revert spontaneously to photochromogeny. Six auxotrophic mutants could be used due to their low frequency of spontaneous reversions. The frequency of STM-resistant mutants increased on an average seven-fold after the mutagenic treatment as compared with the spontaneous frequency.     

The mutagenic effect of ethyl methanesulfonate on a non-acid-fast strain ofMycobacterium phlei

Ethyl methanesulfonate was used for the induction of three types of mutants in a non-acidfast strain ofMycobacterium phlei. A total of 20 auxotrophie mutants was isolated. The mutants were isolated mostly when using doses yielding higher survival of the cells of the basic suspension. The auxotrophic mutants isolated required mostly amino acids, two mutants required purines and three mutants required vitamins. By determining the frequency of spontaneous reversions, it was found that 9 auxotrophic mutants could be used for further genetic studies. These included the following phenotypes: isoleucine⁻, leucine⁻, lysine⁻, nicotinic acid⁻, pyridoxine⁻, and xanthine⁻. Seven scotochromogenic mutants were isolated after ethyl methanesulfonate treatment. One was ochre, the remaining six were orange. Six achromogenic mutants were detected. Spontaneous auxotrophic mutants, scotochromogenic and achromogenic mutants were not isolated. The treatment with 0.2m ethyl methanesulfonate resulted in an increase in the frequency of STM-resistant mutants, the maximum phenotypic expression taking place after 72 hours cultivation in a liquid medium without the drug. The frequency of induced STM-resistant mutants varied within the range of 8.6.10⁻⁵–1.0.10⁻⁴ as compared with the frequency of spontaneous mutants 5.8.10⁻⁶–8.8.10⁻⁶.     

Mutagenic effect of N-Methyl-N-nitrosourea onMycobacterium phlei

N-Methyl-N-nitrosourea was used to induce auxotrophic, scotochromogenic and isonicotinic acid hydrazide resistant mutants inMycobacterium phlei and its effect was compared with that of nitrosoguanidine. Seventeen auxotrophic mutants requiring arnino acids or vitamins and 52 sootochromosgenic mutants with orange colonies were induced. The frequency of isonicotinic acid hydrazide-resistant mutants increased by two orders of magnitude.     

The mutagenic effect of nitrosoguanidine onMycobacterium phlei PA

The effect of nitrosoguanidine on the induction of three types of mutagenic changes inMycobacterium phlei PA was studied. The mutagenic changes included: change of prototrophy to auxotrophy, conversion of sensitivity to isoniazide to resistańce and sensitivity to streptomycin to resistance. The induction of auxotrophic mutants was successful especially when using NTG at a concentration of 1000 μg/ml. A total of 100 auxotrophs was obtained out of which only 13 were sufficiently stable to be used in further studies. Amino acid requirements especially the glycine⁻(serine⁻) type characterized more than half of all auxotrophic mutants obtained. A group of mutants requiring purines also included a high number of mutants. A considerable spontaneous reversion frequency was found in both groups of auxotrophs. The kinetics of the induction of INH-resistant mutants by nitrosoguanidine at a concentration of 1000 μg/ml was studied and a high induction of these mutants, particularly at high lethal effect of the mutagen found. The mutability of the STM^r marker was relatively low in the present model microorganism as compared with the two markers mentioned earlier.     

Mycobacterium rhodesiae sp. nov.

A number of rapid-growing scotochromogenic mycobacteria were isolated from the sputum specimen of Rhodesian patients with pulmonary disease and recognized as a new species. This was given the name Mycobacterium rhodesiae sp. nov. A comparison between M. rhodesiae, M. parafortuitum, M. aurum and M. vaccae was done, and distinguishing characters serving for differentiation between these 4 species of rapid-growing scotochromogenic mycobacteria were described.     

Serological Typing of 31 Achromogenic and 40 Melanogenic Pseudomonas aeruginosa Strains

Thirty-one achromogenic and 40 melanogenic Pseudomonas aeruginosa strains were studied with 10 monovalent typing sera (3). Twenty-one of the achromogenic (67.7%) and seven of the melanogenic (17.5%) strains were agglutinated by one of the 10 typing sera. Ten achromogenic and 33 melanogenic strains were not agglutinated by any of the 10 typing sera. As far as this set of antisera is concerned, the typability of achromogenic and melanogenic P. aeruginosa strains appears to be much lower than that of the chromogenic, nonmelanogenic strains of the species reported previously.     

Creation of a non-mycorrhizal control for a bioassay of AM effectiveness

γ -irradiation of soil by 10 and 3 kGy, and the use of a myc⁻ mutant. The methods were examined on clay and loam. Two management histories were included with both soils to study the ability of the methods to differentiate AM effectiveness. For each soil type, two pot experiments were conducted in field soil, one to investigate the effects of the methods on soil nutrient status, and the other to study the effects on mycorrhization and plant response. The test plants, flax (Linum usitatissimum) and pea (Pisum sativum) myc⁺ and myc⁻ mutants, were grown in 1-l pots for 4 weeks in a growth chamber. To test the ability of the bioassay to reflect differences in AM effectiveness in the field, the mutants and benomyl were also studied in the field from which the loam for the pot experiments was obtained. The bioassay accurately represented the situation in the field and the use of benomyl appeared to be the most appropriate method currently available. The advantages were the ability to use a test plant responsive to AM, the use of less elevated nutrient concentrations than with irradiation, and thus the possibility to use untreated soil as the mycorrhizal treatment. The pea mutants proved unresponsive to AM, and reinoculation to irradiated soil resulted in only half the colonization rate in untreated soil. Benomyl may, however, lead to an underestimation of AM effectiveness because the control is not totally non-mycorrhizal. Its use also carries with it health and environmental risks. Received: 9 February 1999 / Accepted: 27 September 1999     

The use of ethyl methanesulfonate for the induction of mutants inMycobacterium phlei PA

The mutagenic effect of ethyl methanesulfonate in a concentration of 0.2m on a prototrophic, acid-fast strainMycobacterium phlei PA was studied by following the induction of changes of three genetic markers: prototrophy to auxotrophy and sensitivity to two antituberculosis drugs (INH and STM) to resistence. Ethylmethanesulfonate was found to be a very effective mutagen in all three cases. Thirty auxotrophic strains were obtained, out of which eight exhibited a low frequency of spontaneous reversions and could hence be used for further studies. Of the phenotypes induced the glycine⁻ (serine⁻) type was most frequently isolated and represented more than half of all auxotrophs obtained. Requirements for lysine and purines were also observed. The EMS treatment (1% survival of the basic suspension) resulted in a 74-fold increase of the frequency of INH-resistant mutants and a frequency of STM-resistant mutants about 1.1/2 to almost 2 orders of magnitude higher than the spontaneous values     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium. Received: 21 October 1996 / Accepted: 23 September 1997     

Degradation and replenishment of the labile protein fraction in non-growing cells of the asporogenic mutant ofBacillus megaterium

Kinetics of degradation of labelled proteins was followed in two asporogenic mutants ofBacillus megaterium during incubation in a sporulation medium. Both the mutant producing exocellular protease (KM 1prn ⁺) and the mutant not producing the enzyme (KM 12prn) were found to contain a labile protein fraction, whose proportion decreases with prolonged time of labelling and whose half-life is about 1 h. Most proteins were relatively stable and were degraded at a rate of 1 %/h and 2 %/h in strains KM 1 and KM 12, respectively (half life 70–80 h and 35–40 h in strains KM 1 and KM 12, respectively). The intracellular proteolytic activity of the KM 12 mutant remains practically the same during incubation in the sporulation medium or slowly increases. The labile protein fraction practically disappears from the cells after a 3.5-h incubation. When such a culture is then subjected to a shift-up and transferred again to the sporulation medium, the rate of protein turnover temporarily increases. The temporary increase of the turnover rate is caused by a partial replenishment of the labile protein fraction rather than by an accelerated degradation of the relatively stable fraction. The intracellular proteolytic activity does not increase under these conditions. The wild sporogenic strain ofB. megaterium also contains the labile protein fraction. Its half protein life is 1 h or less. However, the second protein fraction is degraded much more rapidly than in the asporogenic mutants and its half life is 6–7 h.     

Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae

Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes α-isopropyl malate synthase I (α-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3′ end of the LEU4 ORF. Six different point mutations – four transitions and two transversions – were found in the remaining mutants. α-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn²⁺-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn²⁺-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity. Received: 30 September 1998 / Accepted: 20 October 1998     

Mutational accessibility of essential genes on chromosome I(left) in Caenorhabditis elegans

We have analyzed a region of approximately 5.4 million base pairs for mutations, which under standard laboratory conditions result in developmental arrest, sterility, or maternal-effect lethality in Caenorhabditis elegans. Lethal mutations were isolated, maintained, and genetically manipulated as homozygotes using sDp2– a duplication of the left half of chromosome I. All of the lethals and rearrangements used in this analysis were balanced by sDp2. Relatively low doses of mutagen, (approximately 15 mM ethylmethane sulfate; EMS), were used so as to limit the occurrence of second-site mutations, thus increasing the probability of recovering single nucleotide substitutions. Treatment of over 32,400 marked chromosomes resulted in 486 analyzed mutations. In this paper, we add 133 previously unidentified let genes, isolated in the EMS screens, and one let gene identified by a γ-ray induced mutation, to our collection of 103 essential genes. We also recovered lethal alleles of genes for which visible mutants already existed. In total, eight deficiencies and alleles of 237 essential genes were identified. Eighty-nine of the previously unidentified let genes are represented by more than one lethal allele. Statistical analysis indicates a minimum estimate of 400 essential genes in the region of chromosome I balanced by sDp2. This region occupies approximately half of chromosome I, and contains over 1135 protein-coding genes predicted from the genomic sequence data. Thus, approximately one-third of the predicted genes are estimated to be essential. Of these approximately 60% are represented by lethal alleles. Less than 2% of the lethal-bearing strains recovered in our analysis, including the eight genetically definable deficiencies, carried more than one lethal mutation. Several screens were used to recover mutations for this analysis. Because all the mutations were isolated using the same balancer, under similar screening conditions, it was possible to compare intervals within the sDp2 region with each other. The fraction of essential genes that present relatively large targets for EMS was highest within the central cluster (dpy-5 to unc-13). Received: 12 July 1999 / Accepted: 6 December 1999     

Gravitropism of hypocotyls of wild-type and starch-deficient Arabidopsis seedlings in spaceflight studies

The major purpose of this spaceflight project was to investigate the starch-statolith hypothesis for gravity perception, and a secondary goal was to study plant growth and development under spaceflight conditions. This research was based on our ground studies of gravity perception in the wild type and three starch-deficient (one starchless and two reduced starch) mutants of Arabidopsis thaliana (L.) Heynh. Dark-grown seedlings that developed in microgravity were given one of several (30 min, 60 min, or 90 min) 1-g stimuli by an on-board centrifuge, and additional controls for seedling development also were performed. These latter control experiments included a morphological study of plants that developed in space in microgravity (F μg), in space on a centrifuge (F 1g), on the ground (G 1g), and on a rotating clinostat on the ground. Since elevated levels of ethylene were reported in the spacecraft atmosphere, additional controls for morphology and gravitropism with added ethylene also were performed. While exogenous ethylene reduced the absolute magnitude of the response in all four strains of Arabidopsis, this gas did not appear to change the relative graviresponsiveness among the strains. The relative response of hypocotyls of microgravity-grown seedlings to the stimuli provided by the in-flight centrifuge was: wild type > starch-deficient mutants. Although the protoplast pressure model for gravity perception cannot be excluded, these results are consistent with a statolith-based model for perception in plants. Received: 12 February 1999 / Accepted: 9 March 1999     

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea

The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV ), ORF17 (eryCIV ) and ORF7 (eryBII ) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates. Received: 29 July 1997 / Accepted: 16 October 1997     

Wild-type and mutant alleles of the Aspergillus nidulans developmental regulator gene brlA: correlation of variant sites with protein function

The DNA sequences of two wild-type and eleven mutant alleles of the developmental regulator gene brlA from Aspergillus nidulans, which encodes a zinc-finger protein, were characterized. Variant sites were located on rescued plasmids or PCR products based either on their meiotic map position or the use of denaturing gradient gel electrophoresis. Mutations in three null mutants, one of which is partially suppressible, encode premature stop codons. Two environmentally sensitive mutants were characterised by substitution of leucines required for stabilisation of α-helices in each of the two putative zinc-finger domains. A third zinc-finger substitution is predicted to disrupt recognition of a guanine residue in the DNA target. The mutations in four other leaky mutants map C-terminal to the zinc fingers; one minimally leaky mutant has a premature stop codon, which results in the removal of the last 38 residues of the protein product. Received: 16 February 1999 / Accepted: 22 July 1999     

Amino Acids that Confer Transport of Raffinose and Maltose Sugars in the Raffinose Permease (RafB) of Escherichia coli as Implicated by Spontaneous Mutations at Val-35, Ser-138, Ser-139, Gly-389 and Ile-391

In order to identify amino acid residues in the Escherichia coli raffinose-H⁺ permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and α-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35→Ala (MT1); one mutant had Ile-391→Ser (MT2); and two mutants had Ser-138→Asp, Ser-139→Leu and Gly-389→Ala (MT3). Transport studies of [³H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P ≤ 0.05) than cells harboring wild-type RafB. However, [¹⁴C]-raffinose uptake was reduced in all mutant cells (P ≤ 0.05) with MT1, MT2 and MT3 mutants compared to cells harboring wild-type RafB. Kinetic analysis showed enhanced apparent K _m values for maltose and reduced V _max/ K _m ratios for raffinose compared to wild-type values. The apparent K _i value of maltose for RafB indicates a competitive relationship between maltose and raffinose. Maltose “uphill” accumulation was greater for mutants (P ≤ 0.05) than for cells with wild-type RafB. Thus, we implicate residues in RafB that are responsible for raffinose transport and suggest that the substituted residues in RafB dictate structures that enhance transport of maltose.     

Genome structures in a lysogenic Rhizobium meliloti system

Summary The genome structure of the temperateRhizobium meliloti phage and the attachment site of this phage on the host chromosome were examined by genetic means. The heat-sensitive mutants used in 2 and 3 point crosses gave a linear chromosome map. There was no evidence for map circularity. The immunity region has a distal position on the phage chromosome. The functional grouping of the used 23 phage mutants was made byin vivo andin vitro complementation tests and 20 cistrons were obtained. The cistrons, near to the immunity region, were identified as early genes, the remaining ones as morphogenetic cistrons. The latter inin vitro complementation tests gave two complementing groups, presumably as head and tail donors. The attachment site of the prophage on the host chromosome was localized by pulse mutagen treatments in synchronously replicating cultures. The sequence of markers are O-str — hs — att ¹⁶⁻³ — T.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998