Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

Genes affecting the major outer membrane proteins of Escherichia coli K-12: Mutations at nmpA and nmpB

Summary Strains of Escherichia coli K-12 carrying mutations at either of two distinct loci (nmpA and nmpB) produce a new outer membrane pore protein which is not present in wild type cells. Mutations at either of these loci result in sensitivity to phage TC45, which can use this new protein as its receptor, and the new protein (the NmpAB protein) appears to be identical in both NmpA and NmpB mutants. In order to determine whether both of these loci contain structural genes for the NmpAB protein, strains carrying secondary mutations at either of these loci which produced altered proteins were sought by selecting for resistance to phage TC45. Mutants which produced proteins with altered electrophoretic mobility and altered peptide maps were isolated from strains carrying both nmpA and nmpB mutations, and these secondary mutations mapped at the same sites as the original mutations leading to production of the NmpAB protein. This suggests that both loci contain structural genes. Strains mutant at nmpB which can no longer produce the protein can mutate at the nmpA locus to produce the new protein, indicating that both genes can exist in the same cell. Since the altered proteins of mutant strains could be distinguished from one another, we attempted to construct strains in which both nmpA and nmpB were expressed. In all cases only the protein produced by the nmpB mutation was produced, indicating some form of cooperative regulation of the two genes.     

Instability at the y-1 locus of Chlamydomonas reinhardtii

Summary Twenty-three spontaneous yellow mutants were isolated from two stable green strains of the unicellular green alga Chlamydomonas reinhardtii. Genetic characterization indicated that 22 of 23 mutants had a mutation at the y-1 locus, and all 22 y-1 alleles were unstable. Crosses designed to follow the inheritance of instability at the y-1 locus showed that instability is caused by a single genetic factor located at the y-1 locus or very close to it.     

Spore control (Sco) mutations in Bacillus subtilis

Summary Morphogenesis of spore control (Sco) mutants of Bacillus subtilis was followed by quantitative electron microscopy. In wild type cultures the morphological stages II, III, IV and V attain their peaks of frequency at 1.5, 3.5, 4.5 and 5 h after t ₀, respectively. Stages II and IV are short, stages III and V occupy the main part of the process. Morphogenesis is slowed down in Sco A1, Sco B2 and Sco12 strains: all the stages are delayed and prolonged. Stage III cells are predominant for a long time, until t ₉ or later. It is suggested that the mechanism which switches off sporulation genes is affected and this leads to overproduction of sporulation-associated products. In other Sco mutants, such as Sco C3, part of the population sporulates normally but a fraction of cells persists for a long time at stages III and V. The pleiotropy of the spore control mutations is discussed.     

Unstable ascospore color mutants of Ascobolus immersus

Summary New unstable mutants of Ascobolus immersus involving the color or size of ascospores were sought among spontaneous mutants. Among the 34 unstable mutants isolated, 31 had white spores, 2 had pink spores and 1 had a large sized spores. The unstable mutants involve 11 loci whose mutation leads to white spores and 2 loci whose mutations give pink spores, among the 19 loci known to be implicated in this character; 1 locus is defined by only one large spore mutant. All these genes are localized on at least 7 different chromosomes. Unstable mutants of the same locus may correspond to several different sites, but the number of these sites is very limited.The frequency of unstable mutations was estimated: among 36 mutants belonging to 8 different genes, 20 were stable and 16 were unstable. This high frequency of unstable mutants is undoubtedly underestimated. The moment of reversion of 23 of these new mutants was also sought: 15 of them revert as does mutant B, previously studied, in the very young mycelium, at high temperature and with a reversion frequency of 0.004 to 0.34, according to the mutant; 5 of them revert as mutant 301, also previously studied, during the development of the fruit-body and with a frequency of 0.009 to 0.035; two of these mutants revert very early in the ascospore as soon as the first mitoses or in the very young mycelium at 22° C, with a very high reversion frequency that may reach 1.0; finally, the last mutant studied reverts in the fruit-body with a frequency reaching 0.40, but with modalities different from mutant 301. The mutants of the same locus may revert with different modalities. The same modality may correspond to different sites of the same gene.In unstable double-mutant strains involving two different genes, the reversion of one is independent of the reversion of the other, whether or not the reversion modailities of each mutant are identical.Results indicate the existence of inducers common to several unstable mutants which present the same modalities of reversion.These data support the previously formulated hypothesis of transposable elements.     

Insertions of Tn5 linked to mutations affecting carotenoid synthesis inMyxococcus xanthus

Summary We have characterized severalMyxococcus xanthus mutants in which carotenoid synthesis is affected. Six of them produce carotenoids in the absence of visible light, an absolute requirement for carotenogenesis in wild-type strains, and thus will be referred to as constitutive mutants. The six corresponding mutations have been mapped by transductional analysis mediated by linked Tn5 insertions. Five of the mutations have been localized to a single locus, closely linked to Tn5 insertion ΩMR136 and loosely linked to ΩDK4611. The sixth mutation, present in strain MR7, is linked to Tn5 insertion ΩMR134. Another Tn5 insertion site (ΩDK2836) has been characterized and found to be linked to the MR7 colour mutation and to ΩMR134. Darkor light-grown cultures of strains carrying the Tn5 insertion ΩDK2836 do not produce carotenoids even if they simultaneously carry any of the constitutive mutations.     

Colletotrichum sublineolum genetic instability assessed by mutants resistant to chlorate

The fungus Colletotrichum sublineolum, causal agent of sorghum anthracnose, presents high variability, genetic instability and host specialization. The aims of the present work were to investigate the mechanisms involved in the genetic instability in this species. Mutants resistant to chlorate and unable to use nitrate (Nit mutants), were obtained spontaneously, isolated and characterized for complementation pattern, reversion frequency and RAPD profile. The results showed that chlorate-resistant mutants could be divided into six phenotypic classes that probably represented mutations in the structural nitrate reductase locus (nit1), in the structural nitrite reductase locus (nit6 and niiA of Neurospora and Aspergillus, respectively), in the specific regulator locus (nit3), in the main regulator locus (nit2), in loci that codified the cofactor containing molybdenum necessary for nitrate reductase activity (NitM), and one or more genes responsible for nitrate intake (crn). In addition, the genetic control of this metabolism in C. sublineolum seems to be similar to other fungi species such as Aspergillus, Neurospora and Fusarium. The high reversion frequency (10⁻⁴ to 10⁻⁵) presented by nit1 mutants suggests that the instability in evaluated strains could be a result of transposable elements activity. The RAPD analysis enabled confirmation that the Nit mutants have a similar genetic background to original strain, and that polymorphism exists among wild-type strains, nit1 mutants and revertants of C. sublineolum. These are important aspects for the later direction of molecular analysis, where these mutants will be used as a tool to isolate the active transposable elements in the C. sublineolum genome.     

Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

Protochlorophyllide photoconversion mutants of Chlamydomonas reinhardtii

Summary We have developed a procedure for the isolation of Chlamydomonas reinhardtii mutants defective in light-dependent protochlorophyllide reduction (photoconversion), a key step in the biosynthesis of chlorophyll. Mutants were isolated by mutagenizing y-1-4, a temperature-sensitive yellow mutant blocked in the alternative light-independent protochlorophyllide reduction pathway, and screening for colonies which failed to green in the light at the restrictive temperature. Seven mutants were isolated which fail to photoconvert protochlorophyllide in photoconversion tests. All seven mutants have a single mutation at the pc-1 locus responsible for the defect in photoconversion. pc-1 maps close to y-5 on nuclear linkage group I. The pc-1 mutation is not itself temperature-sensitive because it blocks photoconversion at the permissive temperature when combined with the non-conditional yellow mutations y-5 and y-7. Cells containing the pc-1 mutation alone synthesize about 52% and 36% of the wildtype chlorophyll level in the dark and light, respectively, demonstrating that the light-independent protochlorophyllide reduction pathway in C. reinhardtii operates in the light.     

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     

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.     

A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana

Summary Monogenic mutants of the early ecotype Landsberg erecta were selected on the basis of late flowering under long day (LD) conditions after treatment with ethyl methanesulphonate or irradiation. In addition to later flowering the number of rosette and cauline leaves is proportionally higher in all mutants, although the correlation coefficient between the two parameters is not the same for all genotypes. Forty-two independently induced mutants were found to represent mutations at 11 loci. The mutations were either recessive, intermediate (co locus) or almost completely dominant (fwa locus). The loci are located at distinct positions on four of the five Arabidopsis chromosomes. Recombinants carrying mutations at different loci flower later than or as late as the later parental mutant. This distinction led to the assignment of eight of the loci to three epistatic groups. In wild type, vernalization promotes flowering to a small extent. For mutants at the loci fca, fve, fy and fpa, vernalization has a large effect both under LD and short day (SD) conditions, whereas co, gi, fd and fwa mutants are almost completely insensitive to this treatment. SD induces later flowering except for mutants at the co and gi loci, which flower with the same number of leaves under LD and SD conditions. This differential response of the mutants to environmental factors and their subdivision into epistatic groups is discussed in relation to a causal model for floral initiation in Arabidopsis thaliana.     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.     

Mutants of Nicotiana plumbaginifolia with specific resistance to auxin

Summary We have isolated nine independent auxin-resistant mutants of Nicotiana plumbaginifolia by culturing M₂ seedlings in the presence of indole-3-acetic acid ethyl ester or 1-naphthaleneacetic acid at concentrations which significantly inhibit hypocotyl elongation of the wild type. The mutations were induced by treating seed with ethyl methanesulphonate and were found in the course of screening 10 000 individual M₂ families. Auxin resistance was in all cases the result of a mutation at a single, nuclear locus. The dominance relationships of two of the mutants could be defined as recessive or dominant; all other mutants showed partial dominance. In contrast to previously described mutants of Arabidopsis and N. plumbaginifolia, all of the present mutants were specifically resistant to auxin; the mutants were cross-resistant to several auxins, but showed no increased resistance to cytokinin, abscisic acid, ethylene or 1-amino-cyclopropane-1-carboxylic acid. The importance of the choice of the selection criterion for the isolation of specific resistance traits is discussed.     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn 49 system of Drosophila melanogaster

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement. Received: 19 February 1997 / Accepted: 8 October 1997     

Translational errors as the cause of mutations in Escherichia coli

Summary The present work suggests that a significant proportion of spontaneous mutations in Escherichia coli are the result of translational errors. This idea is supported by the following observations: (i) Streptomycin can induce the formation of auxotrophic mutants in streptomycin-sensitive cells, but not in rpsL mutants resistant to streptomycin, and (ii) strains having hyper-accurate ribosomes (rpsL999 and rpsL1204 strains) show reduced mutation rates. The implications of these results are discussed with respect to the dogma of randomness of spontaneous mutations and the directed mutation hypothesis.     

Arg-7 mutant x wild-type crosses in Chlamydomonas reinhardi: Study of the enzyme produced in diploid strains

Summary The arg-7 locus is the structural gene for the argininosuccinate lyase (ASL). Interallelic complementation was previously found to occur between several mutants of the locus: this is indicative for the homomultimeric nature of ASL.Two complementing (arg-7-5 and arg-7-7) and two non-complementing (arg-7-1 and arg-7-6) mutants of the arg-7 locus were crossed to the pab-2 strain (which is wild-type for the arg-7 locus). In each cross, heterozygote phenotypically wild-type strains were isolated; their diploid pattern was demonstrated by various criteria: mating type, cell volume, nuclear size.The four heterozygotes were compared to the haploid wild-type and in some experiments, to the diploid strain arg-1xpab-2 homozygous for the arg-7 locus. No difference was found in growth rate and in the Michaelis constant values for ASL. The specific activity of the enzyme produced in the heterozygotes was about 50 percent of the activity found in haploid or diploid wild-type. The heat sensitivity of ASL was also investigated in the different strains: two (containing the complementing mutations arg-7-5 and arg-7-7) of the four heterozygotes produce ASL varieties different from the wild-type enzyme as far as the thermolability is concerned.These results suggest that hybrid ASL can be formed by interaction between the products of wild-type and mutant genes. A clear dominance of the wild-type allele is expected only when the mutant allele has no product of the gene: this could be the case for arg-7-1 and arg-7-6.     

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa

Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.