Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Reduction of N-oxides and sulfoxide by the same terminal reductase inProteus mirabilis

Proteus mirabilis can grow anaerobically on the fermentable substrate, glucose. When the glucose medium was supplemented with an electron acceptor, growth doubled. However, the organism failed to grow anaerobically on the oxidizable substrate glycerol unless the medium was supplemented with an external electron acceptor. Dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nicotinamide N-oxide (NAMO), and nitrate (NO₃) can serve this function. Cell-free extracts ofP. mirabilis can reduce these compounds in the presence of various electron donors. In order to determine whether the same or different terminal reductase(s) are involved in the reduction of these compounds, we isolated mutants unable to grow on glycerol/DMSO medium. When these mutants were tested on glycerol medium containing TMAO, NAMO, and NO₃ as electron acceptors, it was found that there were two groups. Group I mutants were unable to grow with DMSO, TMAO, and NAMO, while their growth was unaffected with NO₃. Group II mutants were unable to grow on any electron acceptor including NO₃. Enzyme assays using reduced benzyl viologen with both groups of mutants were in agreement with growth studies. On the basis of these results, we conclude that the same terminal reductase is involved in the reduction of DMSO, TMAO, and NAMO (group I) and that the additional loss of NO₃ reductase in group II mutants is probably owing to a defect in the synthesis or insertion of molybdenum cofactor.     

Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.

We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae. These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition of 50 mM KCl but not NaCl to the medium.     

A spectinomycin dependent mutant of Escherichia coli.

Summary A mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence. The mutant, termed RD, needs spectinomycin to grow at temperatures of 37° or below; it is unable to grow at 42° in either the presence or absence of spectinomycin. Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10^-6. Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1. Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked.Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E. coli chromosome and was represented by the mutation in S5. The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.     

In vitro and ex vitro growth of grapevine rootstock `5BB' as influenced by number of air exchanges and the presence or absence of sucrose in culture media

Single node cuttings of grapevine rootstock `5BB' were cultured for 5 weeks under different numbers of air exchange (0, 0.8, 1.5, 2.5 and 4.4 h ^–1) with or without sucrose in the medium. To determine the effect of number of air exchanges related to presence or absence of sucrose, in vitro growth, net photosynthetic rate, stomata characteristics and ex vitro growth were investigated. Increasing numbers of air exchanges accelerated plantlet growth regardless of the presence or absence of sucrose in the medium; under the same number of air exchanges, plantlet growth and net photosynthesis were greater in sucrose-containing medium compared with sucrose-free medium. However, a high number of air exchanges (4.4 h^–1) inhibited the growth and photosynthesis in sucrose-containing medium compared to sucrose-free medium. A higher number of stomata was observed in sucrose-containing medium, and larger size stomata was observed in sucrose-free medium. These results emphasize the importance of ventilation (increased number of air exchanges) during in vitro culture, for ex vitro plantlet survival was greatly affected by with or without air exchanges. Without air exchanges ex vitro plantlet survival was less than 70%, while air exchanges increased ex vitro survival by more than 90% with greater growth.     

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.     

Coordinated alterations in ribosomes and cytoplasmic membrane in sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli.

Alterations in cytoplasmic membrane and ribosomes from sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli, mutants that are resistant to spectinomycin in the presence of 20% sucrose but sensitive in the absence of sucrose, were studied. The protein composition of cytoplasmic membrane was analyzed by gel electrophoresis on polyacrylamide gel containing 8 M urea and 0.5% sodium dodecyl sulfate, which assured the reproducible separation of 28 protein bands. A major protein band, I-19, was missing in all cytoplasmic membrane preparations from 10 Sucd-Spcr mutants. Besides protein I-19, proteins I-13 and I-24 were missing in some mutants. On the other hand, the protein composition of cytoplasmic membrane from a sucrose-independent spectinomycin-resistant mutant was indistinguishable from that from the wild-type strain. The polypeptide synthetic activity of ribosomes from Sucd-Spcr mutants was resistant to spectinomycin. Studies on a revertant obtained from one of these mutants without any selection for sensitivity to spectinomycin revealed that a single mutation was responsible for both the ribosomal alteration, i.e., spectinomycin resistance, and the lack of protein I-19 in the cytoplasmic membrane. Studies on a transductant obtained with a Sucd-SPcr mutant as the donor also confirmed the single-mutation concept. It was concluded that in Sucd-SPcr mutants an alteration in the ribosomes caused the deletion of protein I-19 from cytoplasmic membrane.     

Characterization of AAT1: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae

A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified. These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine). This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested. Leucine uptake through the leucine-specific permease is inhibited to less than 35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions. aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement.     

Novel hybrid maturases in unstable pseudorevertants of maturaseless mutants of yeast mitochondrial DNA.

Unstable pseudorevertants of mitochondrial mutants of Saccharomyces cerevisiae lacking the maturase function encoded by the fourth intron of the cytochrome b gene (bI4) were isolated. They were found to be heteroplasmic cells owing their regained ability to respire (and grow on glycerol medium) to the presence of a rearranged (rho-) mtDNA that contains an in-frame fusion of the reading frames of the group I introns bI4 and intron 4 alpha of the coxl gene encoding subunit I of cytochrome c oxidase (aI4 alpha). The products of those gene fusions suppress the bI4 maturase deficiency still present in those heteroplasmic cells. Similar heteroplasmic pseudorevertants of a group II maturaseless mutant of the first intron of the coxI gene were characterized; they result from partial deletion of the coxI gene that fuses the reading frames of introns 1 and 2. These heteroplasms provide independent support for the existence of RNA maturases encoded by group I and group II introns. Also, since the petite/mit- heteroplasms arise spontaneously at very high frequencies they provide a system that can be used to obtain mutants unable to form or maintain heteroplasmic cells.     

Isolation and characterization of biochemical and morphological mutants in chlamydomonas smithii

Mutants of Chlamydomanas smithii resistant to chlorate were induced after UV irradiation. Some were unable to grow on nitrate and suspected to be defective in nitrate reductase. One of them (nit-a) was analyzed and shown to complement nit-1 nit-2 of C. reinhardtii.Mutants resistant to streptomycin, spectinomycin or erythromycin selected from nit-a pre-grown in the presence of 5-fluorodeoxyridine. Two spectinomycin resistant strains were genetically analyzed from crosses with C. reinhardtii carrying different chloroplast mutations. The transmission of resistance was shown to be non-Mendelian and similar to that obtained in intraspecific crosses involving C. reinhardtii chloroplast mutants.Mutants lacking a cell wall (CW⁻) were selected after UV treatment on the basis of the particular flat amoeboid shape of the colonies. Seven of them were tested for complementation with CW15 and CW_d mutants of C. reinhardtii: none was found to be allelic with these two mutations.     

蔗糖诱导拟南芥幼苗胚轴维管束细胞数量增多

为揭示蔗糖能否引起植物胚轴维管束细胞数量增多，将拟南芥播种于添加88mmol·L-1蔗糖和不添加糖的MS培养基上，对生长在不同培养基上的幼苗胚轴横切，显微镜下统计切片上维管束细胞数量。结果显示，与不加糖相比，加糖条件下萌发4d后幼苗维管束细胞总数增加约70％，维管薄壁细胞和导管分子都增加100％以上，筛管分子增加约90％，中柱鞘细胞数量不变。显然，蔗糖不仅使维管束薄壁细胞数量增多，也使筛管分子和导管分子数量增多。因此认为，添加蔗糖对拟南芥幼苗胚轴维管束具有双重效应，既引起维管薄壁细胞增殖，又促进维管薄壁细胞分化，从而使导管分子和筛管分子数量增多。     

Mass selection of conditional mating mutants of Tetrahymena thermophila

The mating reaction in Tetrahymena thermophila includes a starvation period and two distinct cell interactions, co-stimulation and cell pairing, before the cells are cytoplasmically joined as conjugants. A selection procedure for harvesting mutants unable to mate at a restrictive temperature has been developed. A conjugant pair consisting of one cycloheximide-resistant cell and one wild-type cell (cycloheximide-sensitive) was itself sensitive to the drug. By adding cycloheximide and nutrient medium to a cross made at the restrictive and grow. Repetition of the selection procedure enriched for cells unable to conjugate at the restrictive temperature. The selected cells were able to grow at 38 degrees C and could conjugate at 28 degrees C. This procedure may be narrowed to select specifically for cell interaction mutants.     

Isolation and characterization of mutants of Streptococcus mutans using selective removal of wild-type cells by agglutination with an agglutinin from Persea americana

Persea americana agglutinin (PAA), a substance known to bind basic proteins and inhibit the sucrose-independent adherence of Streptococcus mutants to saliva = coated hydroxyapatite (Staat et al., 1980) was used to selectively enrich for mutants defective in a variety of cell surface associated virulence characteristics from cultures UAB62 (PS14 Riff, serotype c), UAB66 (6715 Strr Spcr, serotype g) and UAB77 (GS5, serotype c). Following mutagenesis and growth for segregation and phenotypic expression, washed cells of each strain were exposed to PAA overnight at 37 degrees C. Aggregated cells were removed by low-speed centrifugation and cells remaining in the supernatant fluids were concentrated, grown to stationary phase and the enrichment with PAA repeated. Mutants isolated following enrichment were phenotypically diverse and included strains defective in one or more of the following characteristics: adherence to glass in a sucrose-containing medium, aggregation with sucrose, dextran or PAA. dextranase production, colony morphology, cell or chain morphology, fermentation of sorbitol, lactose, galactose, raffinose, melibiose, or fructose, and production of surface protein antigen A (SpaA). The diversity of mutant phenotypes identified along with the observation that PAA could still cause aggregation (with a lower efficiency) of all mutants leads us to infer that the interaction of this agglutinin with proteins on the S. mutans cell surface is relatively nonspecific and that the observed inhibition of S. mutants attachment to saliva-coated hydroxyapatite caused by PAA is not due to a highly specific unique interaction of PAA with the protein(s) responsible for sucrose-independent adherence.     

Effect of Sucrose on Growth and Chlorophyll Synthesis of Teak Shoots in Mixotrophic Culture

Clonally propagated shoots of teak (Tectona grandis Linn) were cultured in vitro under photomixotrophlc (sucrose 10-40 g l^-1) and photoautotrophic (sucrose-free medium) conditions in MS medium containing kinetin and benzyl amino purine (0.1 mg l^-1 each). Sucrose concentrations were gradually depleted in mixotrophic cultures. Growth and fresh weight of shoot chlorophyll a, b and total chlorophyll content of leaves were estimated. In sucrose-free medium, growth and chlorophyll synthesis decreased after limited period of 2-3 subcultures, whereas they got stimulated under photomixotrophic condition with 10-30 g l^-1 sucrose; optimum being the medium with 30 g l^-1 sucrose. Higher concentration of sucrose (40 g l^-1) inhibited shoot growth. Shoots can tolerate gradual depletion of sucrose upto a limit of 5 g l^-1 under mixotrophic condition.     

Mapping of mutations in Pseudomonas aeruginosa defective in pyoverdin production.

Twelve mutants of Pseudomonas aeruginosa PAO defective in pyoverdin production were isolated (after chemical and transposon mutagenesis) that were nonfluorescent and unable to grow on medium containing 400 microM ethylenediaminedi(o-hydroxyphenylacetic acid). Four mutants were unable to produce hydroxamate, six were hydroxamate positive, one was temperature sensitive for pyoverdin production, and another was unable to synthesize pyoverdin on succinate minimal medium but was capable of synthesizing pyoverdin when grown on Casamino Acids medium (Difco Laboratories, Detroit, Mich.). The mutations were mapped on the PAO chromosome. All the mutations affecting pyoverdin production were located at 65 to 70 min, between catA1 and mtu-9002.     

The isolation and preliminary characterisation of 6-thioguanine-resistant mutants of human diploid fibroblasts

Mutant clones of human diploid fibroblasts deficient in the enzyme, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected by their ability to grow in medium containing the cytotoxic purine analogue, 6-thioguaninine (6TG). The optimal conditions for mutant selectiom were 6TG concentrations between 1 and 5 μg ml^?1 and cell plating densities ～ 10³ cells cm^?2.Nine spontaneous and four radiation-induced 6TG-resistant mutants had <2% of the parental strain HGPRT activity and were unable to grow in medium containing azaserine. These mutants were phenotypically stable during > 25 population doublings in non-selective medium and five mutants that were examined showed no gross change from the normal human karyotype.Evidence is presented to show that 6TG is a better selective agent than 8-azaguanine (8AG) for HGPRT-deficient mutants of human diploid fibroblasts.     

Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium.

Mutations in the ack (acetate kinase) and pta (phosphotransacetylase) genes in Salmonella typhimurium were characterized and determined to be analogous to those of previously described Escherichia coli mutants. We established that in both bacterial species these genes were cotransducible with the neighboring histidine transport operon and were distally located relative to purF. pta mutants were sensitive to the dye alizarin yellow and were unable to grow on medium containing inositol as a carbon source. We selected mutants of both species with deletions covering both the ack and the pta genes; some deletions extended into the histidine transport operon.     

Role of Thin Fimbriae in Adherence and Growth of Acinetobacter calcoaceticus RAG-1 on Hexadecane

Acinetobacter calcoaceticus RAG-1, a hydrocarbon-degrading bacterium which adheres avidly to hydrocarbons and other hydrophobic surfaces, possesses numerous thin fimbriae (ca. 3.5-nm diameter) on the cell surface. MR-481, a nonadherent mutant of RAG-1 which is unable to grow on hexadecane under conditions of limited emulsification and low initial cell density, lacks these fimbriae. Prolonged incubation of MR-481 in hexadecane medium enriched for partial adherence revertants. The reappearance of thin fimbriae was observed in all such revertant strains. RAG-1 cells and partial revertant strains were agglutinated in the presence of antibody, whereas MR-481 cells were not. Another mutant, AB15, which was previously isolated on the basis of its nonagglutinability in the presence of antibody, also lacked thin fimbriae and was conditionally nonadherent. Furthermore, strain AB15 was unable to grow on hexadecane medium. Adherence of RAG-1 cells to hexadecane was considerably reduced after shearing treatment. The material removed from the cell surface by shearing of RAG-1 and the partial revertant strains yielded a single antigenic band in RAG-1 and partial revertant strains, as observed by crossed immunoelectrophoresis. This band was absent in both fimbriae-less mutants, MR-481 and AB15. The data demonstrate that the thin fimbriae of RAG-1 (i) are a major factor in adherence to polystyrene and hydrocarbon, (ii) may be crucial in enabling growth of cells on hexadecane, and (iii) constitute the major cell surface agglutinogen.     

Fermentation characteristics of levansucrase mutants of Zymomonas mobilis

Two mutants, Ls1 and Ls2, of Zymomonas mobilis B-806 unable to produce levan were isolated. With native gel electrophoresis and zymogram analysis it was confirmed that the mutants did not synthesize active levansucrase (E2). However, they produced intracellular sucrase (E1) and extracellular invertase (E3). Comparison of these mutants with the parent strain for alcohol production on glucose, fructose and sucrose (100 g/l each) media revealed that the final ethanol concentration achieved in sucrose medium was only about 5 g/l higher with the mutants than with the wild type. The ethanol yield of the mutants increased from 0.48 g/g to 0.50 g/g on sucrose medium.