Sulfur amino acid auxotrophy in Micrococcus species isolated from human skin.

Since methionine and (or) cysteine are required by a large percentage of natural auxotrophic Micrococcus strains isolated from human skin, investigations were directed to determine the specific enzymes affected in sulfur amino acid biosynthesis. Known intermediates in the interrelated cysteine and methionine biosynthetic pathways were tested as growth stimulants. Based on these growth studies, sulfur amino acid auxotrophs were grouped into three cysteine classes and five methionine classes. Selected auxotrophs of M. luteus had deficiencies in ATP sulfurylase (EC 2.7.7.4) and adenosine-5-sulfatophosphate (APS) kinase (EC 2.7.1.25), sulfite reductase (EC 1.8.1.2), serine transacetylase (EC 2.3.1.30), or beta-cystathionase (EC 4.4.1.8) activity; auxotrophs of M. lylae had deficiencies in sulfite reductase and serine transacetylase, beta-cystathionase, or N5, N10-methyltetrahydrofolate reductase (EC 1.1.1.68) activity; all auxotrophs of M. sedentarius tested had deficiencies in N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. nishinomiyaensis had deficiencies in adenosine-3-phosphate-5-sulfatophosphate (PAPS) reductase, sulfite reductase, serine transacetylase, or N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. varians had deficiencies in APS kinase, PAPS reductase, sulfite reductase, homoserine omicron-transsuccinylase, beta-cystathionase, or N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. kristinae had deficiencies in serine transacetylase or cystathionine-gamma-synthase (EC 4.2.99.9) activity; auxotrophs of M. roseus had deficiencies in PAPS reductase, sulfite reductase, or serine transacetylase activity. Results of studies with various mutagens suggested that sulfur amino acid auxotrophy was primarily the result of a single base substitution in usually one or two of the genes controlling biosynthesis. A preliminary study of the amino acid composition of sweat suggested that this important source of nutrients does not contain adequate amounts of cysteine for the growth of cysteine auxotrophs but contains methionine that may be utilized in place of cysteine.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

Transformation of Aspergillus parasiticus with a homologous gene (pyrG) involved in pyrimidine biosynthesis.

The lack of efficient transformation methods for aflatoxigenic Aspergillus parasiticus has been a major constraint for the study of aflatoxin biosynthesis at the genetic level. A transformation system with efficiencies of 30 to 50 stable transformants per microgram of DNA was developed for A. parasiticus by using the homologous pyrG gene. The pyrG gene from A. parasiticus was isolated by in situ plaque hybridization of a lambda genomic DNA library. Uridine auxotrophs of A. parasiticus ATCC 36537, a mutant blocked in aflatoxin biosynthesis, were isolated by selection on 5-fluoroorotic acid following nitrosoguanidine mutagenesis. Isolates with mutations in the pyrG gene resulting in elimination of orotidine monophosphate (OMP) decarboxylase activity were detected by assaying cell extracts for their ability to convert [14C]OMP to [14C]UMP. Transformation of A. parasiticus pyrG protoplasts with the homologous pyrG gene restored the fungal cells to prototrophy. Enzymatic analysis of cell extracts of transformant clones demonstrated that these extracts had the ability to convert [14C]OMP to [14C]UMP. Southern analysis of DNA purified from transformant clones indicated that both pUC19 vector sequences and pyrG sequences were integrated into the genome. The development of this pyrG transformation system should allow cloning of the aflatoxin-biosynthetic genes, which will be useful in studying the regulation of aflatoxin biosynthesis and may ultimately provide a means for controlling aflatoxin production in the field.     

Induction of Symbiotically Defective Auxotrophic Mutants of Rhizobium fredii HH303 by Transposon Mutagenesis

Symbiotically defective auxotrophic mutants were isolated by transposon Tn5 mutagenesis of Rhizobium fredii HH303, a fast-growing microsymbiont of North American commercial soybean cultivars such as Glycine max cv. Williams. Three different Tn5-carrying suicide vectors, pBLK1-2, pSUP1011, and pGS9, were used for mutagenesis with transposition frequencies of 4 x 10, 3 x 10, and 1 x 10, respectively, while the frequency of background mutation resistant to 500 mug of kanamycin per ml was 1 x 10. From 2,600 Tn5-induced mutants, 14 auxotrophic mutants were isolated and classified in seven groups including adenosine (four), aspartate (two), cysteine or methionine (two), isoleucine and valine (two), nicotinic acid (one), pantothenic acid (one), and uracil (two). All the auxotrophs induced nodulation on soybean, but the symbiotic effectiveness of each mutant was different. Three auxotrophs (two cysteine or methionine and one pantothenic acid) formed effective nodules similar to those of the wild type. Three auxotrophs (one nicotinic acid and two aspartate) produced mature nodules like those of the wild type, but the nodules lacked the characteristic pink color inside and were unable to fix nitrogen. Four auxotrophs (two adenosine and two uracil) induced pseudonodules unable to fix nitrogen. The other four auxotrophs repeatedly induced both effective and ineffective nodules, but bacteroids isolated from the effective nodules were prototrophic revertants. The symbiotic phenotype and the degree of effectiveness of the auxotrophic mutants varied with the type of mutation.     

Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti

Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs were sulfite reductase mutants and each of these auxotrophs had a mutation in cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants. One hundred per cent co-transfer of Tn5-induced kanamycin resistance and auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant cell most likely had a single Tn5 insertion. However, the presence of more than one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules induced by cysteine auxotrophs were fully effective like those of the parental strain-induced nodules, whereas the nodules induced by methionine auxotrophs were completely ineffective. The supplementation of methionine to the plant nutrient medium completely restored symbiotic effectiveness to the methionine auxotrophs. These results indicated that the alfalfa host provides cysteine but not methionine to rhizobia during symbiosis. Histological studies showed that the defective symbiosis of methionine auxotrophs with alfalfa plants was due to reduced number of infected nodule cells and incomplete transformation of bacteroids.     

新生隐球菌Cap59缺陷株ura5突变株的筛选方法

改进筛选新生隐球菌Cap59荚膜缺陷株ura5突变株的方法。采用硫酸二乙酯化学诱导新生隐球菌Cap59荚膜缺陷株,利用5-氟乳清酸(5-FOA)反筛选法筛选ura5尿嘧啶合成基因突变株。用新方法筛选到2株Cap59荚膜缺陷株ura5突变株。建立了一种筛选新生隐球菌荚膜缺陷株ura5突变株的简易方法。     

Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

Selective enrichment of aromatic amino acid auxotrophs in Hansenula polymorpha.

Aromatic amino acid auxotrophs of the methanol-utilizing yeast Hansenula polymorpha were effectively selected by the use of nystatin and a medium that inhibits the growth of tyrosine auxotrophs. The procedure resulted in a frequency of aromatic auxotrophs of 2% of survivors and an enrichment of 20-fold. The new procedure also takes less time than traditional procedures. Of the auxotrophic mutants isolated, two-thirds required tyrosine and the remainder were tyrosine-phenylalanine double auxotrophs.     

A red/white selection for Saccharomyces cerevisiae auxotrophs

Red pigment production in yeast cells with adel or ade2 mutations has been exploited to develop a method of identifying specific amino acid auxotrophs. Amino acid auxotrophs carrying mutations in adel or ade2 show delayed pigment production at sub-optimal amino acid levels. This delay allows selection of amino acid auxotrophs following mutagenesis, since red pigment is produced in prototrophs whilst auxotrophs remain white. This differential colour reaction has been applied to select leucine, lysine and serine auxotrophs. Large numbers of colonies could easily be screened without the need for extensive replica plating.     

Induction of nutritional mutants ofMicrococcus glutamicus and their amino acid accumulation

Micrococcus glutamicus ATCC 13032, a glutamic acid-producing organism, was treated with 0.2M ethylmethane sulfonate, the auxotrophs isolated showing varied patterns of extracellular amino acids. Eighty auxotrophic strains were obtained, out of which 31 excreted 1.0-4.0 mg threonine per ml and all the auxotrophs required biotin for growth and production of the amino acid. Eleven auxotrophs produced 1.5 to 3.0 mg alanine per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin-requiring auxotroph Micrococcus glutamicus EM with gamma rays resulted in the isolation of 89 auxotrophic strains, out of which 28 excreted threonine (up to 5.0 mg per ml) higher than the parent auxotroph. Exposure to X-rays yielded 97 auxotrophs, out of these 35 producing 1.0-3.0 mg methionine per ml and requiring biotin for growth and production of the amino acid. Other auxotrophs produced alanine (0.5 to 2.0 mg per ml) and threonine (2.0 to 3.3 mg per ml). Irradiation with gamma rays favoured the development of threonine producing auxotrophs while X-rays favoured methionine-producing auxotrophs.     

Intergenic Complementation of Glucoamylase and Citric Acid Production in Two Species of Aspergillus

All auxotrophs of Aspergillus foetidus and all but two auxotrophs of A. niger which we isolated yield glucoamylase and citric acid, respectively, at levels below that of the prototrophic strain from which they were derived. Results of representative heterokaryon tests suggest that the nucleus was principally responsible for the inheritance of citric acid or glucoamylase production. Most somatic diploid strains of A. foetidus gave rise to higher yields of glucoamylase when compared to their haploid component strains. Both heterokaryons and somatic diploid strains of A. niger synthesized between auxotrophs which were simultaneously reduced in citric acid yields also gave rise to enhanced yields when compared with their haploid components. The yields of a heterokaryon and somatic diploid synthesized between two high producers of citric acid were not higher than those of respective haploid components. We concluded from these results that gene dosage (or ploidy) does not increase the yield of citric acid. The apparent enhancement in yields observed in diploids or heterokaryons synthesized between auxotrophs with reduced yields in both species can be interpreted as resulting from intergenic complementation.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens

Summary Eighteen nutritional mutants have been isolated in the haploid, monoecious moss, Physcomitrella patens: five nicotinic acid auxotrophs, four p-aminobenzoic acid auxotrophs, four adenine auxotrophs, two amino acid requiring mutants and three nitrate non-utilising mutants. Seventeen of them were obtained using total isolation; one was isolated selectively. Strains resistant to the amino acid analogues, D-serine and p-fluorophenyl-alanine, and the purine analogue, 8-azaguanine, have been selected. Many of the auxotrophs are self-sterile. Crosses between auxotrophic strains have been effected and the progeny analysed. No linkage has been detected. Nicotinic acid auxotrophy has resulted from mutation in at least two genes. Self-sterility segregates as a pleiotropic effect of four mutations which produce nutritional dependence. A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.     

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Uridine 5′-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5′-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5′-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations.     

Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus.

Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid. The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T. flavus mutant strains. It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced. The deduced polypeptide sequence extends over 183 amino acids. Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene.     

The development of a negative selection system for the isolation of plant temperature-sensitive auxin auxotrophs

Summary A protocol has been developed for the negative selection of plant auxotrophs using the nucleoside analogues BUdR and FUdR. The protocol was optimised using nitrogen-starved protoplast-derived cells of Nicotiana plumbaginifolia to simulate auxotrophy. The present results represent a significant improvement over previous reports in that: 1) The background of colonies escaping BUdR/FUdR kill is low and reproducible. 2) The protocol was improved to the point where background survival was 0.03% for non-starved cultures and 0.09% for auxin-starved cultures. 3) It was shown that UV irradiation decreases BUdR sensitivity of dividing cells and that this is overcome by increased exposure to BUdR. 4) Application of the method to auxin-starved haploid protoplast-derived cell suspensions resulted, for the first time, in the selection of temperature-sensitive (ts) auxin auxotrophs. 5) It could be demonstrated, for the first time, that the method in practice enriches for auxotrophs, in this case by a factor of 10 for auxin auxotrophs and at least 60 for ts auxin auxotrophs.Abbreviations BUdR 5-bromodeoxyuridine - FUdR 5-fluoro-deoxyuridine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthaleneacetic acid - BAP 6-benzylamino-purine - CFE colony forming efficiency - PE plating efficiency - ts temperature sensitive     

Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus

Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei. Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts. Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA. Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy. These results indicate the integrated DNA was stably inherited.     

Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn916 mutagenesis

Abstract Fourteen Tn 916 mutants of Neisseria meningitidis strain NMB were identified as auxotrophs. Among these were eight amino acid auxotrophs, with five different phenotypes, and three isolates restricted in carbon source utilization, growing in the presence of glucose but not on L-lactate, D-lactate, pyruvate, or casamino acids as principal carbon sources.     

Conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine and relationships of A. tumefaciens mutants to crown-gall tumor induction

Optimum mutagenesis of Agrobacterium tumefaciens by N-methyl-N′-nitro-N-nitrosoguanidine occurred at pH 6.5 using 250 μg/ml of the mutagen for 3 h at 30°. Antibiotic-resistant mutants and amino acid auxotrophs were selected and scored for crown-gall tumor-inducing ability on Helianthus annuus (sunflower). Mutants resistant to neomycin, kanamycin or rifampicin were not directly affected in their tumor-inducing ability. Mutants that were resistant to neomycin were also resistant to kanamycin and vice versa. Various amino acid auxotrophs varied in virulence. Some of the auxotrophs that required histidine, leucine or tryptophan had simultaneously lost their virulence. The alteration of virulence of the organism is not dependent on its growth since the avirulent auxotrophs when supplemented with the amino acid requirement grew in vivo almost as well as the prototrophic strains and yet remained avirulent.     

Dependence of Diaminopurine Utilization on the Mutational Site of Purine Auxotrophy in Bacillus subtilis II. Tracer Experiments

Tracer experiments were carried out in an attempt to explain why guanineless auxotrophs can use diaminopurine as a guanine replacement but nonexacting purine auxotrophs cannot do so. Cell suspensions of the nonexacting purineless Bacillus subtilis MB-1356 incorporated more radioactivity from diaminopurine-2-¹⁴C into nucleic acid than did guanineless B. subtilis MB-1517. The radioactivity in MB-1356 ribonucleic acid (RNA) was distributed in both adenine and guanine nucleotides, thus eliminating the possibility that the deamination of diaminopurine to guanine occurred predominantly on the level of nucleoside di- or triphosphates. Strain MB-1517 incorporated adenine-8-¹⁴C into nucleic acids extremely poorly. This correlated with results obtained with cell-free extracts; strain MB-1517 showed much less adenosine monophosphate (AMP) pyrophosphorylase activity than did MB-1356. Likewise, guanineless MB-1517 converted diaminopurine to its nucleotide much more slowly than did the nonexacting purine auxotroph. The results indicated that the lack of growth of nonexacting auxotrophs on diaminopurine alone is due not to an inability to convert the analogue to nucleic acid adenine but to the greater capacity of the nonexacting auxotrophs to convert diaminopurine to its 5′-ribonucleotide. Presumably, this compound, or a coenzyme analogue produced from it, inhibits growth of mutants which cannot make AMP de novo and only when the medium is devoid of adenine.