Methods for Isolation of Auxotrophic Mutants of Methanobacterium ivanovii and Initial Characterization of Acetate Auxotrophs

To develop a biochemical genetic approach to understanding cell carbon synthesis or metabolic pathways in methanogens, Methanobacterium ivanovii was selected as a model organism for genetic manipulation studies. The organism displayed a colony size of 3 to 6 mm in less than 2 weeks and had a plating efficiency of about 90%, which made it suitable for replica plating. Mutagenesis and selection techniques were developed for selection of acetate auxotrophs. Chemical mutagenesis with ethyl methanesulfonate, followed by enrichment with bacitracin as a selective agent, resulted in stable acetate auxotrophs. M. ivanovii was very sensitive to UV, but UV-induced acetate auxotrophs were unstable and reverted within two to four transfers. The acetate auxotrophs were analyzed in relation to wild type for carbon monoxide dehydrogenase enzyme activity.     

Isolation and characterization of yeast mutants auxotrophic for 2''-deoxythymidine 5''-monophosphate.

Summary Mutant strains of Saccharomyces cerevisiae auxotrophic for deoxythymidine monophosphate (dTMP) were isolated and characterized. Two distinct classes of auxotrophs were obtained. One class had a simple requirement for dTMP and was analogous to thymine-requiring bacteria. The second class required dTMP, adenine, histidine and methionine and this complex nutritional phenotype was due to defects in folate metabolism. The dTMP-dependent growth of respiratory-competent grande auxotrophs was found to be markedly affected by media composition and carbon source. In the absence of dTMP thymineless death occurred in both mutant classes.     

Isolation and characterization of pyridoxine auxotrophs of Escherichia coli

Dempsey, Walter B. (University of Florida, Gainesville), and P. F. Pachler. Isolation and characterization of pyridoxine auxotrophs of Escherichia coli. J. Bacteriol. 91:642-645. 1966.-Pyridoxine auxotrophs of Escherichia coli B have been consistently produced by a modified penicillin enrichment method. This modified penicillin technique included a 6-hr growth period in the absence of any pyridoxine followed by a 2-hr treatment with penicillin at 1,000 units per ml. Penicillin was removed from these cultures by membrane filtration and the process was repeated. A minimum of three cycles was found necessary to isolate auxotrophs. Different phenotypes within the group of pyridoxine auxotrophs were distinguished by their responses to feeding with the various forms of pyridoxine, as well as by cross-feeding tests. Two auxotrophs were also differentiated by their frequency of reversion to prototrophy. Cross-feeding tests indicated that seven of the phenotypes fed in a linear sequence. These phenotypes had a very low frequency of reversion. The auxotrophs with a high frequency of reversion cross-fed in a linear sequence and fed the first five of the other seven auxotrophs. One of the auxotrophs isolated was a pyridoxal auxotroph.     

Prototrophic hybrids of bakers yeast. I. Selection of haploids and diploids of prototrophic yeast

153 haploids, including 8 prototrophs, 12 biotin prototrophs and 4 biotin auxotrophs were isolated from Y, F and FB strains of the baker's yeast Saccharomyces cerevisiae. Remaining haploids were dependent on vitamins of B group other than biotin. Obtained haploids were characterized by large cell sizes, a or α mating type, the ability to fermentation of sugars and the assimilation of non-fermented carbon sources. Haploids obtained from the yeast of Y and FB strains a good growth in the synthetic medium. Prototrophic haploids, prototrophic haploids with biotin auxotrophs and biotin prototrophs with biotin auxotrophs were crossed. 89 prototrophic hybrids capable of growth in synthetic medium without vitamins were selected out of 178 hybrids obtained. Hybrids are characterized by features typical for baker's yeast; however, not all of them are capable of sporulation. As result of selection, prototrophic hybrids and 16 hybrids characterized by a good increase of biomass in molasses medium were chosen. The efficiency of the biomass of hybrids designated as YY 3040/2, YY 3040/3 and FFB 1910/2 is considerably higher than one obtained from the cultivation of the industrial strain French Mautner (Mf). All hybrids possess adequate enzymatic activity in anaerobic metabolism of saccharose and maltose. Selected prototrophic hybrids were sent out to two yeast factories in the country for experimental propagation.     

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.     

Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae

Summary We have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Pyrimidine biosynthesis in Pseudomonas oleovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity. METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source. Two uracil auxotrophs of P. oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. oleovorans enzyme. CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps. oleovorans. Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps. oleovorans to increase the synthesis of polyhydroxyalkanoates.     

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.     

Role of vitamin B 6 biosynthetic rate in the study of vitamin B 6 synthesis in Escherichia coli

Nutritional auxotrophs of Escherichia coli synthesize vitamin B(6) compounds at a rate of 1 x 10(-10) to 2 x 10(-10) moles per hr per mg (dry weight) of cells when they are suspended in minimal medium lacking their required nutrients. A few auxotrophs have been found to stop or reduce vitamin B(6) synthesis during such an experiment. These include thiamineless, citrate synthaseless, and pyridoxineless mutants as well as mutants which require four carbon compounds for growth. Glycolaldehyde was found to restore vitamin B(6) synthesis in the last named of these mutants without restoring normal growth. A class of pyridoxineless mutants which responded with normal growth to 0.4 mm glycolaldehyde or 0.15 x 10(-3) mm pyridoxol was also found. The results suggest that a thiamine pyrophosphate-requiring step as well as glycolaldehyde may be involved in pyridoxal phosphate biosynthesis.     

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.     

Starvation survival response of Mycobacterium tuberculosis

The ability of Mycobacterium tuberculosis auxotrophs to survive long-term starvation was measured. Tryptophan and histidine auxotrophs did not survive single-amino-acid starvation, whereas a proline auxotroph did. All three auxotrophs survived complete starvation. THP-1 cells were also able to restrict the growth of the tryptophan and histidine auxotrophs.     

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.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

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.     

Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.     

Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere.

Cysteine auxotrophs of Salmonella typhimurium mutated in cysB, cysI or cysJ grew with sulphate as a sulphur source when incubated under a hydrogen/carbon dioxide atmosphere. Yields obtained under these conditions were equivalent to those characteristic of wild-type S. typhimurium. The same mutants failed to grow with sulphate as a sulphur source when incubated aerobically. Auxotrophs mutated in cysA, cysC, cysD, cysE, cysG and cysH required cysteine for growth under both incubation conditions. The results suggest that mutations in cysB (regulation of the several cys operons) and also cysI and cysJ (sulphite reductase activity) can be circumvented during anaerobic growth under hydrogen.     

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.     

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.