Use of chemostat for selection ofStreptomyces hygroscopicus mutants altered in regulation of maltose utilization

Summary Streptomyces hygroscopicus mutants showing altered fermentation kinetics were isolated using a selection procedure in a chemostat. Several mutants were obtained which differed in their capacity to produce the macrolide antibiotic turimycin.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

Oxygen sensitivity of nitrogenase is not always a limiting factor of growth under nitrogen-fixing conditions inAzotobacter vinelandii

Summary The effect of a combined nitrogen source to oxygen sensitive mutants was examined. For some oxygen sensitive mutants, oxygen sensitivity was not restored by the addition of nitrogen compounds to their medium. One of these mutants showed oxygen resistant nitrogenase activity similar to that of a wild strain. Results imply that oxygen sensitivity of nitrogenase is not always a limiting factor of growth under aerobic nitrogen-fixing conditions inAzotobacter vinelandii.     

Mutants ofMethylobacterium organophilum unable to synthesize PQQ

The phenotype of mutants unable to synthesize PQQ is analyzed for different categories of methylotrophic bacteria. The advantages offered by strains dissimilating methylamine through methylated amino-acids are discussed. InM.organophilum, 40% of the mutants unable to grow in methanol medium but with normal methylamine utilization, were affected in PQQ metabolism. The genetic properties ofM.organophilum useful to study PQQ mutants are discussed, mainly the use of pSUP106 to create insertion mutations in the bacterial chromosome and to replace wild-type genes by modified genes. An example is given of the possibility to create R plasmids containing large fragments ofM.organophilum DNA. Some physiological properties of a PQQ mutant are described, regarding growth kinetics, PQQ uptake and accumulation.     

Strain selection criteria for Penicillium funiculosum in enzymic hydrolysis of lignocellulosics

Summary The mutants ofPenicillium funiculosum viz. N-4, BU-36 and Cu-1 producing different proportions of cellulase components have been isolated. Results of saccharification experiments using various lignocellulose substrates with culture filtrates of these mutants suggested criteria for their selection.     

A simple procedure for quick identification of tryptophanase positive recombinant clones and tryptophanase regulatory mutants of bacteria

Summary A simple and rapid technique for quick identification of tryptophanase regulatory mutants and tryptophanase positive clones in a bacterial population is described. This method was used for the detection of tryptophanase regulatory mutants of Vibrio cholerae and tryptophanase positive recombinant clones of Escherichia coli.     

Visual detection of β-fructofuranosidase (inulase) —Regulatory mutants ofKluyveromyces fragilis

Summary Inulase constitutive mutant cells of the yeastKluyveromyces fragilis were enumerated in continuous culture cell populations. After cloning and growth on glycerol agar plates, mutant colonies stained red when exposed to a mixture of sucrose and a chromogenic reagent for glucose.Mutants with improved inulase production on glucose were isolated from opaque agar plates containing undissolved inulin. Mutant colonies were surrounded with clearing zones. Attempts to isolate similar mutants by selection for 2-deoxyglucose resistance proved unsuccessful withK. fragilis.     

Production of mold lactase

A process for production of mold lactase was developed. Tests were carried out in pilot and industrial scale with an Aspergillus niger strain selected after screening a number of molds.A computer coupled autoanalyzer system was used for monitoring enzyme formation in the pilot fermentor. Lactase production was investigated using different pH- and temperature-profiles. A. niger lactase has an acid pH optimum, a high temperature optimum and good stability. It does not require any metal ions. It is suitable for immobilization for hydrolysis of lactose in acid whey.Three-fold enhancement of lactase production was obtained by mutagenizing A. niger using NTG as mutagenic agent.The lactases produced by the mutants have the same pH and temperature optima and stability but the growth properties of the mutants were different from those of the original strain.Sufficient specific activity of the enzyme preparation for immobilization was obtained by purifying the enzyme by selective adsorption on Na-Ca-silicate.     

Selection aid properties of hyperproducing D-serine deaminase mutants of Escherichia coli

Summary Hyperproducing constitutive D-serine deaminase mutant E. coli were selected by a chemostat method. The specific activity of the enzyme in these mutants is 25fold higher in comparison with the fully induced parental strain.     

Use of chemostat for selection ofStreptomyces chrysomallus mutants altered in the induction of D-glucose isomerase

Summary D-glucose isomerase ofStreptomyces chrysomallus PL45 is inducible by D-xylose only. In mutants obtained by means of a selection procedure in a chemostat the isomerase was induced in xylose-free medium containing glucose as carbon source.     

Characterization of a cytochrome oxidase-deficient yeast mutant which accumulates porphyrins

A cytochrome oxidase-deficient mutant (pop4) of Saccharomycescerevisiae which accumulates porphyrins has been characterized. The pop4 mutation is recessive and affects a single nuclear gene. The bulk of the accumulated porphyrins consists of uroporphyrin and its partial decar?ylation products, suggesting that the mutation causes a block at the level of uroporphyrinogen decar?ylase. However, pop4 is not allelic to hem6 or pop3, putative decar?ylase structural-gene mutants. These results suggest that there may be a uroporphyrinogen decar?ylase isoenzyme specifically involved in heme a production.     

Photosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Mutants with Reduced Chloroplast Number

We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2–15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005–1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.     

Ethanol and biomass production of wild strains and respiratory deficient mutants of Saccharomyces cerevisiae under anaerobic and aerobic conditions

Summary In order to test the possibility of producing ethanol under aerobic conditions, 4 mitochondrial mutants of Saccharomyces cerevisiae lacking the capacity to respire were assayed for ethanol and biomass yield. As controls the corresponding wild strains were tested under anaerobic and aerobic conditions. In the latter case respiration was blocked by catabolite repression. The data show that the respiratory deficient mutants yield slightly less ethanol than the anaerobically grown wild strains, but more than those grown aerobically. Therefore, if for technical reasons aerobic fermentation is necessary, the use of mitochondrial mutants would be economically advantageous.     

Glucose and glycerol repression of α-amylase in Streptomyces kanamyceticus and isolation of deregulated mutants

Summary Glucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.     

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

Summary Two mutants, unable to utilize fructose (Fru^–) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru^– mutants among survivors of mutagenesis was 1 in 10⁴. The two Fru^– mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.     

Bile acid catabolites accumulated by transposon-induced mutants ofPseudomonas putida: Production of hydroxylated 1,4-androstadiene-3,17-diones

Summary Transposon mutagenesis of a bile acid-utilizingPseudomonas putida strain generated 5 classes of mutants based on their ability to accumulate steroild catabolites. Bile acids (up to 5%) could be fermented using mutant strains, to the appropriate hydroxy-1,4-androstadiene-3,17-dione type product in yields close to theoretical.     

Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Effects of site-directed mutagenesis of <Emphasis Type="Italic">mglA</Emphasis> on motility and swarming of <Emphasis Type="Italic">Myxococcus xanthus</Emphasis>

Background  

The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized.     

Improved sensitivity of genetic complementation of nitrate reductase deficient mutants via protoplast fusion

An improved rapid assay for complementation testing of mutants of Nicotiana tabacum deficient in nitrate reductase is described. The test is based on measurement of in vivo nitrate reductase activity in 7 to 10 day old cultures derived from fusion-treated protoplast mixtures of the respective mutants as a criterion for complementation. It allows to detect complementing hybrids induced by the conventional droplet fusion technique in small numbers of protoplasts per assay (8×10⁴).Abbreviations NR nitrate reductase - 2,4-D dichlorophenoxy-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - PEG polyethylene glycol