Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine     

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit⁻, unable to grow in nitrate, and 28 Nit⁺, able to grow in nitrate). All the Nit⁻ strains lacked MoCo activity. Diploid complementation of Nit⁻, MoCo⁻ strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit⁺ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c⁺) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c⁺ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.     

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.     

Effects of chemical components and nitrogen sources on zygospore development inPhycomyces blakesleeanus

We examined the effects of chemical components and nitrogen sources on zygospore development, using 62 different ingredients based on Sutter's synthetic medium Sl, which has been widely used for studies of sexual physiology inPhycomyces. An increase of inorganic microelements such as ZnSO₄, NaMoO₄ and CaCl₂ promoted an increase in the number of zygospores per unit area. Glutamate (Glu) contained in Sl as the sole nitrogen source was indispensable for sexual development, and replacement of Glu with NH₄ ⁺ (Am) strongly inhibited it, mainly because of growth inhibition. However, zygospore production was enhanced 1.8-fold by equivalent amounts of both Glu and Am as compared with Glu alone. A newly developed medium, mSl+Am, enriched with Am and the above-mentioned effective microelements doubled the number of zygospores formed per unit area (density), compared with Sutter's original Sl, and increased both the density and the weight (volume) of zygospores, 1.6- and 2-fold, respectively, compared with potato-dextrose-agar medium enriched with yeast extract and casitone (PDAYC). Sexual stimulation by mSl+Am was also observed in the mating of a pair of β-carotene-deficient mutants. Methionine sulfoxime, an inhibitor of glutamine synthetase, strongly inhibited the progress of mating without significant growth inhibition. The first and the second authors contributed equally to this work.     

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.     

A Transplasma Membrane Redox System in Phycomyces blakesleeanus: Properties of a Ferricyanide Reductase Activity Regulated by Iron Level and Vitamin K3

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.     

Subcellular localization of calcium in sporangiophores of Phycomyces blackesleeanus

The in situ localization of Ca²⁺ in stage I sporangiophores of the fungus Phycomyces blakesleeanus was achieved with the potassium pyroantimonate technique. Precipitates of calcium-antimonate were present in mitochondria, vacuoles, endoplasmic reticulum and adjacent cytoplasm, Golgi-like bodies, and nuclei but not cell walls. Material treated with the calcium chelator EGTA lacked these precipitates. The preferential localization of Ca²⁺ in mitochondria, endoplasmic reticulum and vacuoles suggests that these organelles modulate the level of this cation in sporangiophores of P. blakesleeanus.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N, tetraacetic acid     

Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana

Summary Chlorate resistant mutants of Arabidopsis thaliana were isolated, of which 10 exhibited a lowered nitrate reductase activity and 51 were chlorate-resistant because of an impaired uptake of chlorate. The 51 mutants of this type are all affected in the same gene. The mutants with a lowered nitrate reductase activity fall into 7 different complementation groups. Three of these mutants grow poorly on media with nitrate as the sole nitrogen source, while the others apparently can reduce sufficient nitrate to bring about growth. In all cases a low nitrate reductase activity coincides with an enhanced nitrite reductase activity. After sucrose gradient centrifugation of wildtype extracts nitrate reductase is found at the 8S position, whereas cytochrome-c reductase is found both at 4 and 8S positions. It is suggested that the functional nitrate reductase is a complex consisting of 4S subunits showing cytochrome-c reductase activity and a Mo-bearing cofactor. All mutants except B25 are capable of assembling the 4S subunits into complexes which for most mutants have a lower S value and exhibit a lower nitrate reductase activity than the wildtype complexes. Since the mutants B25 and B73 exhibit a low xanthine dehydrogenase activity, the Mo-bearing cofactor is probably less available in these mutants than in the wildtype. B73 appears to be the only mutant which is partly repaired by excessive Mo. The possible role of several genes is discussed.     

Isolation and characterization of mutants of mesophilic methanogenic bacteria resistant to analogues of DNA bases and nucleosides

Spontaneous mutants of mesophilic Methanobacterium, Methanobrevibacter and Methanosarcina species resistant to 6-mercaptopurine, 5-fluorouracil, 8-azaguanine, 6-azauracil or 5-fluorodeoxyuridine were isolated. Low level resistant mutants were unstable but highly resistant strains (resistance factor greater than 10-fold) were stable and showed growth characteristics comparable to the parent. Wild type strains showed linear uptake of hypoxanthine and uracil into cells, but guanine uptake was only detected in Methanosarcina mazei. 6-Mercaptopurine-resistant clones of Methanobacterium and Methanobrevibacter species and 8-azaguanine-resistant clones of Methanosarcina mazei showed reduced uptake of hypoxanthine and guanine respectively, but no evidence for altered permeability of 5-fluoro-and 6-azauracil-resistant strains to uracil was obtained. Double resistant mutants of Methanobacterium sp. strain FR-2 were characterised. Although these generally exhibited reduced specific growth rates, several were selected which showed similar growth to the parent.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany - MJC minimum inhibitory concentration - cfu colony forming unit - MP 6-mercaptopurine - FU 5-fluorouracil - FDU 5-fluorodeoxyuridine - AG 8-azaguanine - AU 6-azauracil - DA l-deazaadenosine     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Isolation and characterization of valine-resistant mutants of Nicotiana plumbaginifolia

Summary Haploid mesophyll protoplasts of Nicotiana plumbaginifolia were mutagenized by UV-irradiation. Protoplast-derived colonies were then selected for valine resistance on a medium containing 5 or 10 mM valine. From the resistant calli, plants were regenerated. Resistance was inherited as a recessive Mendelian character in seven clones. Mutations conferring valine resistance were shown to be allelic. Protoplast-derived cells of L-valine-resistant plants were also resistant to L-threonine. Resistance to valine was based on a reduced valine uptake rate.     

Isolation and genetic characterization of streptomycin and chloramphenicol resistant mutants in the mossPhyscomitrella patens

Summary Mutant strains of the mossPhyscomitrella patens that were resistant to the antibiotics streptomycin and chloramphenicol were isolated following UV irradiation. Mutants resistant to streptomycin at 1.7×10⁻⁴ M showed both dominant and recessive Mendelian and inheritance patterns. Mutants resistant to streptomycin at 1.7 ￠ 10⁻⁴ M and to chloramphenicol at 3.1×10⁻⁴ M were, with one exception, cytoplasmically inherited.     

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

Summary Two temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1–33.8±0.7 forgs(1)N26 and 1–39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of King's stages of oogenesis.     

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.     

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.     

Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity

Pichia stipitis strain NRRL Y-11,543 was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) to improve xylanolytic activity. A total of 20,000 mutants were screened for xylanase overproduction by observing the clear zones around the colonies on remazol-briliant-blue-xylan (RBB-xylan)-containing agar. Of 94 mutants isolated 11 of them were found to have enhanced xylanase activity compared to the parental strain. The most active mutant NP54376 had superior properties to the wild type which included: double the enzyme activity of wild type, a shorter generation time of 2.22 h compared to 3.13 h when grown on xylan, and an enhanced growth and yield of xylanase when low levels of xylose were added to the medium. Zymogram analysis of the crude enzyme preparations from both NP54376 and the wild type by isoelectric focusing showed multiple bands ranging between pI 4.2 and 7.4. No significant difference was observed in the K _m and V _max values of the parental strain and NP54376. K _m and V _max values of xylanase for birchwood xylan were 4.2 mg ml⁻¹ and 0.08 μmol min⁻¹ mg⁻¹ of protein, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa

Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c+o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s).Abbreviations TMPD Tetramethyl-p-phenylenediamine - MD minimal Davis     

Isolation and characterization of thiosulfate reductases from the green alga Chlorella fusca

Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR III_a, 55000; TSR III_b, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, l-cysteine, and -mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR III_a, 8.5; and for TSR III_b, 9.5. The apparent-K_m data for DTE and thiosulfate were determined to: TSR I, 0.164 mmol·l^-1 and TSR II, 0.156 mmol·l^-1; K_mDTE TSR I, 1.54 mmol·l^-1 and TSR II 1.54 mmol·l^-1. The thiosulfate reductases III_a and III_b were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.Abbreviations DTE dithioerythritol - TSR thiosulfate reductase Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday