Ferricyanide reductase of rose plasma membranes is regulated by nitrogen supply

Summary The ability of suspension-cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells to reduce ferricyanide is decreased by 50% during an overnight incubation in a low-nutrient (1 mM CaCl₂, 0.1 mM KCl) solution. This loss is not observed when nitrate and/or glutamate is added to the low-nutrient medium, but it occurs in medium containing all the components needed for normal growth except nitrate plus glutamate. Thus, the cells possess both constitutive and inducible enzymes for the reduction of ferricyanide, and nitrate or glutamate is both necessary and sufficient to stimulate the production of the inducible enzyme.     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone     

Bacterial 2,3-butanediol dehydrogenases

Enterobacter aerogenes, Aeromonas hydrophila, Serratia marcescens and Staphylococcus aureus possessing L(+)-butanediol dehydrogenase produced mainly meso-butanediol and small amounts of optically active butanediol; Acetobacter suboxydans, Bacillus polymyxa and Erwinia carotovora containing D(-)-butanediol dehydrogenase produced more optically active butanediol than meso-butanediol. Resting and growing cells of these organisms oxidized only one enantiomer of racemic butanediol. The D(-)-butanediol dehydrogenase from Bacillus polymyxa was partially purified (30-fold) with a specific activity of 24.5. Except NAD and NADH no other cofactors were required. Optimum pH-values for oxidation and reduction were pH 9 and pH 7, respectively. The optimum temperature was about 60°C. The molecular weight was 100000 to 107000. The K _m-values were 3.3 mM for D(-)-butanediol, 6.25 mM for meso-butanediol, 0.53 mM for acetoin, 0.2 mM for NAD, 0.1 mM for NADH, 87 mM for diacetyl, 38 mM for 1,2-propanediol; 2,3-pentanedion was not a substrate for this enzyme. The L(+)-butanediol dehydrogenase from Serratia marcescens was purified 57-fold (specific activity 22.3). Besides NAD or NADH no cofactors were required. The optimum value for oxidation was about pH 9 and for reduction pH 4.5. The optimum temperature was 32–36°C. The molecular weight was 100000 to 107000. The K _m-values were 5 mM for meso-butanediol, 10 mM for racemic butanediol, 6.45 for acetoin, 1 mM for NAD, 0.25 mM for NADH, 2.08 mM for diacetyl, 16.7 mM for 2,3-pentanedion and 11.8 mM for 1,2-propanediol.Abbreviations Bud 2,3-butanediol - DH dehydrogenase     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Molecular weight and quaternary structure of ribulose bisphosphate carboxylase from the cyanobacterium,Synechococcus sp.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH ₄ ⁺ . Light-dependent C₂H₂ reduction and H₂ production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H₂-palladised asbestos prior to assay, it only partially restricted C₂H₂ reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H₂-dependent photoreduction of ¹⁴CO₂; (c) -glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor).Metronidazole (1 mM) completely inhibited C₂H₂ reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C₂H₂ reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

The Role of GIGANTEA Gene in Mediating the Oxidative Stress Response and in Arabidopsis

The Arabidopsis GIGANTEA (GI) gene has been shown to be involved in the regulation of the oxidative stress response; however, little is known about the mechanism by which GI gene regulates the oxidative stress response. We show here that enhanced tolerance of the gi-3 mutant to oxidative stress is associated, at least in part, with constitutive activation of superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes. The gi-3 plants were more tolerant to parquart (PQ) or hydrogen peroxide (H₂O₂)-mediated oxidative stress than wild-type plants. Analyses of concentrations of endogenous H₂O₂ and superoxide anion radicals as well as lipid peroxidation revealed that enhanced tolerance of gi-3 plants to oxidative stress was not due to defects in the uptake of PQ or the sequestration of PQ from its site of action, and that the gi-3 mutation alleviated oxidative damage of plant cells from PQ stress. Moreover, the gi-3 mutant showed constitutive activation of cytosolic Cu/ZnSOD and plastidic FeSOD as well as cytosolic APX1 and stromal APX genes, which at least in part contributed to constitutive increases in activities of anti-oxidative enzymes SOD and APX, respectively. To our knowledge, we demonstrate, for the first time, that GI gene regulates the oxidative stress response, at least in part, through modulation of SOD and APX genes.     

Direct conversion of cellulose to methane by anaerobic fungus Neocallimastix frontalis and defined methanogens

Co-cultures of N. frontalis with a formate-utilizing methanogen, Methanobacterium formicicum and/or an aceticlastic methanogen, Methanosaeta concilii, were performed for methane production from cellulose. In the co-culture with M. formicicum, ca. 16 mM CH₄ was produced after 7 days without accumulation of H₂ and formate. In the co-culture with M. concilii, 12 mM CH₄ was produced after 17 days with decreasing acetate production. In the tri-culture of N. frontalis with M. formicicum and M. concilii, 24 mM CH₄ was produced after 17 days where acetate still remained at 23 mM, but production of lactate and ethanol decreased. When a 4-times concentrated culture broth of M. concilii was inoculated in this tri-culture system in a bioreactor, 150 mM CH₄ was produced after 24 days by feeding of cellulose, although 57 mM acetate still accumulated.     

Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate

Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K _s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K _s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.     

Protein synthesis by Beggiatoa alba B18LD in the presence and absence of sulfide

The interaction of sulfide oxidation and protein synthesis by Beggiatoa alba B18LD was investigated using the incorporation of radiolabeled leucine to estimate protein synthesis. Leucine was assimilated into whole cells in the presence of 6.1 mM acetate at a rate of 0.6 nmol · min^-1 · mg protein^-1, 43% of which was incorporated into the protein fraction. Protein synthesis by B. alba was unaffected by 1 mM sulfide, whether or not the cells had been preincubated with sulfide. B. alba oxidized radioactive sulfide to sulfur within 30 s of addition of the label, whether or not the organism was preinduced by sulfide. Furthermore, chloramphenicol, which inhibited protein synthesis, did not significantly inhibit sulfide oxidation by sulfide-induced or uninduced B. alba. This indicates that sulfide oxidation is a constitutive process. Enrichments of sulfur inclusions from B. alba B18LD that were analyzed by polyacrylamide gel electrophoresis demonstrated two enriched peptides with M_r values of 13,000 and 15,000. The 13,000 and 15,000 M_r peptide bands were more evident in cells grown in a medium containing sulfide than in cells from a medium lacking sulfide. Although sulfide did not increase the rate of overall protein synthesis, the synthesis of a few peptides was increased by the addition of sulfide to the growth medium. Among those, the 15,000 M_r peptide was one of the most distinctive.Non-standard abbreviations SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - PPO 2,5-diphenyloxazole - POPOP 1,4-bis [5-phenyl-2-oxazolyl]-benzene - BSS basal salts solution - BH Beggiatoa heterotrophic (medium) - BSO Beggiatoa sulfide oxidation (medium) - CM chloramphenicol - TCA trichloroacetic acid - M_r molecular mass     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Uptake of sulphate,taurine, cysteine and methionine by symbiotic and free-living dinoflagellates

Sulphate uptake by Amphidinium carterae, Amphidinium klebsii and Gymnodinium microadriaticum grown on artificial seawater medium with sulphate, cysteine, methionine or taurine as sulphur source occurred via an active transport system which conformed to Michaelis-Menten type saturation kinetics. Values for K _m ranged from 0.18–2.13 mM and V _max ranged from 0.2–24.2 nmol · 10⁵ cells^–1 · h^–1. K _m for symbiotic G. microadriaticum was 0.48 mM and V _max was 0.2 nmol · 10⁵ cells^–1 · h^–1. Sulphate uptake was slightly inhibited by chromate and selenate, but not by tungstate, molybdate, sulphite or thiosulphate. Cysteine and methionine (0.1 mM), but not taurine, inhibited sulphate uptake by symbiotic G. microadriaticum, but not by the two species of Amphidinium. Uptake was inhibited 45–97% under both light and dark conditions by carbonylcyanide 3-chlorophenylhydrazone (CCCP); under dark conditions sulphate uptake was 40–60% of that observed under light conditions and was little affected by 3-(3,4-dichlorophenyl) 1,1-dimethylurea (DCMU).The uptake of taurine, cysteine and methionine by A. carterae, A. klebsii, cultured and symbiotic G. microadriaticum conformed to Michaelis-Menten type saturation kinetics. K _m values of taurine uptake ranged from 1.9–10 mM; for cysteine uptake from 0.6–3.2 mM and methionine from 0.001–0.021 mM. Cysteine induced a taurine uptake system with a K _m of 0.3–0.7 mM. Cysteine and methionine uptake by all organisms was largely unaffected by darkness or by DCMU in light or darkness. CCCP significantly inhibited uptake of these amino acids. Thus energy for cysteine and methionine uptake was supplied mainly by respiration. Taurine uptake by A. carterae was independent of light but was inhibited by CCCP, whereas uptake by A. klebsii and symbiotic G. microadriaticum was partially dependent on photosynthetic energy. Taurine uptake by cultured G. microadriaticum was more dependent on photosynthetic energy and was more sensitive to CCCP. Cysteine inhibited uptake of methionine and taurine by cultured and symbiotic G. microadriaticum to a greater extent than in the Amphidinium species. Methionine did not greatly affect taurine uptake, but did inhibit cysteine uptake. Taurine did not affect the uptake of cysteine or methionine.     

Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC₅₀ values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Kinetic study of the toxicity of zinc and lead ions to the heavy metals accumulating fungus Paecilomyces marquandii

Studies have been carried out to determine the toxicity of zinc and lead ions to germinating spores and hyphal growth of heavy metal accumulating fungus Paecilomyces marquandii (former Verticillium marquandii). Inhibitive concentration (IC₅₀) of zinc and lead ions was assayed by three different methods: image analysis, nephelometric on-line measurement and microcalorimetry. A kinetic model of spore germination and germ tube elongation was formulated and used as an auxiliary tool to determine IC₅₀ values upon image analysis data. The inhibitive effect of Zn²⁺ and Pb²⁺ to P. marquandii spores was mathematically described by the Edwards equation. Comparing the obtained IC₅₀ values, lead ions occurred to be more toxic to the germinating spores of P. marquandii than zinc ions (2.80 and 5.20 mM, respectively), although zinc ions induced a more significant delay in the development of the hyphae (13.84 h for 5 mM of Zn²⁺ and 9.30 h for 5 mM of Pb²⁺), which was demonstrated by the lengthened lag-phase (spore-swelling phase).