RESUMEN
Cell wall bound calcium constitutes a significant fraction (25%) of total mycelia calcium in Neurospora crassa. Wall bound calcium increases as a function of growth and calcium concentration, while cell wall bound calcium decreases in Ca-free medium. Removal of wall bound calcium causes its rapid replacement from intracellular pool, inhibited by verapamil, nifedipine, concanamycin A, and wortmanin in a vacuolar mutant (Vma-5), but is unaffected by trifluoropyrazine, and calmidizoluim in a calcineurin mutant (Cnb-1) of N. crassa. Ca(2+) removal from surface with EGTA resulted in leakage of periplasmic enzymes invertase and alkaline phosphatase. Scanning and transmission electron microscopy revealed gross abnormalities represented by giant vacuoles. Toxic metal ions bound to wall fraction by displacing calcium. Our data underline the physiological importance of wall bound calcium in N. crassa.
Asunto(s)
Calcio/metabolismo , Pared Celular/química , Pared Celular/fisiología , Neurospora crassa/química , Neurospora crassa/fisiología , Pared Celular/metabolismo , Pared Celular/ultraestructura , Homeostasis , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Neurospora crassa/metabolismo , Neurospora crassa/ultraestructuraRESUMEN
Removal of radioactive cobalt at trace levels (approximately nM) in the presence of large excess (10(6)-fold) of corrosion product ions of complexed Fe, Cr, and Ni in spent chemical decontamination formulations (simulated effluent) of nuclear reactors is currently done by using synthetic organic ion exchangers. A large volume of solid waste is generated due to the nonspecific nature of ion sorption. Our earlier work using various fungi and bacteria, with the aim of nuclear waste volume reduction, realized up to 30% of Co removal with specific capacities calculated up to 1 microg/g in 6-24 h. In the present study using engineered Escherichia coli expressing NiCoT genes from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), we report a significant increase in the specific capacity for Co removal (12 microg/g) in 1-h exposure to simulated effluent. About 85% of Co removal was achieved in a two-cycle treatment with the cloned bacteria. Expression of NiCoT genes in the E. coli knockout mutant of NiCoT efflux gene (rcnA) was more efficient as compared to expression in wild-type E. coli MC4100, JM109 and BL21 (DE3) hosts. The viability of the E. coli strains in the formulation as well as at different doses of gamma rays exposure and the effect of gamma dose on their cobalt removal capacity are determined. The potential application scheme of the above process of bioremediation of cobalt from nuclear power reactor chemical decontamination effluents is discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Radioisótopos de Cobalto/metabolismo , Descontaminación/métodos , Escherichia coli/metabolismo , Ingeniería Genética , Reactores Nucleares , Proteínas Bacterianas/genética , Biodegradación Ambiental , Proteínas de Transporte de Catión/genética , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Rayos gamma , Expresión Génica , Residuos Radiactivos , Rhodopseudomonas/metabolismoRESUMEN
A cobalt-resistant wall-less mutant (slime) of Neurospora crassa was obtained by repeated sub-culturing of the sensitive wall-less mutant (W-sl) on agar medium containing toxic concentrations of cobalt. Resistance was stable on culturing Cor-sl on cobalt-free medium up to 15 weekly subcultures. Cor-sl is 10-fold more resistant to cobalt when compared to W-sl. It is also cross-resistant to Cu (10-fold) and Ni (3-fold). Cobalt accumulated by Cor-sl during growth and in short-term uptake experiments was lower when compared to W-sl. Cells previously loaded with cobalt was released into medium in both mutants, while in case of Cor-sl most of cobalt taken up (> 80%), was released back into the medium when compared to W-sl. Metabolic inhibitor (Sodium azide) and magnesium ions inhibited cobalt uptake in both the mutants. Fractionation of cell-free extracts showed that most of the cobalt (70%) taken up by Cor-sl was bound to an inducible protein fraction which bound to DEAE-Cellulose, while in W-sl only 20% of cobalt was associated with this fraction. Subcellular localization of cobalt in W-sl indicated most of it to be cytoplasmic (70%) while nuclei and mitochondria had 10% and 5% respectively. In case of Cor-sl, mitochondrial cobalt accounted for only 2% while no significant differences were noted for other fractions. Our data implicate both transport block and intracellular sequestration of cobalt to play a major role in resistance.
Asunto(s)
Pared Celular/fisiología , Cobalto/farmacología , Farmacorresistencia Fúngica , Mutación/genética , Neurospora crassa/efectos de los fármacos , Azidas/farmacología , Transporte Biológico/efectos de los fármacos , Cinética , Magnesio/farmacología , Neurospora crassa/citología , Neurospora crassa/genéticaRESUMEN
The susceptibility of proteins in the myelin membrane to proteases was studied. Lyophilized rat brain myelin suspended in water was subjected to controlled proteolytic digestion with pure trypsin (N-tosyl-L-phenylalanine chloromethyl ketone treated, 5 units/mg of myelin), and proteins remaining in the pellet were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under these conditions, large basic protein (LBP) was completely hydrolyzed in 5-10 min, proteolipid proteins remained largely intact until 60 min, whereas Wolfgram protein (WP) was progressively degraded from 10 min onward with the simultaneous appearance of a new protein band with a molecular weight of 35K. A similar pattern was obtained on treatment with chymotrypsin or subtilisin. The 35K protein band was shown to be derived from WP by its immunological cross-reactivity with WP antibodies. Western blot analysis showed that 35K protein is the only major breakdown product of WP under these conditions. Treatment with higher concentrations of trypsin (greater than 20 units/mg of myelin) resulted in the degradation of all myelin proteins. Essentially all the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) activity was observed in the myelin pellet after controlled or drastic digestion with trypsin. It is concluded that the major fragment of WP (35K) is located in the hydrophobic milieu of the bilayer, relatively inaccessible to trypsin, whereas a portion (20K) of the WP is exposed to the cytoplasmic side (major dense line), like LBP, and that peptide fragments (less than 14K) that remained in the myelin membrane lipid bilayer after trypsin digestion could exhibit CNP activity.
Asunto(s)
2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Encéfalo/metabolismo , Proteínas de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Péptido Hidrolasas/metabolismo , Hidrolasas Diéster Fosfóricas , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Animales , Electroforesis en Gel de Poliacrilamida , Inmunodifusión , Cinética , Peso Molecular , Proteína Básica de Mielina/metabolismo , Ratas , Tripsina/metabolismoRESUMEN
Three different Ni2+-resistant strains of Neurospora crassa (NiR1, NiR2 and NiR3) have been isolated. All are stable mutants and are fourfold more resistant to Ni2+ than the parent wild-type strain. NiR1 and NiR2 are also sixfold more resistant to Co2+, whereas NiR3 is only twice as resistant to Co2+; the former two are also twofold more resistant to Zn2+, but NiR3 is not. These three strains also differ in sensitivity to Cu2+. Toxicities and concomitant accumulation patterns of Ni2+, Co2+ and Cu2+ have been examined in these strains. NiR1 and NiR2, despite quantitative individual differences, generally accumulate very high amounts of Ni2+ and Co2+, and Mg2+ reverses the toxicities of these two ions by different mechanisms; Ni2+ uptake is suppressed, but not that of Co2+. In NiR3, Mg2+ controls uptake of both Ni2+ and Co2+. Studies indicate that two kinds of Ni2+-resistant strains of N. crassa exist; one kind is resistant because it can tolerate high intracellular concentrations of heavy-metal ions, whereas the other is resistant because it can control metal-ion accumulation.