RESUMO
The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Delta mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V(max) of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Delta mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Delta mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade.
Assuntos
Glucose/metabolismo , Glicosídeo Hidrolases/genética , Proteína Quinase C/fisiologia , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Regulação Fúngica da Expressão Gênica , Mutação , Proteína Quinase C/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica , beta-FrutofuranosidaseRESUMO
The structural complexity of the nitrogen sources strongly affects biomass production and secretion of hydrolytic enzymes in filamentous fungi. Fusarium oxysporum and Aspergillus nidulans were grown in media containing glucose or starch, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids), peptides (peptone) and protein (gelatin). In glucose, when the initial pH was adjusted to 5.0, for both microorganisms, higher biomass production occurred upon supplementation with a nitrogen source in the peptide form (peptone and gelatin). With a close to neutrality pH, biomass accumulation was lower only in the presence of the ammonium salt. When grown in starch, biomass accumulation and secretion of hydrolytic enzymes (amylolytic and proteolytic) by Fusarium also depended on the nature of the nitrogen supplement and the pH. When the initial pH was adjusted to 5.0, higher growth and higher amylolytic activities were detected in the media supplemented with peptone, gelatin and casamino acids. However, at pH 7.0, higher biomass accumulation and higher amylolytic activities were observed upon supplementation with peptone or gelatin. Ammonium sulfate and casamino acids induced a lower production of biomass, and a different level of amylolytic enzyme secretion: high in ammonium sulfate and low in casamino acids. Secretion of proteolytic activity was always higher in the media supplemented with peptone and gelatin. Aspergillus, when grown in starch, was not as dependent as Fusarium on the nature of nitrogen source or the pH. The results described in this work indicate that the metabolism of fungi is regulated not only by pH, but also by the level of structural complexity of the nitrogen source in correlation to the carbon source.
Assuntos
Amilases/metabolismo , Aspergillus nidulans/enzimologia , Biomassa , Fusarium/enzimologia , Nitrogênio/química , Peptídeo Hidrolases/metabolismo , Aspergillus nidulans/crescimento & desenvolvimento , Meios de Cultura , Fusarium/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Hidrólise , Nitrogênio/metabolismoRESUMO
A chemically defined medium was developed for the production of intracellular malate dehydrogenases by Streptomyces aureofaciens NRRL-B 1286. The composition of the medium (per liter) was as follows: 50 g of starch, 4 g of ammonium sulfate, 7.32 g of l-aspartic acid, 13.8 g of MgSO(4) . 7H(2)O, 1.7 g of K(2)HPO(4), 0.01 g of ZnSO(4) . 7H(2)O, 0.01 g of FeSO(4) . 7H(2)O, 0.01 g of MnSO(4) . H(2)O, and 0.005 g of CoSO(4) . 7H(2)O. The pH of the medium was adjusted to 6.7 to 7.0 after sterilization. The activity of the intracellular malate dehydrogenases of the crude cell extract was greatest after 40 h of mycelium growth in a rotary shaker at 30 degrees C. The best temperature for the enzyme reactions was approximately 35 degrees C for NAD activity at pH 9.7 and 40 degrees C for NADP -linked enzyme at pH 9.0. The NAD activity required Mg, and both activities were sensitive to SH-group reagents. The NADP -dependent activity remained completely stable, and the NAD -dependent activity decreased to a very low residual level after 30 min at 60 degrees C.