RESUMEN
Killer spectra were investigated to select killer yeast strains to inhibit the growth of wild yeasts that cause aerobic deterioration of silage. Kluyveromyces lactis IFO 1267 was characterized by its rapid killing activity and wide spectrum against the target yeasts. Crude killer protein produced by K. lactis IFO 1267 or inoculation of the cell itself inhibited the growth of the target strain, Saccharomyces cerevisiae IFO 0304 (in liquid or in solid culture), particularly when lactose was used as carbon source instead of glucose.
Asunto(s)
Kluyveromyces/fisiología , Ensilaje , Aerobiosis , División Celular , Glucosa/metabolismo , Lactatos/metabolismo , Ácido Láctico , Micotoxinas/farmacología , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The G1 cell cycle arrest imposed by Kluyveromyces lactis zymocin on Saccharomyces cerevisiae requires a functional RNA polymerase II (pol II) TOT/Elongator complex. In a study of zymocin's mode of action, genetic scenarios known to impair transcription or affect the pol II machinery itself were found to elicit hypersensitivity to zymocin. Thus, mutations in components of SAGA, SWI/SNF, Mediator and Ccr4-Not, complexes involved in transcriptionally relevant functions such as nucleosome modification, chromatin remodelling and formation of the preinitiation complex, make yeast cells hypersensitive to the lethal effects of zymocin. The defects at the level of transcriptional elongation displayed by rtf1Delta, ctk1, fcp1 and rpb2 mutants also result in zymocin hypersensitivity. Intriguingly, inactivation of histone deacetylase (HDAC) activity, which is expected to reduce the demand for the histone acetyltransferase (HAT) function of TOT/Elongator, also reduces sensitivity to zymocin. Thus, zymocin interferes with pol II-dependent transcription, and this effect requires the HAT function of TOT, presumably while the Elongator complex is associated with pol II.
Asunto(s)
Fase G1/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Micotoxinas/farmacología , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Transcripción Genética , Farmacorresistencia Fúngica , Genes Fúngicos/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Factores Asesinos de Levadura , Kluyveromyces , Mutación/genética , ARN Polimerasa II/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK; ATP-dependent) was cloned from the Kluyveromyces lactis genome using a PCR amplicon from Saccharomyces cerevisiae PCK1 gene as a probe. A DNA fragment of about 4.8 kb containing KlPCK1 complemented PEPCK activity of the mutant of S. cerevisiae defective in PEPCK. The KlPCK1 gene has an open reading frame of 1629 bp (543 amino acids). The KlPCK1 nucleotide sequence and deduced amino acid sequence showed 76% and 84% homologies to those of S. cerevisiae PCK1, respectively. Multiple alignment of ATP-dependent PEPCK genes shows highly conserved regions.
Asunto(s)
Genes Fúngicos , Kluyveromyces/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Prueba de Complementación Genética , Kluyveromyces/enzimología , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
In this study, we propose a new process of adding a genetically modified killer yeast to improve the aerobic stability of silage. Previously constructed Kluyveromyces lactis killer strain PCK27, defective in growth on lactic acid due to disruption of the gene coding for phosphoenolpyruvate carboxykinase, a key enzyme for gluconeogenesis, inhibited the growth of Pichia anomala inoculated as an aerobic spoilage yeast and prevented a rise in pH in a model of silage fermentation. This suppressive effect of PCK27 was not only due to growth competition but also due to the killer protein produced. From these results, we concluded that strain PCK27 can be used as an additive to prolong the aerobic stability of maize silage. In the laboratory-scale experiment of maize silage, the addition of a killer yeast changed the yeast flora and significantly reduced aerobic spoilage.