RESUMEN
Actinobacteria are believed to play a major role in organic matter degradation and humification processes in composts. In this study, the effects of different temperature regimes on the succession of Actinobacteria populations during composting were investigated in a laboratory reactor. Phospholipid fatty acid (PLFA) was used to investigate quantitative changes in the overall microbial biomass and community structure, and in the size of Actinobacteria populations. Qualitative changes were determined using PCR-DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rRNA genes with Actinobacteria-specific primers. The peak in total microbial biomass was roughly twice as high and delayed in trials where the maximum temperature was 40 degrees C, compared to those where it was 55 or 67 degrees C. There was a shift from members of Corynebacterium, Rhodococcus and Streptomyces at the onset to species of thermotolerant Actinobacteria in the cooling phase, e.g. Saccharomonospora viridis, Thermobifida fusca and Thermobispora bispora. In conclusion, temperature was an important selective factor for the development of Actinobacteria populations in composts, and they constituted a substantial part of the community in the later compost stages.
Asunto(s)
Actinobacteria/clasificación , Actinobacteria/crecimiento & desarrollo , Microbiología del Suelo , Temperatura , Actinobacteria/química , Actinobacteria/genética , Secuencia de Bases , Biomasa , Recuento de Colonia Microbiana/métodos , Dermatoglifia del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Fosfolípidos/análisis , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , SueloRESUMEN
Two genes involved in protein secretion, encoding the Rab protein YPT1/YPTA and the general fusion factor NSFI/NSFA, were characterized from two filamentous fungi, Trichoderma reesei and Aspergillus niger var. awamori. The isolated genes showed a high level of conservation with their Saccharomyces cerevisiae and mammalian counterparts, and T. reesei ypt1 was shown to complement yeast Ypt1p depletion. The transcriptional regulation of the T. reesei ypt1, nsf1, and sar1 genes, involved in protein trafficking, was studied with mycelia treated with the folding inhibitor dithiothreitol (DTT) and with brefeldin A, which inhibits membrane traffic between the endoplasmic reticulum and Golgi complex. The well-known inducer of the yeast and T. reesei unfolded protein response (UPR), DTT, induced the nsf1 gene and the protein disulfide isomerase gene, pdi1, in both of the experiments, and sar1 mRNA increased in only one experiment under strong UPR induction. The ypt1 mRNA did not show a clear increase during DTT treatment. Brefeldin A strongly induced pdi1 and all of the intracellular trafficking genes studied. These results suggest the possibility that the whole secretory pathway of T. reesei could be induced at the transcriptional level by stress responses caused by protein accumulation in the secretory pathway.
Asunto(s)
Aspergillus niger/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Trichoderma/fisiología , Proteínas de Unión al GTP rab/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Aspergillus niger/genética , Aspergillus niger/metabolismo , Medios de Cultivo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , Trichoderma/genética , Trichoderma/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/genéticaRESUMEN
The 14-3-3 proteins are highly conserved, ubiquitously expressed proteins taking part in numerous cellular processes. Two genes encoding 14-3-3 proteins, ftt1 and ftt2, were isolated and characterised from the filamentous fungus Trichoderma reesei. FTTI showed the highest sequence identity (98% at the amino acid level) to the Trichoderma harzianum protein Th1433. FTTII is relatively distinct from FTTI, showing approximately 75% identity to other fungal 14-3-3 proteins. Despite their sequence divergence, both of the T. reesei ftt genes were equally able to complement the yeast bmh1 bmh2 double disruption. The T. reesei ftt genes were also found to be quite closely linked in the genomic DNA. A C-terminally truncated version of ftt1 (ftt1DeltaC) was first isolated as a multicopy suppressor of the growth defect of the temperature-sensitive yeast secretory mutant sec15-1. Overexpression of ftt1DeltaC also suppressed the growth defect of sec2-41, sec3-101, and sec7-1 strains. Overexpression of ftt1DeltaC in sec2-41 and sec15-1 strains could also rescue the secretion of invertase at the restrictive temperatures, and overexpression of full-length ftt1 enhanced invertase secretion by wild-type yeast cells. These findings strongly suggest that the T. reesei ftt1 has a role in protein secretion.