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Evidence of cAMP involvement in cellobiohydrolase expression and secretion by Trichoderma reesei in presence of the inducer sophorose.

Nogueira KM, Costa Mdo N, de Paula RG, Mendonça-Natividade FC, Ricci-Azevedo R, Silva RN - BMC Microbiol. (2015)

Bottom Line: Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner.Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources.These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil. karolmvnogueira@hotmail.com.

ABSTRACT

Background: The signaling second messenger cyclic AMP (cAMP) regulates many aspects of cellular function in all organisms. Previous studies have suggested a role for cAMP in the regulation of gene expression of cellulolytic enzymes in Trichoderma reesei (anamorph of Hypocrea jecorina).

Methods: The effects of cAMP in T. reesei were analyzed through both activity and expression of cellulase, intracellular cAMP level measurement, western blotting, indirect immunofluorescence and confocal microscopy.

Results: To elucidate the involvement of cAMP in the cellulase expression, we analyzed the growth of the mutant strain ∆acy1 and its parental strain QM9414 in the presence of the inducers cellulose, cellobiose, lactose, or sophorose, and the repressor glucose. Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner. The expression cel7a, and cel6a genes was higher in the presence of sophorose than in the presence of cellulose, lactose, cellobiose, or glucose. Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources. Concomitantly, our immunofluorescence microscopy and western blot data suggest that in the presence of sophorose, cAMP may regulate secretion of cellulolytic enzymes in T. reesei.

Conclusions: These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei. Finally, our data may help develop new strategies to improve the expression of cel7a and cel6a genes, and therefore, favor their application in several biotechnology fields.

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Related in: MedlinePlus

Total cellulolytic activity in different carbon sources. T. reesei QM9414 strain was grown in cellulose, lactose, or cellobiose for 24, 48, and 72 h, in glucose for 24, and 48 h, and in sophorose for 2, 4, and 6 h. Activity was measured using the azure-Cellulose method and is expressed as the ratio between growth factor and the absorbance obtained in the standard curve equation, per minute, and per amount of enzyme added to the reaction
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Fig2: Total cellulolytic activity in different carbon sources. T. reesei QM9414 strain was grown in cellulose, lactose, or cellobiose for 24, 48, and 72 h, in glucose for 24, and 48 h, and in sophorose for 2, 4, and 6 h. Activity was measured using the azure-Cellulose method and is expressed as the ratio between growth factor and the absorbance obtained in the standard curve equation, per minute, and per amount of enzyme added to the reaction

Mentions: Similar to qPCR-RT results, the QM9414 strain exhibited higher cellulolytic activity in cultures with cellulose, sophorose, and lactose (Fig. 2). However, different to what was observed in the expression analysis, which showed an increase of cel7a and cel6a expression after 6 h, cellulolytic activity in sophorose increased mainly at 2 h in culture (24.5 U/mL). A similar pattern was observed growing QM9414 in lactose, where cellulolytic activity reached high levels at 72 h (22.5 U/mL). Cellulolytic activity in cellulose was higher at 48 and 72 h (24.5 U/mL and 20.7 U/mL, respectively). On the contrary, hydrolytic activity in glucose was lower than in other carbon sources, being 6, 5, and 4.8 times lower than in cellulose, sophorose, lactose and cellobiose, respectively. Interestingly, our results showed no correlation between the expression profile and cellulolytic activity in cellobiose. The cellulolytic activity showed a steady increase from 24 h and a maximum at 72 h (19.8 U/mL) (Fig. 2). This result may be explained from the fact that the azure-Cellulose® method detects total cellulolytic activity without distinguishing specific cellulases.Fig. 2


Evidence of cAMP involvement in cellobiohydrolase expression and secretion by Trichoderma reesei in presence of the inducer sophorose.

Nogueira KM, Costa Mdo N, de Paula RG, Mendonça-Natividade FC, Ricci-Azevedo R, Silva RN - BMC Microbiol. (2015)

Total cellulolytic activity in different carbon sources. T. reesei QM9414 strain was grown in cellulose, lactose, or cellobiose for 24, 48, and 72 h, in glucose for 24, and 48 h, and in sophorose for 2, 4, and 6 h. Activity was measured using the azure-Cellulose method and is expressed as the ratio between growth factor and the absorbance obtained in the standard curve equation, per minute, and per amount of enzyme added to the reaction
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590280&req=5

Fig2: Total cellulolytic activity in different carbon sources. T. reesei QM9414 strain was grown in cellulose, lactose, or cellobiose for 24, 48, and 72 h, in glucose for 24, and 48 h, and in sophorose for 2, 4, and 6 h. Activity was measured using the azure-Cellulose method and is expressed as the ratio between growth factor and the absorbance obtained in the standard curve equation, per minute, and per amount of enzyme added to the reaction
Mentions: Similar to qPCR-RT results, the QM9414 strain exhibited higher cellulolytic activity in cultures with cellulose, sophorose, and lactose (Fig. 2). However, different to what was observed in the expression analysis, which showed an increase of cel7a and cel6a expression after 6 h, cellulolytic activity in sophorose increased mainly at 2 h in culture (24.5 U/mL). A similar pattern was observed growing QM9414 in lactose, where cellulolytic activity reached high levels at 72 h (22.5 U/mL). Cellulolytic activity in cellulose was higher at 48 and 72 h (24.5 U/mL and 20.7 U/mL, respectively). On the contrary, hydrolytic activity in glucose was lower than in other carbon sources, being 6, 5, and 4.8 times lower than in cellulose, sophorose, lactose and cellobiose, respectively. Interestingly, our results showed no correlation between the expression profile and cellulolytic activity in cellobiose. The cellulolytic activity showed a steady increase from 24 h and a maximum at 72 h (19.8 U/mL) (Fig. 2). This result may be explained from the fact that the azure-Cellulose® method detects total cellulolytic activity without distinguishing specific cellulases.Fig. 2

Bottom Line: Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner.Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources.These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil. karolmvnogueira@hotmail.com.

ABSTRACT

Background: The signaling second messenger cyclic AMP (cAMP) regulates many aspects of cellular function in all organisms. Previous studies have suggested a role for cAMP in the regulation of gene expression of cellulolytic enzymes in Trichoderma reesei (anamorph of Hypocrea jecorina).

Methods: The effects of cAMP in T. reesei were analyzed through both activity and expression of cellulase, intracellular cAMP level measurement, western blotting, indirect immunofluorescence and confocal microscopy.

Results: To elucidate the involvement of cAMP in the cellulase expression, we analyzed the growth of the mutant strain ∆acy1 and its parental strain QM9414 in the presence of the inducers cellulose, cellobiose, lactose, or sophorose, and the repressor glucose. Our results indicated that cAMP regulates the expression of cellulase in a carbon source-dependent manner. The expression cel7a, and cel6a genes was higher in the presence of sophorose than in the presence of cellulose, lactose, cellobiose, or glucose. Moreover, intracellular levels of cAMP were up to four times higher in the presence of sophorose compared to other carbon sources. Concomitantly, our immunofluorescence microscopy and western blot data suggest that in the presence of sophorose, cAMP may regulate secretion of cellulolytic enzymes in T. reesei.

Conclusions: These results allow us to better understand the role of cAMP and expand our knowledge on the signal transduction pathways involved in the regulation of cellulase expression in T. reesei. Finally, our data may help develop new strategies to improve the expression of cel7a and cel6a genes, and therefore, favor their application in several biotechnology fields.

Show MeSH
Related in: MedlinePlus