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Multiple signals modulate the activity of the complex sensor kinase TodS.

Silva-Jiménez H, Ortega Á, García-Fontana C, Ramos JL, Krell T - Microb Biotechnol (2014)

Bottom Line: In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione.The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways.This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, C/ Prof. Albareda 1, Granada, 18008, Spain.

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Expression from PttgGHI and PtodX in response to menadione.A. Estimation of the pleiotropic effect of menadione. Pseudomonas putida DOT-T1E bearing pANA96 (PttgG::’lacZ) (Rojas et al., 2003) was grown on LB medium with 1 mM of toluene in the presence of increasing menadione concentrations.B. Effect of menadione on the expression form PtodX. Pseudomonas putida DOT-T1E ΔtodST bearing pMIR66 (containing todST) and pMIR77 (PtodX::′lacZ) was grown in LB medium with 1 mM toluene in the absence or presence of 0.25 mM menadione. At an OD660 = 0.8, β-galactosidase activities were determined.
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fig05: Expression from PttgGHI and PtodX in response to menadione.A. Estimation of the pleiotropic effect of menadione. Pseudomonas putida DOT-T1E bearing pANA96 (PttgG::’lacZ) (Rojas et al., 2003) was grown on LB medium with 1 mM of toluene in the presence of increasing menadione concentrations.B. Effect of menadione on the expression form PtodX. Pseudomonas putida DOT-T1E ΔtodST bearing pMIR66 (containing todST) and pMIR77 (PtodX::′lacZ) was grown in LB medium with 1 mM toluene in the absence or presence of 0.25 mM menadione. At an OD660 = 0.8, β-galactosidase activities were determined.

Mentions: We have then studied the influence of menadione on PtodX promoter activity. Initial experiments were aimed at determining the pleiotropic effect of menadione on gene expression. As a control, we have used the PttgG promoter that has been extensively studied in our group. It is controlled by the TtgV repressor but not by the TodS/TodT system (Rojas et al., 2003; Guazzaroni et al., 2004; 2005). In analogy to the TodS/TodT system, TtgV responds to different hydrocarbons like toluene (Guazzaroni et al., 2005). Using a transcriptional fusion of promoter PttgG to the lacZ gene, we have assessed the effect of toluene and menadione on gene expression. The β-galactosidase activity in the presence of toluene and absence of menadione was of approximately 1000 Miller units (Fig. 5A), and a similar activity was observed in the presence of 0.25 mM menadione. A reduction in expression at menadione concentrations starting from 0.5 mM was observed. We have therefore studied PtodX expression at a menadione concentration of 0.25 mM. As shown in Fig. 5B, literally no expression from PtodX occurs in the absence of toluene whereas around 10 000 Miller units were observed in its presence. However, in the presence of menadione, the toluene-mediated upregulation of PtodX activity was reduced by around one third (Fig. 5B). This demonstrates that the inhibitory effect of menadione is also observed in vivo.


Multiple signals modulate the activity of the complex sensor kinase TodS.

Silva-Jiménez H, Ortega Á, García-Fontana C, Ramos JL, Krell T - Microb Biotechnol (2014)

Expression from PttgGHI and PtodX in response to menadione.A. Estimation of the pleiotropic effect of menadione. Pseudomonas putida DOT-T1E bearing pANA96 (PttgG::’lacZ) (Rojas et al., 2003) was grown on LB medium with 1 mM of toluene in the presence of increasing menadione concentrations.B. Effect of menadione on the expression form PtodX. Pseudomonas putida DOT-T1E ΔtodST bearing pMIR66 (containing todST) and pMIR77 (PtodX::′lacZ) was grown in LB medium with 1 mM toluene in the absence or presence of 0.25 mM menadione. At an OD660 = 0.8, β-galactosidase activities were determined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321377&req=5

fig05: Expression from PttgGHI and PtodX in response to menadione.A. Estimation of the pleiotropic effect of menadione. Pseudomonas putida DOT-T1E bearing pANA96 (PttgG::’lacZ) (Rojas et al., 2003) was grown on LB medium with 1 mM of toluene in the presence of increasing menadione concentrations.B. Effect of menadione on the expression form PtodX. Pseudomonas putida DOT-T1E ΔtodST bearing pMIR66 (containing todST) and pMIR77 (PtodX::′lacZ) was grown in LB medium with 1 mM toluene in the absence or presence of 0.25 mM menadione. At an OD660 = 0.8, β-galactosidase activities were determined.
Mentions: We have then studied the influence of menadione on PtodX promoter activity. Initial experiments were aimed at determining the pleiotropic effect of menadione on gene expression. As a control, we have used the PttgG promoter that has been extensively studied in our group. It is controlled by the TtgV repressor but not by the TodS/TodT system (Rojas et al., 2003; Guazzaroni et al., 2004; 2005). In analogy to the TodS/TodT system, TtgV responds to different hydrocarbons like toluene (Guazzaroni et al., 2005). Using a transcriptional fusion of promoter PttgG to the lacZ gene, we have assessed the effect of toluene and menadione on gene expression. The β-galactosidase activity in the presence of toluene and absence of menadione was of approximately 1000 Miller units (Fig. 5A), and a similar activity was observed in the presence of 0.25 mM menadione. A reduction in expression at menadione concentrations starting from 0.5 mM was observed. We have therefore studied PtodX expression at a menadione concentration of 0.25 mM. As shown in Fig. 5B, literally no expression from PtodX occurs in the absence of toluene whereas around 10 000 Miller units were observed in its presence. However, in the presence of menadione, the toluene-mediated upregulation of PtodX activity was reduced by around one third (Fig. 5B). This demonstrates that the inhibitory effect of menadione is also observed in vivo.

Bottom Line: In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione.The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways.This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, C/ Prof. Albareda 1, Granada, 18008, Spain.

Show MeSH
Related in: MedlinePlus