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Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation.

Riboldi GP, Bierhals CG, Mattos EP, Frazzon AP, d'Azevedo PA, Frazzon J - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis.The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions.Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Cocos Gram-positivos e Microbiologia Molecular, Departamento de Microbiologia, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brasil.

ABSTRACT
The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

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Related in: MedlinePlus

: comparative critical threshold (∆∆Ct) data after normalisation relativeto the expression profiles of the constitutive genes gyrase beta chain(gyrB) (upper and lower left panels) or RNA polymerase betachain (rpoB) (upper and lower right panels). Graphicsrepresent experiments performed with the Enterococcus faecalisX1 strain upon increasing conditions of iron depletion [2’,2’-dipyridyl (DIP):0.2-1.0 mM; upper left and right panels] or oxidative stress [hydrogen peroxide(H2O2): 2.0-40 mM; lower left and right panels]. Error bars are also includedto indicate data variance.
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f03: : comparative critical threshold (∆∆Ct) data after normalisation relativeto the expression profiles of the constitutive genes gyrase beta chain(gyrB) (upper and lower left panels) or RNA polymerase betachain (rpoB) (upper and lower right panels). Graphicsrepresent experiments performed with the Enterococcus faecalisX1 strain upon increasing conditions of iron depletion [2’,2’-dipyridyl (DIP):0.2-1.0 mM; upper left and right panels] or oxidative stress [hydrogen peroxide(H2O2): 2.0-40 mM; lower left and right panels]. Error bars are also includedto indicate data variance.

Mentions: Oxidative stress enhances sufCDSUB expression dramatically - To studythe expression pattern of the E. faecalis SUF operon under challengingconditions, cultures were subjected to oxidative stress by being exposed to increasingquantities of H2O2 and were separately grown under iron limitationwith increasing concentrations of the iron chelator, DIP. As demonstrated in Fig. 3, qPCR revealed extremely negative∆∆Ct values for all H2O2 concentrations tested.These values were statistically significant when compared to the control group andindicated a substantial transcriptional increase of these genes, as illustrated byrelative gene expression (2-∆∆Ct) in the heat-map shown in Fig. 4. kat, a gene whose expressionpattern is indicative of oxidative stress, was included in the experiment as a positivecontrol to indicate this growth challenge. As expected, kat expressionlevels were up regulated in all conditions tested, which confirmed that the cells wereunder oxidative stress. The sufCDSUB genes showed a significanttranscriptional induction in all H2O2 concentrations tested, withan up to 10,000-fold increase in gene expression, when compared to control growthconditions (Fig. 4).


Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation.

Riboldi GP, Bierhals CG, Mattos EP, Frazzon AP, d'Azevedo PA, Frazzon J - Mem. Inst. Oswaldo Cruz (2014)

: comparative critical threshold (∆∆Ct) data after normalisation relativeto the expression profiles of the constitutive genes gyrase beta chain(gyrB) (upper and lower left panels) or RNA polymerase betachain (rpoB) (upper and lower right panels). Graphicsrepresent experiments performed with the Enterococcus faecalisX1 strain upon increasing conditions of iron depletion [2’,2’-dipyridyl (DIP):0.2-1.0 mM; upper left and right panels] or oxidative stress [hydrogen peroxide(H2O2): 2.0-40 mM; lower left and right panels]. Error bars are also includedto indicate data variance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155840&req=5

f03: : comparative critical threshold (∆∆Ct) data after normalisation relativeto the expression profiles of the constitutive genes gyrase beta chain(gyrB) (upper and lower left panels) or RNA polymerase betachain (rpoB) (upper and lower right panels). Graphicsrepresent experiments performed with the Enterococcus faecalisX1 strain upon increasing conditions of iron depletion [2’,2’-dipyridyl (DIP):0.2-1.0 mM; upper left and right panels] or oxidative stress [hydrogen peroxide(H2O2): 2.0-40 mM; lower left and right panels]. Error bars are also includedto indicate data variance.
Mentions: Oxidative stress enhances sufCDSUB expression dramatically - To studythe expression pattern of the E. faecalis SUF operon under challengingconditions, cultures were subjected to oxidative stress by being exposed to increasingquantities of H2O2 and were separately grown under iron limitationwith increasing concentrations of the iron chelator, DIP. As demonstrated in Fig. 3, qPCR revealed extremely negative∆∆Ct values for all H2O2 concentrations tested.These values were statistically significant when compared to the control group andindicated a substantial transcriptional increase of these genes, as illustrated byrelative gene expression (2-∆∆Ct) in the heat-map shown in Fig. 4. kat, a gene whose expressionpattern is indicative of oxidative stress, was included in the experiment as a positivecontrol to indicate this growth challenge. As expected, kat expressionlevels were up regulated in all conditions tested, which confirmed that the cells wereunder oxidative stress. The sufCDSUB genes showed a significanttranscriptional induction in all H2O2 concentrations tested, withan up to 10,000-fold increase in gene expression, when compared to control growthconditions (Fig. 4).

Bottom Line: In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis.The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions.Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Cocos Gram-positivos e Microbiologia Molecular, Departamento de Microbiologia, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS, Brasil.

ABSTRACT
The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

Show MeSH
Related in: MedlinePlus