Selective and sensitive colorimetric detection of stringent alarmone ppGpp with Fenton-like reagent

Zheng, Lin Ling; Huang, Cheng

doi:10.1039/c4an01632g

“…To directly test whether polyP, through complexing iron, mitigates the Fenton reaction, we monitored the H 2 O 2 /FeSO 4 -mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) ( 42 ) in the presence of increasing amounts of polyP ( Fig. 5A ).…”

Section: Resultsmentioning

confidence: 99%

“…The ABTS assay was performed as previously described in reference 42 . In brief, 250 μM ABTS was mixed with 5 μM FeSO 4 and 200 μM H 2 O 2 in the absence or presence of increasing concentrations of polyP 300 (see figure legends for details).…”

Section: Methodsmentioning

confidence: 99%

Polyphosphate Functions In Vivo as an Iron Chelator and Fenton Reaction Inhibitor

Beaufay

¹

,

Quarles

²

,

Franz

³

et al. 2020

mBio

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Maintaining cellular iron homeostasis is critical for organismal survival. Whereas iron depletion negatively affects the many metabolic pathways that depend on the activity of iron-containing enzymes, any excess of iron can cause the rapid formation of highly toxic reactive oxygen species (ROS) through Fenton chemistry. Although several cellular iron chelators have been identified, little is known about if and how organisms can prevent the Fenton reaction. By studying the effects of cisplatin, a commonly used anticancer drug and effective antimicrobial, we discovered that cisplatin elicits severe iron stress and oxidative DNA damage in bacteria. We found that both of these effects are successfully prevented by polyphosphate (polyP), an abundant polymer consisting solely of covalently linked inorganic phosphates. Subsequent in vitro and in vivo studies revealed that polyP provides a crucial iron reservoir under nonstress conditions and effectively complexes free iron and blocks ROS formation during iron stress. These results demonstrate that polyP, a universally conserved biomolecule, plays a hitherto unrecognized role as an iron chelator and an inhibitor of the Fenton reaction. IMPORTANCE How do organisms deal with free iron? On the one hand, iron is an essential metal that plays crucial structural and functional roles in many organisms. On the other hand, free iron is extremely toxic, particularly under aerobic conditions, where iron rapidly undergoes the Fenton reaction and produces highly reactive hydroxyl radicals. Our study now demonstrates that we have discovered one of the first physiologically relevant nonproteinaceous iron chelators and Fenton inhibitors. We found that polyphosphate, a highly conserved and ubiquitous inorganic polyanion, chelates iron and, through its multivalency, prevents the interaction of iron with peroxide and therefore the formation of hydroxyl radicals. We show that polyP provides a crucial iron reservoir for metalloproteins under nonstress conditions and effectively chelates free iron during iron stress. Importantly, polyP is present in all cells and organisms and hence is likely to take on this crucial function in both prokaryotic and eukaryotic cells.

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“…In vitro Fenton reaction and DNA damage assay -The ABTS assay was performed as previously described in (Zheng & Huang, 2014). In brief, 250 µM ABTS was mixed with 5 µM FeSO4 and 200 µM H2O2 in the absence or presence of increasing concentrations of polyP 300 (see figure legends for details).…”

Section: Methodsmentioning

confidence: 99%

“…To directly test whether polyP, through complexing iron, mitigates the Fenton reaction, we monitored the H2O2 / FeSO4 -mediated oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Zheng & Huang, 2014) in the presence of increasing amounts of polyP (Fig. 5A).…”

Section: Polyp Protects Against Fe-mediated Dna Damage In Vitromentioning

confidence: 99%

Polyphosphate FunctionsIn Vivoas Iron Chelator and Fenton Inhibitor

Beaufay¹,

Quarles²,

Franz³

et al. 2020

Preprint

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Maintaining cellular iron homeostasis is critical for organismal survival. Whereas iron depletion negatively affects the many metabolic pathways that depend on the activity of iron-containing enzymes, any excess of iron can cause the rapid formation of highly toxic reactive oxygen species (ROS) through Fenton chemistry. Although several cellular iron chelators have been identified, little is known about if and how organisms can prevent the Fenton reaction. By studying the effects of cisplatin, a commonly used anticancer drug and effective antimicrobial, we discovered that cisplatin elicits severe iron stress and oxidative DNA damage in bacteria. We found that both of these effects are successfully prevented by polyphosphate (polyP), an abundant polymer consisting solely of covalently linked inorganic phosphates.Subsequent in vitro and in vivo studies revealed that polyP provides a crucial iron reservoir under nonstress conditions, and effectively complexes free iron and blocks ROS formation during iron stress.These results demonstrate that polyP, a universally conserved biomolecule, plays a hitherto unrecognized role as an iron chelator and an inhibitor of the Fenton reaction.

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“…[14,15] Some colorimetric and fluorescent probes have also been developed. [16][17][18][19][20][21] However, even though these chemosensors enable real-time monitoring of in vitro ppGpp synthesis using E. coli ribosomal complex, they are limited for solution-based detection. There is still a void in detecting ppGpp in live cells, especially at the single-cell level for studying cell-to-cell variations.…”

mentioning

confidence: 99%

Live-Cell Imaging of (p)ppGpp with RNA-based Fluorescent Sensors

Sun

¹

,

Wü

²

,

Zhao

³

et al. 2021

Preprint

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Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to directly image and measure cellular (p)ppGpp. Here, we report a type of RNA-based fluorescent sensors for live-cell imaging of (p)ppGpp. Our sensor is engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels can now be obtained under different nutritional conditions. We predict that these RNA-based sensors will be broadly adapted to study bacterial stringent response.

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Selective and sensitive colorimetric detection of stringent alarmone ppGpp with Fenton-like reagent

Cited by 14 publications

References 48 publications

Polyphosphate Functions In Vivo as an Iron Chelator and Fenton Reaction Inhibitor

Polyphosphate Functions In Vivo as an Iron Chelator and Fenton Reaction Inhibitor

Polyphosphate FunctionsIn Vivoas Iron Chelator and Fenton Inhibitor

Live-Cell Imaging of (p)ppGpp with RNA-based Fluorescent Sensors

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