Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp

Rhee, Hyun‐Woo; Lee, Chang-Ro; Cho, Seung-Hyon; Song, Mi-Ryung; Cashel, Michael; Choy, Hyon E.; Seok, Young-Jae; Hong, Jong‐In

doi:10.1021/ja0759139

Cited by 92 publications

(52 citation statements)

References 26 publications

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“…The presence of (p)ppGpp was detected with a fluorescent chemosensor, PyDPA, selective for (p)ppGpp (44). To ensure that PyDPA can be used with L. pneumophila, the WT and the relA spoT double mutant were grown to exponential phase in rich medium (AYE).…”

Section: Methodsmentioning

confidence: 99%

“…To test this hypothesis, we determined the level of (p)ppGpp in the rpoS mutant. The (p)ppGpp assay was performed using PyDPA, a selective fluorescent chemosensor for (p)ppGpp (44). Extraction of nucleotides from bacterial cells was carried out with 13 M formic acid as described previously (45,46), further purified with phenol-chloroform (46), and concentrated by freeze-drying (47).…”

Section: Figmentioning

confidence: 99%

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A Regulatory Feedback Loop between RpoS and SpoT Supports the Survival of Legionella pneumophila in Water

Trigui

Dudyk

et al. 2015

Appl Environ Microbiol

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bLegionella pneumophila is a waterborne pathogen, and survival in the aquatic environment is central to its transmission to humans. Therefore, identifying genes required for its survival in water could help prevent Legionnaires' disease outbreaks. In the present study, we investigate the role of the sigma factor RpoS in promoting survival in water, where L. pneumophila experiences severe nutrient deprivation. The rpoS mutant showed a strong survival defect compared to the wild-type strain in defined water medium. The transcriptome of the rpoS mutant during exposure to water revealed that RpoS represses genes associated with replication, translation, and transcription, suggesting that the mutant fails to shut down major metabolic programs. In addition, the rpoS mutant is transcriptionally more active than the wild-type strain after water exposure. This could be explained by a misregulation of the stringent response in the rpoS mutant. Indeed, the rpoS mutant shows an increased expression of spoT and a corresponding decrease in the level of (p)ppGpp, which is due to the presence of a negative feedback loop between RpoS and SpoT. Therefore, the lack of RpoS causes an aberrant regulation of the stringent response, which prevents the induction of a successful response to starvation. L egionella pneumophila is the causative agent of Legionnaires' disease. It is widely distributed in natural freshwater systems (1) and readily colonizes man-made water systems such as cooling towers and water distribution systems (2). L. pneumophila persists in aquatic environments thanks to its ability to adapt to a variety of different ecological niches, either as an intracellular parasite of amoebae or ciliate protozoans, as a free-living member of complex biofilm communities, or as planktonic cells (3, 4). Amoebae support intracellular multiplication of L. pneumophila, and protect against suboptimal growth conditions and exposure to chlorine (5-7). Infection of HeLa cells and Tetrahymena tropicalis leads to the differentiation of L. pneumophila into mature infectious forms (MIFs), characterized by ultrastructural changes and accumulation of poly-␤-hydroxybutyrate (8-10). MIFs are highly infectious and are more resistant to antibiotics and detergent than other forms (9, 11). Increased resistance to antibiotics was also described after incubation of L. pneumophila in Acanthamoeba castellanii buffer for 16 h (12). MIFs are able to resist starvation better than stationary-phase forms in encystment buffer (11), but both forms show similar levels of resistance in distilled water (8).In the free-living state outside the amoebal host, L. pneumophila encounters stressful conditions, such as limited nutrient availability in aquatic systems (1, 13). Nevertheless, previous studies have shown that L. pneumophila is able to survive for long periods (up to 1.5 years) in sterilized tap, surface and estuarine waters (14-17). The genetic determinants underlying the ability of L. pneumophila to survive in its natural habitat of low-nutrient water for a...

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Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

A Regulatory Feedback Loop between RpoS and SpoT Supports the Survival of Legionella pneumophila in Water

Trigui

Dudyk

et al. 2015

Appl Environ Microbiol

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“…It is characterized by a monomer emission at 370-380 nm and an excimer emission at 460-480 nm. To date, many research groups have reported pyrene-based probes for metal ions, 2 phosphates 55 or phosphate-containing biomolecules, such as bacterial alarmone (p)ppGpp 56 and adenosine-5′-triphosphate (ATP). 57 …”

Section: Monomer-excimer Based Ratiometric Sensingmentioning

confidence: 99%

Small molecule-based ratiometric fluorescence probes for cations, anions, and biomolecules

2015

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Quantitative determination of specific analytes is essential for a variety of applications ranging from life sciences to environmental monitoring. Optical sensing allows non-invasive measurements within biological milieus, parallel monitoring of multiple samples, and less invasive imaging. Among the optical sensing methods currently being explored, ratiometric fluorescence sensing has received particular attention as a technique with the potential to provide precise and quantitative analyses. Among its advantages are high sensitivity and inherent reliability, which reflect the self-calibration provided by monitoring two (or more) emissions. A wide variety of ratiometric sensing probes using small fluorescent molecules have been developed for sensing, imaging, and biomedical applications. In this research highlight, we provide an overview of the design principles underlying small fluorescent probes that have been applied to the ratiometric detection of various analytes, including cations, anions, and biomolecules in solution and in biological samples. This highlight is designed to be illustrative, not comprehensive.

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“…These results suggest that DksA and ppGpp play roles in regulating cell growth under both nutrient-rich and nutrient-limited conditions. Intracellular ppGpp levels in E. amylovora WT and mutant strains were quantified in HMM by using the selective and sensitive fluorescent chemosensor PyDPA for ppGpp as reported previously (44,45). PyPDA contains pyrene (Py) and bis(Zn 2ϩ -dipicolylamine) (DPA), the latter of which is well known for its strong binding to pyrophosphate groups in water (45).…”

Section: Growth Of Erwinia Amylovora Is Regulated By Ppgpp and Dksamentioning

confidence: 99%

“…Intracellular ppGpp levels in E. amylovora WT and mutant strains were quantified in HMM by using the selective and sensitive fluorescent chemosensor PyDPA for ppGpp as reported previously (44,45). PyPDA contains pyrene (Py) and bis(Zn 2ϩ -dipicolylamine) (DPA), the latter of which is well known for its strong binding to pyrophosphate groups in water (45). We found that the ppGpp level was slightly increased (10% higher) in the spoT mutant, while the ppGpp level was about 7 times lower in the relA mutant than in the WT.…”

Section: Growth Of Erwinia Amylovora Is Regulated By Ppgpp and Dksamentioning

confidence: 99%

The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

et al. 2015

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The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp 0 ) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp 0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp 0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and ␣-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.E rwinia amylovora causes a devastating fire blight disease of apples and pears, which results in severe economic losses to growers around the world (1, 2). E. amylovora is closely related to members of the Enterobacteriaceae family, including many important human pathogens, such as Escherichia coli, Yersinia pestis, and Salmonella enterica (3). Studies have revealed that the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a major pathogenicity factor in E. amylovora (4-7). The hrp T3SS genes are carried on a pathogenicity island (8), and the alternative sigma factor HrpL, a member of the ECF subfamily of sigma factors, serves as the master regulator to control the expression of the structural and effector genes by binding to a consensus sequence known as the hrp box (9-13)...

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Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp

Cited by 92 publications

References 26 publications

A Regulatory Feedback Loop between RpoS and SpoT Supports the Survival of Legionella pneumophila in Water

A Regulatory Feedback Loop between RpoS and SpoT Supports the Survival of Legionella pneumophila in Water

Small molecule-based ratiometric fluorescence probes for cations, anions, and biomolecules

The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

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