One-Step High-Throughput Assay for Quantitative Detection of β-Galactosidase Activity in Intact Gram-Negative Bacteria, Yeast, and Mammalian Cells

Vidal-Aroca, Faustino; Giannattasio, Michele; Brunelli, Elisa; Vezzoli, Alessandro; Plevani, Paolo; Muzi-Falconi, Marco; Bertoni, Giovanni

doi:10.2144/000112145

Cited by 49 publications

(41 citation statements)

References 20 publications

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“…Following incubation, the optical density (600 nm) and the fluorescence intensity (excitation, 360 nm; emission, 460 nm) were measured using a Synergy HT Biotek plate reader. ␤-Galactosidase activity was normalized (24,25). The experiment was performed in triplicate and was repeated three times independently.…”

Section: Methodsmentioning

confidence: 99%

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP, Which Contribute to Type I Interferon Induction, Play a Role in Cell Wall Stress

Zeevi

Shafir

Shaham

et al. 2013

Journal of Bacteriology

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The intracellular bacterial pathogen Listeria monocytogenes activates a robust type I interferon response upon infection. This response is partially dependent on the multidrug resistance (MDR) transporter MdrM and relies on cyclic-di-AMP (c-di-AMP) secretion, yet the functions of MdrM and cyclic-di-AMP that lead to this response are unknown. Here we report that it is not MdrM alone but a cohort of MDR transporters that together contribute to type I interferon induction during infection. In a search for a physiological function of these transporters, we revealed that they play a role in cell wall stress responses. A mutant with deletion of four transporter genes (⌬mdrMTAC) was found to be sensitive to sublethal concentrations of vancomycin due to an inability to produce and shed peptidoglycan under this stress. Remarkably, c-di-AMP is involved in this phenotype, as overexpression of the c-di-AMP phosphodiesterase (PdeA) resulted in increased susceptibility of the ⌬mdrMTAC mutant to vancomycin, whereas overexpression of the c-di-AMP diadenylate cyclase (DacA) reduced susceptibility to this drug. These observations suggest a physiological association between c-di-AMP and the MDR transporters and support the model that MDR transporters mediate c-di-AMP secretion to regulate peptidoglycan synthesis in response to cell wall stress.

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Section: Methodsmentioning

confidence: 99%

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP, Which Contribute to Type I Interferon Induction, Play a Role in Cell Wall Stress

Zeevi

Shafir

Shaham

et al. 2013

Journal of Bacteriology

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“…Reporter gene assays were conducted using a method similar to that published previously (Vidal-Aroca et al 2006;Nelson et al 2013). Bacterial cell cultures were initiated from a single colony and grown overnight in 3 mL of medium (lysogeny broth, LB) with the appropriate antibiotics at 37°C with shaking.…”

Section: -Methylumbelliferyl β-D-galactopyranoside (4-mug) Assay Formentioning

confidence: 99%

The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

Hwang

Stav

et al. 2016

RNA

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The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or "azaaromatics," trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.

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“…Because ONPG hydrolysis is rate limited by its passive diffusion through the OM, we chose to test this hypothesis using a modied b-gal assay on whole E. coli cells stained with DSSN+. 43,[48][49][50] Cells were therefore incubated with varying concentrations of DSSN+ for 5 hours, washed, and then monitored for ONP production as a function of time. The time dependence of the background-corrected A 420 nm versus time provides a measure of product formation (Fig.…”

Section: Whole-cell Hydrolysismentioning

confidence: 99%

Membrane permeabilization by conjugated oligoelectrolytes accelerates whole-cell catalysis

2016

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One-Step High-Throughput Assay for Quantitative Detection of β-Galactosidase Activity in Intact Gram-Negative Bacteria, Yeast, and Mammalian Cells

Cited by 49 publications

References 20 publications

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP, Which Contribute to Type I Interferon Induction, Play a Role in Cell Wall Stress

Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP, Which Contribute to Type I Interferon Induction, Play a Role in Cell Wall Stress

The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

Membrane permeabilization by conjugated oligoelectrolytes accelerates whole-cell catalysis

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