Reporter proteins for in vivo fluorescence without oxygen

Drepper, Thomas; Eggert, Thorsten; Circolone, Franco; Heck, Achim; Krauß, Ulrich; Guterl, Jan‐Karl; Wendorff, Marion; Losi, Aba; Gärtner, Wolfgang; Jaeger, Karl‐Erich

doi:10.1038/nbt1293

Cited by 342 publications

(429 citation statements)

References 17 publications

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“…C62S, in contrast to C62A, cannot form the FMN triplet state (Ref. 44 and data not shown). Earlier in vivo studies of YtvA, in which the illumination conditions were not specified, showed that neither C62A nor C62S could activate B with light, not even upon overproduction of YtvA (15).…”

Section: Discussionmentioning

confidence: 99%

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Ávila-Pérez¹,

Vreede

Tang

et al. 2009

Journal of Biological Chemistry

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LOV2 domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted J␣ (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8,9). Stress via the addition of salt or ethanol (for a review, see Ref.10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13,14). YtvA, which mediates light activation of B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16). When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9,14,17,18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180 o upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central ␤-sheet and the C-terminal linker region, the J␣-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavinbinding pocket, the ␤-sheet of the LOV domain, and the GTPbinding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approac...

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Section: Discussionmentioning

confidence: 99%

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Ávila-Pérez¹,

Vreede

Tang

et al. 2009

Journal of Biological Chemistry

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“…To generate new reporter cell lines we stably transfected mouse fibroblast cells (MEF) with the reporter plasmid pNF-κB-hrGFP which has a GFP reporter gene under the control of 4 κB binding sites and a hygromycin resistance gene. Hygromycin selected cells were stimulated with TNFα [9] (R3 in Fig. 1C) or LPS (LN#) and the high responders were sorted by FACS.…”

Section: Gfp Reporter Responds Differently To Different Nf-κb Stimulimentioning

confidence: 99%

“…Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its various variants [18] now form a cornerstone of both in vivo imaging of cells and tissues and in vitro fluorescence labeling. GFP-fusion proteins can be used to analyze the expression, localization, movement, interaction of proteins in addition to studying enhancers and promoters [7,9]. GFP is unique in that the GFP fluorophore spontaneously forms intracellularly [12].…”

Section: Introductionmentioning

confidence: 99%

Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

El-Guendy

Sinai

2008

Mol Cell Biochem

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The nuclear factor-κB (NF-κB) family of transcription factors plays a central role in numerous physiological processes including development, cell survival, immunity and inflammation. We generated a series of stable clonal lines in mouse embryonic fibroblasts carrying NF-κB-GFP plasmid as a reporter. These cell lines were selected by flow cytometry for their high responsiveness to tumor necrosis factor (TNFα) or lipopolysaccharide (LPS), two classic NF-κB inducing stimuli. Although all clones were generated from the same parental cell line, they each had a distinctive pattern of response to NF-κB stimuli. While exhibiting distinct profiles with regard to the GFP reporter, analysis of endogenous NF-κB downstream targets did not always show the same variability. This suggests that in the absence of confirmation of the signaling outcomes using endogenous outputs, considerable caution must be exercised in the interpretation of data using stable reporter systems.

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“…This delay could confound the interpretation of the timing of gene expression in certain experiments. Third, in work conducted on E. coli (Drepper et al, 2010), Saccharomyces cerevisiae (Tielker et al, 2009), Candida albicans, Rhodobacter capsulatus (Drepper et al, 2007), Porphyromonas gingivalis (Choi et al, 2011), Bacteroides fragilis (Lobo et al, 2011), Arabidopsis thaliana (Chapman et al, 2008) and mammalian cells (Walter et al, 2012), FbFPs have proven to be suitable fluorophores in anoxia. To date, studies utilizing FbFPs have focused on either aerobic or anaerobic conditions.…”

Section: Introductionmentioning

confidence: 99%

“…However, the oxygen dependence of GFP has stymied efforts to visualize transcriptional dynamics in anoxia (Tsien, 1998). An alternative to GFP is the FbFP [flavin mononucleotide (FMN)-based fluorescent protein] family of fluorophores that require FMN for maturation, a common metabolic oxidizing agent, rather than oxygen (Drepper et al, 2007). One member of this family, EcFbFP, derived from the light oxygen voltage (LOV) domain of the YtvA protein from Bacillus subtilis, was codon-optimized for expression in Escherichia coli, and fluoresces in the blue spectrum in both aerobic and anaerobic conditions.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

et al. 2014

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Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates nitrogen-fixing heterocysts when available combined nitrogen is limiting. Growth under diazotrophic conditions results in a mixture of 'new' (recently differentiated) and 'old' (mature) heterocysts. The microoxic environment present in heterocysts makes the interpretation of gene expression using oxygen-dependent fluorophores, including GFP, difficult. The work presented here evaluates the transcriptional dynamics of three developmental genes in mature heterocysts utilizing EcFbFP, a flavin mononucleotide-dependent fluorophore, as the reporter. Expression of both GFP and EcFbFP from the heterologous petE promoter showed that, although GFP and EcFbFP fluoresced in both vegetative cells and new heterocysts, only EcFbFP fluoresced in old heterocysts. A transcriptional fusion of EcFbFP to the late-stage heterocyst-specific nifB promoter displayed continued expression beyond the cessation of GFP fluorescence in heterocysts. Promoter fusions of the master regulator of differentiation, hetR, and its inhibitors, patS and hetN, to GFP and EcFbFP were visualized to determine their role(s) in heterocyst function after morphogenesis. The expression of hetR and hetN was found to persist beyond the completion of development in most heterocysts, whereas patS expression ceased. These data are consistent with a model of heterocyst patterning in which patS is involved in de novo pattern formation, hetN is required for pattern maintenance, and hetR is needed for all stages of development.

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Reporter proteins for in vivo fluorescence without oxygen

Cited by 342 publications

References 17 publications

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

In Vivo Mutational Analysis of YtvA from Bacillus subtilis

Potential problems inherent in cell-based stable NF-κB–GFP reporter systems

Transcriptional dynamics of developmental genes assessed with an FMN-dependent fluorophore in mature heterocysts of Anabaena sp. strain PCC 7120

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