Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

Zweerink, Susanne; Kallnik, Verena; Ninck, Sabrina; Nickel, Sabrina; Verheyen, Julia; Blum, M C; Wagner, Alexander; Feldmann, Ingo; Sickmann, Albert; Albers, Sonja‐Verena; Bräsen, Christopher; Kaschani, Farnusch; Siebers, Bettina; Kaiser, Markus

doi:10.1038/ncomms15352

Cited by 51 publications

(48 citation statements)

References 55 publications

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“…In order to confirm the functionality of the system we cloned the previously described esterase saci_1116 (Zweerink et al, 2017) via the NcoI and XhoI restriction site in pSVAaraFX-SH10 and pSVAaraFX-NtH10S with C-terminal Strep-tagII-10xHistag and N-terminal 10xHis-Strep-tagII, respectively. Cells were grown in Brock medium supplemented with 0.1% N-Z-Amine and dextrin or D-xylose, respectively (Supplementary Figure S5).…”

Section: Promotor Activity Assay On Different Carbon Sourcesmentioning

confidence: 99%

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression

Kolk

Wagner

et al. 2020

Front. Microbiol.

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The thermophilic archaeon Sulfolobus acidocaldarius can use different carbon sources for growth, including the pentoses D-xylose and L-arabinose. In this study, we identified the activator XylR (saci_2116) responsible for the transcriptional regulation of the pentose transporter and pentose metabolizing genes in S. acidocaldarius. A xylR deletion mutant showed growth retardation on D-xylose/L-arabinose containing media and the lack of transcription of the respective ABC transporter. In contrast to so far used promoters for expression in S. acidocaldarius, the xylR responsive promoters have a very low background activity. Finally, two XylR dependent promoters next to the long-established maltose inducible promotor were used to construct a high-throughput expression vector system for S. acidocaldarius to efficiently clone and express proteins in S. acidocaldarius.

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Section: Promotor Activity Assay On Different Carbon Sourcesmentioning

confidence: 99%

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression

Kolk

Wagner

et al. 2020

Front. Microbiol.

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“…Serine hydrolases proceed through an acyl enzyme intermediate during processing of a substrate, thus a range of serine reactive electrophiles can be used to covalently target these enzymes 17 . In fact, flurophosphonate-based probes have been used to globally profile serine hydrolases in animal tissues 11 , 18 , human cell lines 19 , the pathogenic bacteria Mycobacterium tuberculosis 20 , 21 and Vibrio cholerae 22 and in archaea 23 . Here, we describe a functional proteomic screen in live S. aureus under biofilm-promoting conditions using the serine-reactive activity-based probe (ABP) fluorophosphonate-tetramethylrhodamine (FP-TMR) 12 .…”

Section: Introductionmentioning

confidence: 99%

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

et al. 2018

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Serine hydrolases play diverse roles in regulating host-pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here, we describe a chemical proteomic screen that identified 10 previously uncharacterized S. aureus serine hydrolases that mostly lack human homologues. We termed these enzymes Fluorophosphonate-binding hydrolases (FphA-J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of the fphB gene confirms that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduces infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

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“…Also, like host-associated communities, environmental communities have seen limited application of ABPP. ABPP has been used to study axenic cultures of environmental microbes such as nitrifying bacteria, extremophilic archaea, and cellulolytic bacteria (Chauvigne-Hines et al 2012; Bennett et al 2016; Zweerink et al 2017; Sadler et al 2014; Ansong et al 2014). Because ABPP does not require the ability to genetically manipulate organisms or the use of antibodies, it may prove especially useful in the study of microbial communities where those tools are often not available.…”

Section: Biological Applications For Abppmentioning

confidence: 99%

Activity-Based Protein Profiling—Enabling Multimodal Functional Studies of Microbial Communities

Whidbey

Wright

2018

Current Topics in Microbiology and Immunology

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Microorganisms living in community are critical to life on Earth, playing numerous and profound roles in the environment and human and animal health. Though their essentiality to life is clear, the mechanistic underpinnings of community structure, interactions, and functions are largely unexplored and in need of function-dependent technologies to unravel the mysteries. Activity-based protein profiling offers unprecedented molecular-level characterization of functions within microbial communities and provides an avenue to determine how external exposures result in functional alterations to microbiomes. Herein, we illuminate the current state and prospective contributions of ABPP as it relates to microbial communities. We provide details on the design, development, and validation of probes, challenges associated with probing in complex microbial communities, provide some specific examples of the biological applications of ABPP in microbes and microbial communities, and highlight potential areas for development. The future of ABPP holds real promise for understanding and considerable impact in microbiome studies associated with personalized medicine, precision agriculture, veterinary health, environmental studies, and beyond.

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Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

Cited by 51 publications

References 55 publications

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression

Identification of XylR, the Activator of Arabinose/Xylose Inducible Regulon in Sulfolobus acidocaldarius and Its Application for Homologous Protein Expression

Identification of a S. aureus virulence factor by activity-based protein profiling (ABPP)

Activity-Based Protein Profiling—Enabling Multimodal Functional Studies of Microbial Communities

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