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Practical observations on the use of fluorescent reporter systems in Clostridioides difficile

Oliveira Paiva, Ana M. ; Friggen, Annemieke H. ; Douwes, Roxanne ; Wittekoek, Bert ; Smits, Wiep Klaas

Antonie van Leeuwenhoek, 2022-02, Vol.115 (2), p.297-323 [Periódico revisado por pares]

Cham: Springer International Publishing

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Título:
Practical observations on the use of fluorescent reporter systems in Clostridioides difficile
Autor: Oliveira Paiva, Ana M. ; Friggen, Annemieke H. ; Douwes, Roxanne ; Wittekoek, Bert ; Smits, Wiep Klaas
Assuntos: Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biomedical and Life Sciences ; C-Terminus ; Clostridioides ; Clostridioides difficile ; Clostridioides difficile - genetics ; Cytosol ; Fluorescence ; Fluorescence microscopy ; Gene Expression Regulation, Bacterial ; Histones ; Labeling ; Life Sciences ; Medical Microbiology ; Microbiology ; Microscopy ; Original Paper ; Oxygen ; Phenotypes ; Plant Sciences ; Proteins ; Soil Science & Conservation ; Transcription
É parte de: Antonie van Leeuwenhoek, 2022-02, Vol.115 (2), p.297-323
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
Descrição: Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile , the fluorescent proteins CFP opt , mCherry Opt and phiLOV2.1, and the self-labelling tags SNAP Cd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A . However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherry Opt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile . Due to independence of molecular oxygen and fluorescent signal, SNAP Cd appears the most suitable candidate for live-cell imaging in C. difficile to date.
Editor: Cham: Springer International Publishing
Idioma: Inglês

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Practical observations on the use of fluorescent reporter systems in Clostridioides difficile

Oliveira Paiva, Ana M. ; Friggen, Annemieke H. ; Douwes, Roxanne ; Wittekoek, Bert ; Smits, Wiep Klaas

Cham: Springer International Publishing

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