title: A Fluorescent Reporter for Single Cell Analysis of Gene Expression in Clostridium difficile.
abstract: Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.
authors: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal.
authors: Carolina Piçarra
authors: CP
authors: Cassona
authors: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal.|Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, University of Vienna, Althanstr. 14, 1090, Vienna, Austria.
authors: Fátima
authors: F
authors: Pereira
authors: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal.
authors: Mónica
authors: M
authors: Serrano
authors: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, Estação Agronómica Nacional, 2780-157, Oeiras, Portugal. aoh@itqb.unl.pt.
authors: Adriano O
authors: AO
authors: Henriques
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000693:Anaerobiosis / Q000235:genetics; D001483:Base Sequence; D001593:Benzyl Compounds / Q000737:chemistry / Q000378:metabolism; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000447:Aldehydes; D001593:Benzyl Compounds; C092350:FM 4-64; D005456:Fluorescent Dyes; D007211:Indoles; D010455:Peptides; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C111472:mitotracker green FM; C064976:O(6)-benzylguanine; C007293:DAPI; D006147:Guanine; D003596:Cytosine; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: CLIP-tag; Clostridium difficile; Gene expression; SNAP-tag; Single-cell analysis