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.