title: An alkaline phosphatase reporter for use in Clostridium difficile.
abstract: Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Adrianne N
authors.initials: AN
authors.lastname: Edwards
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Pascual
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Kevin O
authors.initials: KO
authors.lastname: Childress
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Nawrocki
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Woods
authors.affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA. Electronic address: shonna.mcbride@emory.edu.
authors.forename: Shonna M
authors.initials: SM
authors.lastname: McBride
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics*; D016360:Clostridioides difficile / Q000235:genetics*; D004789:Enzyme Activation; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D000469:Alkaline Phosphatase
keywords: AP; Alkaline phosphatase; BCIP; Clostridium difficile; Reporter; XP; pNP; phoZ