title: Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis.
abstract: In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies.
authors.affiliation: Instituto de Microbiologia Paulo de Gois, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Leandro A
authors.initials: LA
authors.lastname: Lobo
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Smith
authors.forename: Edson R
authors.initials: ER
authors.lastname: Rocha
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D001441:Bacteroides fragilis / Q000235:genetics*; D002460:Cell Line; D003067:Coenzymes / Q000378:metabolism*; D005285:Fermentation; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D008264:Macrophages / Q000382:microbiology; D008320:Maltose / Q000378:metabolism; D051379:Mice
chemical_list: D003067:Coenzymes; D008164:Luminescent Proteins; D008320:Maltose; D005486:Flavin Mononucleotide; D005947:Glucose