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 selleck screening library 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. The use of fluorescent proteins in biomedical research started over 10 years ago (Chalfie et al., 1994). Since then, fluorescent proteins proved to be extremely useful as reporter tools in several cellular processes such as tracking protein movements in the cell, monitoring mitochondrial redox potential and transcriptional reporters (Wachter, 2006). In bacteria, green fluorescent proteins (GFPs) can be used to survey microorganisms in complex biological systems such as biofilms, soil and to visualize interactions of bacteria with plant or animal host
tissues (Rosochacki & Matejczyk, 2002; Larrainzar et al., 2005; Hoppe et al., 2009; Chudakov et al., 2010). Furthermore, Inhibitor Library cell line GFPs can be transcriptionally and translationally fused to bacterial genes and expressed in vivo as an alternative to immunofluorescence. It can also be
used to examine the function and localization of the gene products (Margolin, 2000). Currently, GFPs are a cornerstone tool used in in vivo imaging, fluorescence resonance energy transfer and quantitative transcriptional analysis. Several GFP-like derivatives have been engineered for better fluorescence and photostability P-type ATPase (Heim et al., 1995) as well as different color emissions (Shaner et al., 2007). However, in the catalytic formation of the chromophore, GFP requires the presence of molecular oxygen (Heim et al., 1994), thus rendering the protein colorless in anaerobic environments, making GFP unsuitable for use as a reporter gene in obligate anaerobic organisms. Recent efforts to create a protein reporter for in vivo labeling and fluorescence either in the presence or in the absence of oxygen led to development of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) (Drepper et al., 2007, 2010). Commercial FbFPs are derived from the blue-light photoreceptors YtvA from Bacillus subtilis and SB2 from Pseudomonas putida that contain the light oxygen voltage (LOV) domains. The LOV domains were first identified in plant phototrophins (Huala et al., 1997) where they regulate several physiological processes such as phototropism, chloroplast relocation and stomatal opening (Briggs & Christie, 2002; Celaya & Liscum, 2005).