Abstract
The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.
Highlights
Rhodamine B (RB) is a xanthene dye commonly used in the textile industry [1], and it is used as a stain in microbiology [2,3], histology and pathology applications [4,5]
Our detailed study of this unexpected discovery revealed that the presence of rhodamine B in the screening medium and a metagenomicderived rsfp gene expression in E. coli cells are the key factors for the pink fluorescence occurring under screening conditions
Expression and purification of soluble RSFP protein In our previous study [11], we demonstrated that the rhodamine B presence in the growth media and the expression of rsfp gene in E. coli cells is crucial for pink fluorescence of the E. coli colonies under UV illumination (l = 312 nm)
Summary
Rhodamine B (RB) is a xanthene dye commonly used in the textile industry [1], and it is used as a stain in microbiology [2,3], histology and pathology applications [4,5]. In 1987, Kouker and Jaeger presented a new plate assay dedicated to the detection of lipase producing bacterial species and to quantify lipase activity in culture supernatants [7], based on a medium containing olive oil and the fluorescent dye rhodamine B. The sources of this fluorescence are complexes of rhodamine B with the free fatty acids released from the olive oil by lipases To date, this plate assay is commonly used for detection of lipolytic bacterial strains [8] and for screening of recombinant bacterial colonies with lipolytic activity in genomic DNA or metagenomic DNA libraries [9], respectively. We carried out crystallographic studies of native RSFP protein and RSFP protein crystal soaked with RB
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