An examination of an Antarctic soil metagenomic-derivate putative methylthioadenosine phosphorylase gene as a novel reporter gene for promoter trapping.

The reporter genes are widely used in promotertrapping vectors for easy and rapid identifi cation of the putative promoter sequences in cloned DNA fragments and in some cases to measure the promoter strength. In this aim, the reporter genes such as a gfp gene from Aequorea victoria encoding Green Fluorescent Protein (Miller et al., 2000; Pothier et al., 2007; Rasko et al., 2007), a lacZ gene encoding E. coli ß-galactosidase (Fan et al., 2009; Li et al., 2007; Zhang et al., 2007), and a luxAB gene encoding Vibrio fi scheri luciferase (Aoki et al., 2002; Kunert et al., 2000) were used. Moreover, the genes coding for antibiotic resistance were utilized in screening for the promoter sequences; e.g., a cat gene encoding chloramphenicol acetyltransferase was applied in the isolation of lactococcal (Jeong et al., 2006), mycobacterial (Das Gupta et al., 1993), cyanobacterial (Marraccini et al., 1993) and streptococcal (Kili et al., 1999) promoter sequences. Although the genes mentioned above are commonly used in many promoter-trapping vectors, scientists are still looking for new ones dedicated for searching out promoters in particular hosts. Cui et al. (2004) reported the successful using of the methyl parathion hydrolase gene from Plesiomonas sp. strain M6 as a reporter gene to clone the promoter sequences, with different promoting strength, derived from the Bacillus subtilis 168 genomic DNA. The rsfp gene was discovered during examination of an Antarctic soil-derived metagenome library and encodes a bacterial methylthioadenosine phosphorylase (GenBank accession number GQ202582). In a previous study, we found that E. coli colonies expressing the rsfp gene reveal strong pink fl uorescence after exposure to UV light when grown on culture medium supplemented with rhodamine B (Cie li ski et al., 2009). In contrast to that, the colonies of the E. coli strains, defi cient in the rsfp gene, do not show any fl uorescence under the same experimental conditions. We found that the reason for the fl uorescence phenomenon of E. coli cells expressing the rsfp gene is the binding of the fl uorescent dye rhodamine B (abbrev. RB) molecules on RSFP protein molecules that leads to the increase in the fl uorescence yield of RB molecules excited by UV light and the accumulation of RB molecules in E. coli cells during colony growth (manuscript in preparation). Based on these results we decided to examine the potential of the rsfp gene as a novel reporter gene for identifying the location of the putative promoter sequences in the E. coli TOP10F′ genome fragments J. Gen. Appl. Microbiol., 58, 387‒395 (2012)