Cloning of pProbe-NT EGFP Reporter Plasmid and Determination of Promoter Activity

The genomic region 300 bp upstream of the determined transcriptional start site (TSS) of ano was amplified by PCR (primers ECs2384-SalI-320F and ECs2384-EcoRI-40R) and restriction enzyme cut sites for SalI and EcoRI were introduced. The PCR product was cloned into the plasmid pProbe-NT (Miller et al., 2000) and transformed into E. coli Top10. The plasmid sequence was verified by Sanger sequencing (Eurofins). Overnight cultures of E. coli Top10 + pProbe-NT and E. coli Top10 + pProbe-NT-PromoterTSS were used for 1:100 inoculation of 10 ml 0.5×LB medium with 30 μg/ml kanamycin. Growth in 0.5×LB was investigated for promoter activity using the following conditions: plain medium, at pH 5, at pH 8.2, plus 400 mM NaCl, plus 0.5 mM CuCl₂, plus 2 mM formic acid or plus 2.5 mM malonic acid. Cultures were incubated at 37°C and 150 rpm until an OD₆₀₀ of 0.5 was reached (3 to 8 h, dependent on growth condition). Next, the cells were pelleted, washed once with phosphate-buffered saline (PBS) and resuspended in 1 ml PBS. The OD₆₀₀ was adjusted to 0.3 and 0.6. Four times each 200 μl bacterial suspension were pipetted in a black microtiter plate and the fluorescence was measured (Wallac Victor³, Perkin Elmer Life Science, excitation 485 nm, emission 535 nm, measuring time 1 s). The fluorescence of E. coli Top10 without plasmid was subtracted as background. Promoter activity at anaerobic conditions was determined with the following changes of the protocol: 15 ml 0.5×LB with 30 μg/ml kanamycin (investigated conditions see above) inoculated 1:100 with overnight cultures of E. coli Top10 + pProbe-NT or E. coli Top10 + pProbe-NT-PromoterTSS in tightly closed 15 ml falcon tubes were incubated at 37°C and 150 rpm until an OD₆₀₀ of 0.5 was reached (6–12 h dependent on growth condition). Subsequently, the cultures were transferred into Schott flasks and incubated for 15 min aerobically at 37°C and 150 rpm to allow the EGFP to mature. Cell harvest and measurement of fluorescence intensity was performed as described above. The experiment was performed in triplicate. Significance of changes was calculated by two-tailed Student’s t-test.