Construction of Transcriptional lacZ Fusions and β-Galactosidase Assay

The BPROM prediction server for bacterial promoters (Solovyev and Salamov, 2011) predicted two possible promoters (Supplementary Figure S3) forming two probable promoter regions, a bigger (L) and a smaller (S) one. Both regions were amplified using primers listed in Supplementary Table S3, digested with XhoI and HindIII restriction enzymes, and inserted into the reporter plasmid pSU11 (Malott et al., 2009). The resulting plasmids (pSU11/Lprom-lacZ; pSU11/Sprom-lacZ) were transferred to P. fluorescens Q2-87 by triparental mating using the helper plasmid (pRK2013). The resulting transconjugants were verified with two sets of primers, the forward and reverse primers used for amplification of promoter regions, and with a set including the forward primer from the amplification and reverse primer specific for the pSU11 plasmid (Supplementary Table S3).

The β-galactosidase assay was performed as previously described with minor modifications (Miller and Mekalanos, 1988). Briefly, cells were harvested and resuspended in Z-buffer (see appendix, 1 mL), and the OD₆₀₀ was measured. Cells were lysed using chloroform (25 μL), sodium dodecyl sulfate (SDS; 25 μL, 0.01%) and briefly vortexed. The resulting mixture was incubated for 10 min at 28°C. The reaction was initiated by adding ortho-Nitrophenyl-β-galactosidase (ONPG; 200 μL, 10 mg/mL), and stopped with Na₂CO₃ (500 μL, 1 M). Cell debris was removed by centrifugation (10 min, 15,000 rpm) and the absorbance at 420 nm was measured. The β-galactosidase activity was calculated using the following equation: