LacZ fusion and β-galactosidase assay

The promoter-proximal DNA region of each target gene was cloned into the corresponding restriction endonuclease sites of pHRP309, which harbors a promoterless lacZ reporter gene and a gentamicin resistance gene [22]. The recombinant plasmid was subsequently transferred into the WT strain and the deletion mutants to measure the β-galactosidase activities of the cellular extracts using a β-Galactosidase Enzyme Assay System (Promega, USA) according to the manufacturer’s instructions [15, 20]. Briefly, the assay was performed by adding 30 μL of diluted sample to an equal volume of assay 2 × buffer that containing the substrate o-nitrophenyl-β-d-galactopyranoside. Samples were incubated for approximately 30 min, during which time the β-galactosidase hydrolyzes the colorless substrate to o-nitrophenol, which is yellow. The reaction was terminated by the addition of 90 μL sodium carbonate, and the absorbance was read at OD₄₂₀ and OD₅₅₀ with a spectrophotometer. The number of Miller units (representing the galactosidase activity) was calculated using the following formula: 10⁶ × [(OD₄₂₀ − 1.75 × OD₅₅₀)/(T × V × OD₆₀₀)]. The Miller units represent the change in the OD₄₂₀/min/ml relative to the OD₆₀₀ of the cells.