FIG 5.

Analysis of inducers and substrates of pump SrpABC. (A) Proposed upper pathway for the conversion of biphenyl in strain B6-2. cis-Dihydrodiol can be spontaneously transformed into 2-hydroxybiphenyl (2-HBP) or 3-hydroxybiphenyl (3-HBP), which cannot be degraded by BphB (biphenyl dehydrogenase). In contrast, 2-HBP and 3-HBP can be catalyzed by BphA (biphenyl dioxygenase). BphC, 2,3-dihydroxybiphenyl-1,2-dioxygenase; BphD, hydrolase. (B) Analysis of P_srpA promoter activity. When the optical density of the cells was approximately 0.8, BP and intermediates dissolved in N,N-dimethylformamide (DMF) were added individually at a concentration of 2 mM, except for 0.5 mM 3-HBP. After 2 h, β-galactosidase activity was measured. (C) Mutants were grown in LB medium at 30°C for 14 h with 200 rpm shaking. The cells were washed and resuspended in BP medium with 3 mM BP at a final OD₆₂₀ of 4.0 at 30°C with 150 rpm shaking. After 4 h, the samples were taken and the amounts of 2,3-DHBP were determined by HPLC. (D) The mutants were grown overnight, diluted 100-fold in LB medium, and incubated with different concentrations of 2,3-DHBP with 200 rpm shaking at 30°C. After 12 h, bacterial growth was monitored by measuring the optical density at 620 nm. All experiments were performed with triplicate samples.