Functional characterization of the transporter YdgF1 in Bacillus licheniformis 9945a

Abstract:

[Objective] To characterize the function of the transporter encoded by ydgF1 in Bacillus licheniformis 9945a. [Methods] The strain 9945a/pHY300-Shu-ydgF1 with ydgF1 being overexpressed and the strain 9945a ΔydgF1 with ydgF1 knocked out were constructed. Phosphate d-alanine (PDA) medium with d-alanine as the sole nitrogen source was designed to observe the growth ability of strains, and cell assimilating experiments were implemented. Real-time quantitative polymerase chain reaction (RT-qPCR) evaluated the relative expression of ydgF1 in 9945a and 9945a ΔydgF1 at different growth phases in the LB medium. Colony forming units (CFU) of 9945a and 9945a ΔydgF1 at the anaphase in the LB medium were determined by viable plate counting. [Results] In the PDA medium, the specific growth rate of 9945a ΔydgF1 was always lower than that of 9945a, while the maximum specific growth rate of 9945a/pHY300-Shu-ydgF1 was 0.336 h⁻¹, which was 1.98 times that of 9945a/pHY300-Shu. OD₆₀₀ of 9945a/pHY300-Shu-ydgF1 was 3.04 after culturing for 15 h, which was 1.73 times that of 9945a/pHY300-Shu. In cell assimilating experiments, the concentration of d-alanine assimilated by 9945a ΔydgF1 was (0.509±0.055) g/L, which was significantly lower than (0.759±0.038) g/L assimilated by 9945a. 9945a/pHY300-Shu-ydgF1 assimilated (0.821± 0.021) g/L, slightly higher than 9945a. The results of RT-qPCR showed the relative expression of ydgF1 in 9945a increased gradually at the transition and stationary phases, and the relative expression of ydgF1 in 9945a ΔydgF1 was almost zero at all different growth phases. CFU of 9945a and 9945a ΔydgF1 at the anaphase suggested that the knockout of ydgF1 weakened the viability of B. licheniformis 9945a in the anaphase. [Conclusion] YdgF1 assimilates d-alanine in B. licheniformis 9945a, which is beneficial to the long-term survival of B. licheniformis 9945a. In addition, it may also assimilate l-asparagine.