地衣芽孢杆菌9945a转运蛋白YdgF1的功能鉴定

Functional characterization of the transporter YdgF1 in Bacillus licheniformis 9945a

摘要:

【目的】本研究对地衣芽孢杆菌(Bacillus licheniformis) 9945a菌株中ydgF1基因编码的转运蛋白进行功能鉴定。【方法】分别构建ydgF1基因过表达株9945a/pHY300-Shu-ydgF1和敲除株9945aΔydgF1，设计了以d-丙氨酸为唯一氮源的磷酸盐d-丙氨酸(phosphate d-alanine, PDA)培养基来考察菌株的生长能力，并进行细胞吸收实验。利用实时荧光定量PCR (real-time quantitative polymerase chain reaction, RT-qPCR)评价菌株9945a与9945aΔydgF1在LB培养基中不同生长时期ydgF1的相对表达量，然后对9945a与9945aΔydgF1后期的培养基平板活菌计数，计算菌落形成单位(colony-forming units, CFU)。【结果】在PDA培养基中，9945aΔydgF1的比生长速率始终低于9945a，而9945a/pHY300-Shu-ydgF1最大比生长速率为0.336 h^–1，是9945a/pHY300-Shu的1.98倍，并且培养15 h后9945a/pHY300-Shu-ydgF1的OD₆₀₀值为3.04，是9945a/pHY300-Shu的1.73倍。细胞吸收实验中，9945a ΔydgF1吸收d-丙氨酸的浓度为(0.509±0.055) g/L，明显低于9945a吸收的(0.759±0.038) g/L。9945a/pHY300-Shu-ydgF1吸收浓度为(0.821±0.021) g/L，略高于9945a。实时荧光定量PCR的结果表明，9945a的ydgF1相对表达量在转换期和稳定期逐渐提高，9945aΔydgF1的ydgF1相对表达量在不同生长时期几乎都为0。9945a与9945a ΔydgF1后期的CFU显示ydgF1的敲除减弱了地衣芽孢杆菌9945a后期的生存能力。【结论】YdgF1在地衣芽孢杆菌9945a中参与d-丙氨酸吸收，有利于地衣芽孢杆菌9945a的长期生存。此外还发现YdgF1可能参与l-天冬酰胺吸收。

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.