DegU负调控地衣芽胞杆菌普切明酸的合成及分泌

Pulcherriminic acid synthesis and secretion are negatively regulated by DegU in Bacillus licheniformis

普切明酸是地衣芽胞杆菌合成并分泌的一种铁离子螯合剂,是细胞维持铁稳态的重要介质。【目的】揭示转录调控因子DegU在调控普切明酸的合成及分泌过程中的作用。【方法】以地衣芽胞杆菌DW2为出发菌株,构建degU缺失菌株DW2ΔdegU和过表达菌株DW2::Pbay-degU,通过产物检测、转录水平检测、凝胶阻滞分析和GFP报告蛋白表达分析等方法分析DegU对普切明酸合成、分泌及调控因子基因的转录调控机制。【结果】DW2ΔdegU的普切明酸产量相比于DW2提高了56.8%,而DW2::Pbay-degU相比于DW2菌株则下降83.7%。同时,degU缺失后,普切明酸合成酶基因yvmC和转运蛋白基因yvmA的转录水平分别上升为DW2的2.85倍和2.71倍,yvmC和yvmA的负调控因子基因yvmB的转录水平则下降为DW2的0.35倍;而在DW2::Pbay-degU菌株中,yvmC和yvmA的转录水平分别下降为DW2的0.47倍和0.24倍,yvmB的转录水平则上升为DW2的1.78倍。凝胶阻滞分析和GFP报告蛋白表达分析表明,DegU可以直接与PyvmC和PyvmB启动子结合,但是与yvmA基因启动子没有直接相互作用。【结论】DegU通过2种模式调控普切明酸合成基因簇的转录,DegU可以通过直接与yvmC-cypX基因簇启动子结合对其进行负调控,另一方面,DegU也可以通过直接激活普切明酸合成的负调控因子基因yvmB的表达对yvmC-cypX和普切明酸转运蛋白基因yvmA起到间接负调控的作用。

Pulcherriminic acid synthesized and secreted by Bacillus licheniformis is an iron chelator that helps to maintain iron homeostasis by removing Fe3+from the environment.[Objective]This study is to investigate the regulatory mechanisms of DegU in the synthesis and secretion of pulcherriminic acid.[Methods]Based on the native strain B.licheniformis DW2 and its derivative strains DW2ΔdegU(degU deletion)and DW2::Pbay-degU(degU overexpression),this study excavated the regulatory mechanisms of DegU in the synthesis and secretion of pulcherriminic acid by determining pulcherriminic acid content,real-time quantitative polymerase chain reaction(RT-qPCR),electrophoretic mobility shift assay(EMSA),and green fluorescent protein(GFP)expression assay.[Results]The pulcherriminic acid yield of DW2ΔdegU was 56.8%higher than that of DW2,while the yield of DW2::Pbay-degU was 83.7%lower than that of DW2.In addition,after degU deletion,compared with the condition of DW2,the transcriptional levels of pulcherriminic acid synthetase genes yvmC and transporter gene yvmA increased to 2.85 and 2.71 times,respectively,whereas the transcriptional level of yvmB,another negative regulator gene of yvmC and yvmA,decreased to 0.35 times.In DW2::Pbay-degU,the transcription levels of yvmC and yvmA reduced to 0.47 and 0.24 times,respectively,while the transcription level of yvmB rose to 1.78 times that of DW2.EMSA and GFP expression assay showed that DegU could directly bind to PyvmC and PyvmB promoters,but had no direct interaction with the promoter of yvmA(PyvmA).[Conclusion]DegU regulated the synthesis and secretion of pulcherriminic acid in two ways:on the one hand,DegU negatively regulated yvmC by directly binding to the promoter of yvmC-cypX cluster;on the other hand,DegU activated the expression of yvmB and negatively regulated yvmC-cypX and yvmA in an indirect way.