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

Abstract:

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.