Mechanism of pyocyanin abolishment caused by double knockout in PAO1.

MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by double knockout mutant of PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by or complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of and also led to the lower expression of and . Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.