To explore the role and possible mechanism of WalK（S221P）mutation in promoting capsule production of Staphylococcus aureus（S. aureus）.

RNA sequencing（RNA-seq）was performed to detect the effect of WalK（S221P）mutation on gene expression of S. aureus strain XN108. RT-qPCR was used to verify the capsule gene variation caused by WalK（S221P）mutation. Capsule staining and transmission electron microscopy（TEM）were employed to determine capsule biosynthesis of S. aureus. The possible regulatory genes involved in WalK（S221P）regulation were screened by using RT-qPCR. The binding site of WalKR on the regulatory regions of mgrA gene was predicted, and electrophoresis mobility shift assay（EMSA）was conducted to detect the direct binding activity of WalK-activated WalR on the mgrA DNA probe.

RNA-seq revealed that the expression of 196 genes altered after WalK（S221P）mutation in S. aureus XN108, and among these genes, 16 genes involved in capsule biosynthesis were upregulated remarkably. RT-qPCR confirmed the upregulation of capA, capH, and capK in the WalK（S221P）-carried S. aureus strains, including XN108 and K-Newman. Capsule staining showed that XN108 had more capsule production than its counterpart XN108-R. TEM observation demonstrated that XN108 had thicker capsule than XN108-R. RT-qPCR indicated an increasing expression of mgrA gene in the WalK（S221P）-carried XN108 when compared with that in XN108-R. A putative binding site of WalR on the regulatory regions of mgrA gene was predicted, and the direct binding activity of WalK-activated WalR on the mgrA regulatory fragment was verified by EMSA.

WalK（S221P）promotes the production of S. aureus capsule, and the underlying mechanism might be associated with the upregulation of MgrA, a positive regulator for S. aureus capsule biosynthesis.

To construct a new fusion luciferase-labeled Staphylococcus aureus（S. aureus）tracing system.

An S. aureus tracer strain was constructed by using DNA homologous recombination technique. The gene encoding fusion luciferase Antares2 was knocked in the genome of S. aureus strain USA300 by fusing with the enolase（Eno）encoding gene. The bioluminescence intensity was analyzed by mixing with the tracer strain and different substrates. The suitable tracer/substrate system was selected based on the detection of bioluminescence intensity. The sensitivity of the tracer/substrate system in vitro and in vivo was also evaluated.

The knock-in plasmid pBT2-eno-antares2 was constructed. After transformed into S. aureus USA300, the Antares2 knock-in strain was screened out. The tracer strain USA300/Eno-Antares2 was identified by DNA sequencing as well as Western blot detection of Eno-Antares2 fusion proteins. Bacterial growth curve and hemolytic activity tests showed comparable results between USA300/Eno-Antares2 tracer strain and its wild-type USA300. USA300/Eno-Antares2 catalyzed substrates diphenylterazine（DTZ）, furimazine（FUR）, and hydrofurimazine（HFZ）to produce macroscopical bioluminescence. Analysis of different substrates at diverse concentrations revealed that USA300/Eno-Antares2/HFZ system exhibited the best performance. This system enabled reliable bioluminescence tracking of S. aureus with sensitivities of approximately 50 colony forming unit（CFU）in vitro and 100 CFU in vivo.

S. aureus USA300/Eno-Antares2/HFZ tracing system is a good tool, which can be used for the study of S. aureus infection, colonization, and dissemination.