The Essentiality of Staphylococcal Gcp Is Independent of Its Repression of Branched-Chain Amino Acids Biosynthesis

Ting Lei, Junshu Yang, Li Zheng, Todd Markowski, Bruce A. Witthuhn, Yinduo Ji

Our previous studies revealed that the staphylococcal protein Gcp is essential for bacterial growth; however, the essential function of Gcp remains undefined. In this study, we demonstrated that Gcp plays an important role in the modulation of the branched-chain amino acids biosynthesis pathway. Specifically, we identified that the depletion of Gcp dramatically elevated the production of key enzymes that are encoded in the ilv-leu operon and responsible for the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine (ILV) using proteomic approaches. Using qPCR and promoter-lux reporter fusions, we established that Gcp negatively modulates the transcription of the ilv-leu operon. Gel-shift assays revealed that Gcp lacks the capacity to bind the promoter region of ilv. Moreover, we found that the depletion of Gcp did not influence the transcription level of CodY, a known repressor of the ilv-leu operon, while induced the transcription of CcpA, a known positive regulator of the ilv-leu operon. In addition, the depletion of Gcp decreased the biosynthesis of N(6)-threonylcarbamoyladenosine (t6A). To elucidate whether the essentiality of Gcp is attributable to its negative modulation of ILV biosynthesis, we determined the impact of the ilv-leu operon on the requirement of Gcp for growth, and revealed that the deletion of the ilv-leu operon did not affect the essentiality of Gcp. Taken together, our results indicate that the essentiality of Gcp isn't attributable to its negative regulation of ILV biosynthesis in S. aureus. These findings provide new insights into the biological function of the staphylococcal Gcp.