Characterization of a novel essential protein Gcp in Staphylococcus aureus

Abstract

The prevalence of multi-drug resistant Staphylococcus aureus, especially methicillin- and vancomycin-resistant S. aureus has caused serious public health concerns. The limited options for the treatment of multidrug resistant S. aureus infections highlight the urgent need for the identification of novel target and the development of new classes antimicrobial agents. The highly conserved gcp operon is a potential target, as it is essential for all tested bacteria. However, the mechanism behind this essentiality is not clear. In this study, we constructed defined Pspac-regulated gcp or yeaZ expression mutant in wild type MRSA strains and demonstrated the essentiality of Gcp and YeaZ for S. aureus growth in culture. Moreover, we demonstrated that Gcp interacts with YeaZ using both a yeast two-hybrid and a bacterial two-hybrid system, and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on charged or polarized amino acids of the C-terminal segment of Gcp and revealed that the C-terminal Y317-F322 region was critical for Gcp binding to YeaZ as well as for the essentiality of Gcp for growth. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. In addition, we demonstrated that Gcp and YeaZ play important roles in the regulation of branched-chain amino acids biosynthesis pathway. Specifically, using qPCR and an ilv-promoter luciferase reporter fusion, we found that the depletion of Gcp or YeaZ dramatically increased the transcription of the ilv-leu operon that encodes key enzymes responsible for the biosynthesis of branched-chain amino acids. Moreover, gel shift analyses showed that Gcp lacked a DNA-binding capacity, whereas YeaZ was able to bind the ilv promoter region, indicating that Gcp indirectly, but YeaZ directly control the transcription of ilv-leu operon. Furthermore, we found that Gcp and YeaZ are involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A). In addition, the depletion of Gcp or YeaZ enhanced bacterial growth in culture medium lacking ILV. To elucidate whether the tight regulation of branched-chain amino acids biosynthesis pathway contributed to the essentiality of Gcp and YeaZ, we created an ilv-leu operon deletion mutant using the defined Pspac-regulated gcp or yeaZ expression strains and found that the deletion of ilv-leu operon had no impact on the requirement of Gcp and YeaZ for bacterial growth. The above data indicate that the essential nature of Gcp and YeaZ is not attributable to their repression of the ilv-leu operon. These new findings provide new insights into the biological function of these essential proteins, Gcp and YeaZ, as well as the regulatory mechanism of branched-chain amino acids biosynthesis.