Determination of essentiality and regulatory function of staphylococcal YeaZ in branched- chain amino acid biosynthesis

The staphylococcal YeaZ is highly conserved in prokaryotic cells and critical for growth of many bacterial pathogens. However, the essentiality for Staphylococcus aureus growth and the biological function of YeaZ behind its essentiality remain undefined. In this study, we created and characterized a defined Pspac-regulated yeaZ expression mutant in S. aureus and demonstrated the indispensability of YeaZ for S. aureus growth. Moreover, we conducted complementation studies, not only confirmed the requirement of YeaZ for S. aureus growth, but also revealed a similarity of essential function between staphylococcal YeaZ and its E. coli homolog. On the other hand, we explored the biological functions of YeaZ and found that YeaZ is involved in the regulation of the transcription of ilv-leu operon that encodes key enzymes responsible for the biosynthesis of the branched-chain amino acids, including isoleucine, leucine, and valine (ILV). qPCR analysis showed that the 6-fold downregulation of YeaZ dramatically elevated approximately 17- to 289-fold RNA levels of ilvD, leuA and ilvA. We further confirmed the transcriptional regulation of the ilv-leu operon by YeaZ using an ilv-promoter-lux reporter system and gel-shift assays and revealed that YeaZ is able to bind the promoter region of ilv. Furthermore, we established that the regulation of ILV biosynthesis isn’t associated with YeaZ’s essentiality, as the deletion of the ilv-leu operon did not affect the requirement of YeaZ for growth in culture. Our results demonstrate the essentiality of YeaZ for S. aureus growth and suggest that the staphylococcal YeaZ possesses regulatory function.