title: The C-terminal domain of the novel essential protein Gcp is critical for interaction with another essential protein YeaZ of Staphylococcus aureus.
abstract: Previous studies have demonstrated that the novel protein Gcp is essential for the viability of various bacterial species including Staphylococcus aureus; however, the reason why it is required for bacterial growth remains unclear. In order to explore the potential mechanisms of this essentiality, we performed RT-PCR analysis and revealed that the gcp gene (sa1854) was co-transcribed with sa1855, yeaZ (sa1856) and sa1857 genes, indicating these genes are located in the same operon. Furthermore, we demonstrated that Gcp interacts with YeaZ using a yeast two-hybrid (Y2H) system and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on the residues of C-terminal segment of Gcp. We found that the mutations of the C-terminal Y317-F322 region abolished the interaction of Gcp and YeaZ, and the mutations of the D324-N329 and S332-Y336 regions alleviated Gcp binding to YeaZ. More importantly, we demonstrated that these key regions of Gcp are also necessary for the bacterial survival since these mutated Gcp could not complement the depletion of endogenous Gcp. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. Our findings provide new insights into the potential mechanisms and biological functions of this novel essential protein.
authors.affiliation: Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Ting
authors.initials: T
authors.lastname: Lei
authors.forename: Xudong
authors.initials: X
authors.lastname: Liang
authors.forename: Junshu
authors.initials: J
authors.lastname: Yang
authors.forename: Meiying
authors.initials: M
authors.lastname: Yan
authors.forename: Li
authors.initials: L
authors.lastname: Zheng
authors.forename: Bruce
authors.initials: B
authors.lastname: Walcheck
authors.forename: Yinduo
authors.initials: Y
authors.lastname: Ji
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D016296:Mutagenesis; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; C519716:Gcp glycoprotease, Staphylococcus aureus; D010447:Peptide Hydrolases; D000409:Alanine