Screening a library of temperature-sensitive mutants to identify secretion factors in Staphylococcus aureus

Protein secretion is an essential cell process in bacteria, required for cell envelope biogenesis, export of virulence factors, and acquisition of nutrients, among other important functions. In the Sec secretion pathway, signal peptide-bearing precursors are recognized by the SecA ATPase and pushed across the membrane through a translocon channel made of the proteins SecY, SecE, and SecG. The Sec pathway has been extensively studied in the model organism Escherichia coli, but the Sec pathways of other bacteria such as the human pathogen Staphylococcus aureus differ in important ways from this model. Unlike in E. coli, a subset of precursors in S. aureus contains a YSIRK/GXXS (YSIRK) motif in an extended signal peptide. These proteins are secreted into the cross-wall compartment bounded by invaginating septal membranes during cell division. To gain insights into the factor(s) and mechanism(s) enabling protein secretion and spatial specificity in S. aureus, we isolated and screened a collection of temperature-sensitive (ts) mutants. These efforts identified at least one secA(ts) allele as well as mutations in the secG and pepV genes. A SecA pull-down experiment identified SecDF, all ribosomal proteins, several chaperones and proteases, as well as PepV, validating the genetic screen in identifying candidate cofactors of SecA in S. aureus.IMPORTANCEAll organisms use the Sec pathway for protein secretion, and key components of this pathway are essential for viability. The discovery of conditional loss-of-function mutants played an important role in defining the genetic basis of protein secretion in model organisms. In turn, the identification of Sec components facilitated mechanistic studies and revealed general rules for protein secretion but did not answer species-specific intricacies. Gram-positive bacteria, such as Staphylococcus aureus, restrict the secretion of some proteins into the septal membranes that bind their division site at mid-cell. Here, we screen a library of conditional temperature-sensitive mutants to define components of the Sec pathway of S. aureus and factors that may regulate its activity.