Role of the Twin-Arginine Translocation Pathway in Staphylococcus

In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.The Sec pathway is the major secretion system that exports the majority of extracytosolic proteins in pro- and eukaryotes. Proteins are translocated through this pathway in a more or less unfolded state. A second protein export pathway was identified first in the chloroplast thylakoid membrane (8) and later in several bacteria (4, 14, 35). This pathway has been designated the twin-arginine translocation system (Tat), as the preproteins targeted to this pathway carry a characteristic amino acid motif, including two consecutive arginine residues, which are essential for the recognition by the Tat translocon. The Tat pathway operates independently of the Sec pathway and exports exoproteins across the bacterial cytoplasmic membrane, apparently in a fully folded conformation (3). Many of these proteins are complexed with cofactors.Studies of several bacterial species, including Escherichia coli (37), Bacillus subtilis (22, 23), Pseudomonas aeruginosa (31), Legionella pneumophila (10, 11), and Mycobacterium smegmatis (29), have demonstrated that they possess a functional Tat export pathway. In E. coli, the TatA, TatB, and TatC proteins have been demonstrated to be essential for Tat-dependent protein translocation (4). However, several bacterial and archaeal species lack a TatB-like protein. For example, the B. subtilis genome encodes three TatA- and two TatC-like proteins. Thus, at least one copy of the TatA homologue and one copy of the TatC homologue are required for a functional Tat pathway. In Bacillus subtilis, several proteins were predicted that could potentially use the Tat pathway, as their signal peptides (SPs) contain RR or KR motifs. However, proteomic analysis revealed that 13 proteins with potential RR/KR SPs were Tat independent, showing that the Tat machinery does not recognize their RR/KR motifs. In fact, only the phosphodiesterase PhoD and the newly identified YwbN protein were shown to be secreted in a strictly Tat-dependent manner (22). YwbN is part of the YwbLMN operon product and is involved in the uptake of free ferric iron (32).For staphylococci, very little information regarding the function of the Tat system exists. A tatC-deficient mutant showed little difference from the wild type (WT) in its extracellular protein pattern (48). In another study, green fluorescent protein (GFP) was fused with the SP of the E. coli TorA protein and the subcellular localization of the hybrid protein was compared in Staphylococcus carnosus and its tatC mutant (30). The results showed that GFP was secreted in neither the WT nor the tatC mutant but was stuck in the cell wall fraction of the WT. However, no Tat-transported protein has been identified in staphylococci so far.Here we show that the tat operon encoding TatA and TatC is present in Staphylococcus aureus, S. carnosus, and Staphylococcus haemolyticus but is missing in a number of other staphylococcal species. By comparative analysis of WT and tatAC mutant strains, the functionality of the Tat pathway in S. carnosus and S. aureus was demonstrated, and it was found that the iron-dependent peroxidase (FepB) is translocated by the Tat system. The Tat system can efficiently translocate heterologous proteins such as lipase or protein A when it is fused with the SP of FepB. ⁵⁵Fe transport experiments demonstrated that FepABC is involved in iron uptake.