YoeB is a bacterial toxin encoded by the yefM-yoeB toxin-antitoxin system found in various bacterial genomes. Here, we show that Staphylococcus aureus contains two YoeB homologues, both of which function as ribosome-dependent mRNA interferases to inhibit translation initiation in a manner identical to that of YoeB-ec from Escherichia coli.Escherichia coli contains a number of toxin-antitoxin systems (1,12). Among these, MazF(19,20), chpBK (21), and PemK (17) are known to be sequence-specific mRNA interferases (16). Other toxins, such as RelE (4,9,14) and YoeB (3,5,18), also cleave mRNAs. RelE is a ribosome-associating factor and has no endoribonuclease activity by itself (4,9,14). Recently, we have demonstrated that YoeB is a 50S ribosome-associating factor and a potent inhibitor for translation initiation (18). It binds to the A site of the mRNA-ribosome initiation complex to block translation initiation. This results in partial cleavage of the mRNA at 3 bases downstream of the initiation codon regardless of the sequence of the mRNA. YoeB appears to have very weak intrinsic endoribonuclease activity (10), and it has been shown that this activity is not responsible for inhibition of protein synthesis (18). On the basis of these results, YoeB and RelE may be classified as ribosome-dependent mRNA interferases (16). To explore the enzymatic function of YoeB homologues in a clinically significant gram-positive species, we attempted to characterize YoeB homologues from Staphylococcus aureus. Here, we describe that YoeB-sa1 and YoeB-sa2 from S. aureus function as translation initiation inhibitors in an identical manner to that of YoeB-ec from E. coli.S. aureus is the most significant nosocomial pathogen, and methicillin-resistant S. aureus strains, which caused approximately 19,000 deaths in 2005 in the United States (2, 11), have recently emerged in the community, causing an inordinate number of skin and soft tissue infections in otherwise healthy populations (13,15).In contrast to the large array of toxin-antitoxin systems in E. coli and Mycobacterium tuberculosis (1, 12), a BLAST search identified one mazE-mazF-like operon and two yefM-yoeB-like operons in the S. aureus genome (8, 12). A MazF homologue in S. aureus (MazF-sa) has been shown to be an mRNA interferase (7), and recently its pentad recognition cleavage sequence has been determined to be UACAU (22). Its unusual abundance (43 cleavage sites) in the gene sraP, which is one of the crucial adhesive factors involved in adhesion of this pathogen to human platelets, suggests that MazF-sa may be involved in the pathogenicity of this bacterium (22). These yefM-yoeB operons in S. aureus (yefM-yoeB-sa1 and yefM-yoeB-sa2), previously identified as the axe1-txe1 and axe2-txe2 operons, respectively, were shown to be autoregulated and their transcriptions were reported to be stimulated by the addition of antibiotics, such as erythromycin and tetracycline (6).As shown in Fig. 1A, both YefM-sa1 and YefM-sa2 show significant identity (42 and 25%) and homology (57 and 46%)...