Bacterial two-hybrid analysis identified the Staphylococcus aureus RNA degradosome-like complex to include RNase J1, RNase J2, RNase Y, polynucleotide phosphorylase (PNPase), enolase, phosphofructokinase, and a DEAD box RNA helicase. Results also revealed that the recently recognized RNase RnpA interacts with the S. aureus degradosome and that this interaction is conserved in other Gram-positive organisms.Escherichia coli bulk RNA degradation is mediated by a holoenzyme complex, the RNA degradosome, which includes the ribonucleases RNase E and polynucleotide phosphorylase (PNPase), an RNA helicase, RhlB, and the glycolytic enzyme enolase (3,20). RNase E is considered the key component of the E. coli degradosome; it catalyzes the initiation of mRNA degradation and serves as a scaffold for the assembly of the other degradosome components (2). By comparison, most Gram-positive bacteria do not contain an RNase E amino acid ortholog and much less is known about their mRNA degradation machinery. Nonetheless, recent studies suggest that the Gram-positive organism Bacillus subtilis produces two RNase E functional orthologs, ribonucleases J1 (RNase J1) and J2 (RNase J2) (17). Using bacterial two-hybrid analyses, both enzymes have been shown to interact with an RNA degradosome-like complex consisting of PNPase, enolase, and a DEAD box RNA helicase (CshA), as well as phosphofructokinase and RNase Y (6).Staphylococcus aureus is a Gram-positive bacterial pathogen of immense health care concern that is evolutionarily similar, but divergent, from the Bacillus genus. Herein we used bacterial two-hybrid and biochemical analyses to measure interactions between putative members of the S. aureus RNA degradosome and compare the results to those with B. subtilis. Results revealed that although many interactions are conserved between both bacterial species, differences are likely to exist. Moreover, we show that the RNase RnpA interacts with components of the S. aureus RNA degradosome and that this interaction is conserved within B. subtilis.Using bacterial two-hybrid analyses, interactions between putative members of the B. subtilis RNA degradosome were recently measured and used to develop the first model of the Gram-positive holoenzyme complex (Fig. 1A) (6, 13). According to the model, the E. coli RNase E orthologs RNase J1 and RNase J2 interact with one another; RNase J1 also interacts with two additional ribonucleases, PNPase and RNase Y, as well as with the glycolytic enzyme phosphofructokinase, each of which binds to the RNA helicase CshA (Fig. 1A). CshA also binds to a second glycolytic enzyme, enolase. Although the mechanism of B. subtilis cellular RNA degradation has yet to be established, RNases J1 and J2 are bifunctional ribonucleases with endonuclease and 5Ј33Ј exoribonuclease activities (8,16,24). In complex, the enzymes act synergistically and may initiate 5Ј mRNA digestion and/or generate endonucleolytic products that could subsequently be digested by RNA helicase-facilitated 3Ј35Ј PNPase exoribonuclease activity (14,1...