A CsrA-Binding, -Acting sRNA of Is Necessary for Optimal Intracellular Growth and Vacuole Formation during Early Infection of Host Cells.

is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of One of them, CbsR12 (mall NA ), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant CsrA-2, but not CsrA-1, proteins Moreover, through a combination of and cell culture assays, we identified several in mRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation of transcripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of transcripts coding for -adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of transcripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion of -containing vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of a -acting sRNA of and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identified -acting sRNAs that interacts with CsrA. Regulation of metabolism and virulence in is not well understood. Here, we show that small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, in In addition, we demonstrate that CbsR12 binds to CsrA-2 and induces autoaggregation and biofilm formation when transcribed ectopically in , consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.