Coxiella burnetii RpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth.

Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponentialphase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneurnophila, which has a developmental cycle superficially similar to that of C. burnetii. Here, we used a C. burnetii Delta rpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii Delta rpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCVspecific protein ScvA. Genes comprising the dot/km locus, responsible for production of the Dot/lcm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii Delta rpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth. IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.