Characterization of a Stress-Induced Alternate Sigma Factor, RpoS, of Coxiella burnetii and Its Expression during the Development Cycle

Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C. burnetii. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue. The rpoS gene was cloned by complementation of an Escherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY:: lacZ). Expression of C. burnetii rpoS was regulated by growth phase in E. coli (induced upon entry into stationary phase). A glutathione S-transferase‐RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment. Coxiella burnetii is an obligate intracellular bacterium that has developed a unique strategy to permit multiplication and survival in the phagolysosome of eukaryotic host cells. The life cycle of C. burnetii is incompletely characterized, but it has at least two morphologically and physiologically distinct participants, large-cell variants (LCV) and small-cell variants (SCV) (7, 14, 28, 31, 38). These two cell populations can be purified to near homogeneity by equilibrium centrifugation in 32% cesium chloride (14, 50). LCV appear to be similar to typical gramnegative bacteria, as they appear during exponential phase of growth, with a clearly distinguishable outer membrane, periplasmic space, cytoplasmic membrane, and diffuse nucleoid, attaining lengths exceeding 1 mm. In contrast, SCV are 0.2 to 0.5 mm in diameter, with electron-dense, condensed chromatin and condensed cytoplasm. SCV are resistant to osmotic shock, oxidative stress, heat shock, sonication, and pressure, unlike the more fragile LCV (1, 2, 13, 30). Differences in resistance to breakage by osmotic and pressure stress were employed to suggest that LCV have greater metabolic activity than SCV based on their ability to transport and evolve labeled carbon dioxide from [ 14 C]glucose and [ 14 C]glutamate when incubated in axenic media (30). These two cell variants have also been shown to differentially express several proteins. The histonelike protein Hq-1 (14), and a small (;4.5-kDa) basic peptide, ScvA (R. A. Heinzen, R. A., D. Howe, L. P. Mallavia, and T. Hackstadt, presented at the 11th Sesqui-Annual Meeting of the American Society for Rickettsiology and Rickettsial Diseases, St. Simons Island, Georgia, 1994), were detected only in SCV. Elongation factor Tu (EF-Tu) was detected only in LCV, while EF-Ts (45) and the major outer membrane protein P1 (29) were both dramatically upregulated in LCV. These observations were the basis for recently proposed models of C. burnetii development (15, 40). In these models, we speculated that LCV and SCV function like logarithmicphase and stationary-phase bacteria, respectively (40). In Escherichia coli, the transition to an altered physiological state is mediated by a global regulator of gene expression, s s (or RpoS), encoded by the rpoS gene. RpoS is a sigma subunit that confers promoter specific transcriptional initiation by RNA polymerase to genes that are expressed during stationary phase. Although associated with the onset of stationary phase, RpoS is also upregulated in response to various stress conditions. RpoS