ABSTRACT: A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome by microarray at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV) and 21 days (late SCV) post-infection (PI) of Vero epithelial cells. Relative to early LCV, genes down-regulated in the SCV were primarily involved with intermediary metabolism. Up-regulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, cryo-electron microscopy revealed SCV with an unusually dense periplasmic layer that was intimately associated with the outer membrane. In contrast, LCV had a clearly distinguishable periplasm and inner/outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance. Coxiella Vero time series. Coxiella was profiled at day 3, day 5, day 7, day 14 and day 21 post-infection of Vero epithelial cells.
