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RpoS regulates genes associated with development of the small cell variant developmental form of Coxiella burnetii

ABSTRACT: Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from replicating (exponential phase) large cell variant (LCV) forms to a non-replicating, (stationary phase) small cell variant (SCV) forms. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary phase growth in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to C. burnetii. To characterize RpoS function during C. burnetii growth and developmental, we constructed a C. burnetii ∆rpoS mutant to define the effects on intracellular and axenic growth, as well as, gene regulation during SCV development. C. burnetii ∆rpoS exhibited intracellular growth defects in J774 mouse macrophage-like cells, but not in Vero epithelial cells. RNA sequencing of C. burnetii ∆rpoS revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Genes previously shown to have increased expression during SCV generation, including the expression of the SCV-specific protein ScvA and pathways involved in oxidative stress, arginine transport, and peptidoglycan remodeling pathways, were dysregulated in the rpoS mutant. These genes were enriched for a predicted RpoS-binding site. These data were corroborated with independent assays demonstrating that the C. burnetii rpoS mutant had increased sensitivity to hydrogen peroxide and carbenicillin. Collectively, these results demonstrate that RpoS is essential for the regulation of genes involved in SCV development and growth inside macrophage-like cells.

ORGANISM(S): Coxiella burnetii

PROVIDER: GSE110091 | GEO | 2019/02/08

REPOSITORIES: GEO

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Project description:RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA and rpoE) and genes in metabolic pathways (e.g., lysA, lysC and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in LB media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media. Experiment Overall Design: A precise rpoS deletion mutant of MG1655 was constructed using the red recombinase method. Wild type and rpoS mutants were inoculated in triplicate into M63 glucose (0.2%) minimal media at a starting OD of 0.0001 and grown aerobically at 37oC. Cultures were harvested at OD600= 0.3 in exponential phase and at OD600= 1.5 in stationary phase. For RNA extraction, cultures were mixed directly with a boiling lysis buffer containing SDS and EDTA followed by acidic hot phenol (65C) to minimize RNA degradation. RNA samples were hybridized to Affymetrix E. coli Antisense Genome Array according to Affymetrix's standard protocols.

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RpoS regulates genes associated with development of the small cell variant developmental form of Coxiella burnetii

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