Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

Abstract

The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress response or "stringent" response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the stringent response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA+ CgtA+ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the stringent response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.

Fig. 1.

Dependence on arabinose for growth. Indicated strains were grown on LB/0.1% arabinose (A and C) or LB only (B and D–F) and spotted as 10-fold serial dilutions from left to right. (A and B) DR209/pDR291. (C and D) DR210/pDR308. (E) DR214. (F) N16961ΔrelAΔspoT.

Fig. 2.

Down-regulated genes in common in CgtA depletion and stringent response induced cells. (A) Complete set of down-regulated genes. (B) Genes involved in protein synthesis, protein fate, and energy metabolism. Open, genes down-regulated because of CgtA depletion; shaded, genes down-regulated because of treatment with serine hydroxamate; hatched, overlap between these gene sets.

Fig. 3.

CgtA depletion causes increased ppGpp concentration. (A) Strain DR209/pDR291 [ΔcgtA/ P_BAD::cgtA] was arabinose-depleted for 20 min, then cells were harvested and analyzed for nucleotide content by TLC. Mock-depleted cultures were cells washed and reinoculated into LB/0.1% arabinose. The ppGpp concentration is represented as the percentage of ppGpp of the amount of GTP and ppGpp combined. (B) Strain N16961Sm treated with 20 mM serine hydroxamate or an equal volume of dH₂O. The ppGpp concentration was determined as in A.

Fig. 4.

Simplified model for the essential CgtA activity. Nutritional stress induces RelA or SpoT to generate ppGpp by phosphorylation of GTP. SpoT is required to hydrolyze ppGpp to prevent growth inhibition. CgtA is required to maintain normal SpoT ppGpp hydrolysis activity.