G-protein control of the ribosome-associated stress response protein SpoT

Abstract

The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.

FIG. 1.

SpoT associates with ribosomes in E. coli. Cell lysates from MG1655 were sedimented through 7 to 47% sucrose gradients, and the samples were monitored by UV at 254 nm. The subsequent fractions were analyzed by immunoblotting using anti-SpoT, anti-CgtA, or anti-L3, as indicated. The positions of the 30S and 50S subunits, the 70S monosome, and the polysomes are labeled. WT, MG1655 cell extract; Δ, ΔspoT ΔrelA cell extract; L, 1/100 of the total sample loaded onto the gradient.

FIG. 2.

Ribosome association of SpoT and CgtA is mutually independent. (A, C, and D) Cell lysates from CF1693 (ΔspoT::cat ΔrelA::kan) (A), JM3867 (MG1655 plus P_BAD-cgtA) with CgtA expression induced with 0.001% arabinose (C), and JM3907 (ΔcgtA::kan plus P_cgtA-cgtAG80ED85N) (D) were sedimented through 7 to 47% sucrose gradients, and the subsequent fractions were subjected to immunoblotting using anti-CgtA and anti-SpoT antibodies, as indicated. The positions of the 30S and 50S subunits, the 70S monosome, and the polysomes are labeled. WT, MG1655 cell extract; Δ, ΔspoT ΔrelA cell extract; L, 1/100 of the total sample loaded onto the gradient. (B) Immunoblot showing the level of CgtA expression from JM3867 (MG1655 plus P_BAD-cgtA) with various levels of arabinose, as indicated.

FIG. 3.

Neither CgtA nor SpoT associates with the 100S ribosomal particle that accumulates in stationary phase. Overnight-grown E. coli MG1655 cells were diluted 1:100, grown in EP medium for 24 h, lysed, and fractionated on sucrose gradients. The ribosome profile is shown with the positions of the 30S, 50S, 70S, and 100S particles indicated. Fractions were analyzed by immunoblotting with anti-SpoT, anti-CgtA, or anti-S4, as indicated. WT, MG1655 cell extract; Δ, ΔspoT ΔrelA cell extract; L, 1/100 of the total sample loaded onto the gradients.

FIG. 4.

Ribosome association of CgtA and SpoT under amino acid and carbon starvation conditions. Cell lysates from E. coli MG1655 cells grown in MOPS minimal medium (A), MOPS medium treated with SH (1 mg/ml) for 20 min before harvest (B), and MOPS medium treated with α-methylglucoside (2.6%) for 2 min before harvest (C) were fractionated over 7 to 47% sucrose gradients. The subsequent fractions were analyzed by immunoblotting with anti-SpoT or anti-CgtA (1/2,000), as indicated. The positions of the 30S, 50S, and 70S particles and the polysomes are labeled. WT, MG1655 cell extract; Δ, ΔspoT ΔrelA cell extract; L, 1/100 of the total sample loaded onto the gradients.

FIG. 5.

(p)ppGpp steady-state level is increased in a cgtA mutant. (A) JM3903 (ΔcgtA::kan plus P_cgtA-cgtA) and JM3907 (ΔcgtA::kan plus P_cgtA-cgtAG80ED85N) cells were grown at 30°C in low-phosphate medium and uniformly ³²P_i labeled (100 μCi/ml) before being treated with SH (1 mg/ml). Samples were taken immediately before the addition and at 10-min intervals thereafter, as indicated. The formic acid-extracted nucleotides were resolved by one-dimensional thin-layer chromatography on polyethyleneimine-cellulose sheets, autoradiographed, and quantified with a phosphorimager. Unlabeled ATP and GTP were spotted on the plates as markers. (A) Autoradiograms of polyethyleneimine thin-layer chromatography plates with or without SH treatment (20-min time point). Lanes 1 and 2 are JM3903 without and with SH, respectively; lanes 3 and 4 are JM3907 without and with SH, respectively. (B) (p)ppGpp levels normalized according to the GTP levels. Open and closed symbols are nontreated and SH-treated JM3903 (triangles) and JM3907 (squares) samples, respectively. Error bars are standard deviations from triplicate experiments.

FIG. 6.

A cgtA relA spoT triple mutant is not viable. GN5002 (ΔcgtA::kan plus P_BAD-cgtA), JM4977 (ΔrelA ΔspoT::cat), and JM4981 (ΔrelA ΔspoT::cat cgtA::kan plus P_BAD-cgtA) were streaked on LB plates containing chloramphenicol (Cm) with or without 0.1% arabinose (Ara) at 30°C, as indicated.