Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction

Abstract

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.

FIG. 1.

Genetic context of acp genes (A) and spoT, relA, and rsh genes (B) in E. coli, P. aeruginosa, S. pneumoniae, and B. subtilis. acp genes are red, and rel and spo genes are yellow. In each panel, genes that are conserved in the genetic context are blue. The gene designations described previously or the gene annotations are indicated, and the designations that we used in the present study are indicated in parentheses. The designations for the relA genes of S. pneumoniae and B. subtilis are misleading, and these genes were designated rsh_Spn or rsh_Bsu in this study to avoid confusion. The acpA gene of B. subtilis is unique and is designated acp_Bsu.

FIG. 2.

Expression of the recombinant proteins. (A) T18Flag-ACPs. Strain C600 was transformed with the pT18Flag series of plasmids containing the indicated acp genes. After induction for 3 h with 0.5 mM IPTG in LB medium at 37°C, the recombinant proteins were detected by Western blotting using anti-Flag M2 antibody. (B) T18-SpoT, -RelA, and -Rsh proteins. Strain C600 was transformed with the pT18Flag series of plasmids containing the indicated relA, spoT, and rsh genes. After induction for 3 h with 0.5 mM IPTG in LB medium at 37°C, the recombinant proteins were detected by far Western blotting with biotinylated calmodulin and by Western blotting using anti-RelA antibodies. (C) T18Flag- and T25Flag-RelP-like and -RelQ-like proteins. Strain C600 was transformed with the pT18Flag and pT25Flag series of plasmids containing the indicated genes. After induction for 3 h with 0.5 mM IPTG in LB medium at 37°C, the recombinant proteins were detected by Western blotting using anti-Flag M2 antibody.

FIG. 3.

Functionality of the recombinant ACPs. The MG1655acp(Ts) strain was transformed with the indicated pT18Flag-acp plasmids, plated on LB medium plates containing ampicillin, and incubated at 30°C for 3 days (A) or at 42°C for 36 h (B).

FIG. 4.

Functionality of the recombinant SpoT, RelA, and Rsh proteins. (A) Complementation of CF1693. Strain CF1693 (ΔrelA ΔspoT) was transformed with the indicated plasmids. Clones selected on LB agar plates containing ampicillin were replicated on M9 minimal agar plates containing ampicillin without amino acids (MM-AA) and incubated for 48 h at 37°C together with strain CF1652 (ΔrelA spoT⁺) transformed with pT18 as a positive control. (B) Complementation of CF4943. Strain CF4943 (ΔrelA spoT203) was transformed with the indicated plasmids. Clones selected on LB agar plates containing ampicillin were replicated on M9 minimal agar plates containing ampicillin supplemented with amino acids (40 μg/ml each) (MM+AA) and incubated for 24 h at 37°C together with strain CF4941 (ΔrelA spoT⁺) transformed with pT18 as a positive control.

FIG. 5.

Interactions between ACP and the RelA, SpoT, and Rsh proteins determined by bacterial two-hybrid analysis. Interactions between the ACPs fused to the T18Flag domain and the RelA, SpoT, and Rsh proteins fused to the T25 domain were assayed by the two-hybrid method as described in Materials and Methods. Black cells, strong interaction; gray cells, interaction; −, no interaction; ND, not determined. The β-galactosidase activity values (in Miller units) are indicated for the significant interactions.

FIG. 6.

Response to amino acid starvation determined by a rapid (p)ppGpp synthesis assay using SHX medium. Labeling was performed as described in Materials and Methods. Autoradiographs of the thin-layer chromatography plates are shown. In each case, a control experiment with the wild-type MG1655 strain transformed with pT18 was performed (wt). For each experiment, the results obtained at three time points (5, 10, and 15 min) are shown. (A) Strain CF1652 (ΔrelA spoT⁺) transformed with the pT18-relA_Eco, pT18-relA_Pae, pT18-rsh_Spn, and pT18-rsh_Bsu plasmids. (B) Strain CF1693 (ΔrelA ΔspoT) transformed with the pT18-spoT_Eco, pT18-spoT_Pae, pT18-rsh_Spn, and pT18-rsh_Bsu plasmids. (C) Strain CF1652 (ΔrelA spoT⁺) transformed with the pT18-relQ_Bsu, pT18-relP_Bsu, pT18-relQ_Spn, pT18-relA_Eco, and pT18-spoT_Eco plasmids as controls.