Acyl carrier protein/SpoT interaction, the switch linking SpoT-dependent stress response to fatty acid metabolism.
Bacteria respond to nutritional stresses by producing an intracellular alarmone, guanosine 5'-(tri)diphosphate, 3'-diphosphate [(p)ppGpp], which triggers the stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, upon fatty acid or carbon starvation, SpoT enzyme activity switches from (p)ppGpp degradation to (p)ppGpp synthesis, but the signal and mechanism for this response remain totally unknown. Here, we characterize for the first time a physical interaction between SpoT and acyl carrier protein (ACP) using affinity co-purifications and two-hybrid in E. coli. ACP, as a central cofactor in fatty acid synthesis, may be an ideal candidate as a mediator signalling starvation to SpoT. Accordingly, we show that the ACP/SpoT interaction is specific of SpoT and ACP functions because ACP does not interact with the homologous RelA protein and because SpoT does not interact with a non-functional ACP. Using truncated SpoT fusion proteins, we demonstrate further that ACP binds the central TGS domain of SpoT, consistent with a role in regulation. The behaviours of SpoT point mutants that do not interact with ACP reveal modifications of the balance between the two opposite SpoT catalytic activities thereby changing (p)ppGpp levels. More importantly, these mutants fail to trigger (p)ppGpp accumulation in response to fatty acid synthesis inhibition, supporting the hypothesis that the ACP/SpoT interaction may be involved in SpoT-dependent stress response. This leads us to propose a model in which ACP carries information describing the status of cellular fatty acid metabolism, which in turn can trigger the conformational switch in SpoT leading to (p)ppGpp accumulation.
Battesti
Aurélia
A
LISM, IBSM, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille, France.
Bouveret
Emmanuelle
E
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000096:biosynthesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D000213:Acyl Carrier Protein; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D006150:Guanine Nucleotides; C027627:guanosine-3',5'-bis(diphosphate) 3'-pyrophosphatase; D011755:Pyrophosphatases