The Energy‐Dependent Degradation of Guanosine 5′‐Diphosphate 3′‐Diphosphate in Escherichia coli

Abstract

Different inhibitors of the energy metabolism have been assayed in Escherichia coli K12 for their ability to increase the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) as a consequence of a restriction of its degradation. Inhibitors of the respiration and uncouplers of oxidative phosphorylations had effects similar to carbon-source-induced downshifts while the ATPase inhibitor dicyclohexylcarbodiimide was less efficient. The effects of dicyclohexylcarbodiimide and of the uncoupler carbonylcyanide p-fluoro methoxyphenylhydrazone (FCCP) on ppGpp degradation were compared in a drug-permeable envelope mutant. At concentrations of inhibitors sufficient to deplete the pool of ATP by 50 %, only FCCP was able to block ppGpp degradation. Moreover, FCCP also inhibited ppGpp degradation in a ATPase-deficient strain growing on glucose as carbon source while, as expected, it did not change the level of ATP. It is concluded, according to Mitchell's chemiosmotic hypothesis, that, in vivo, the integrity of the transmembrane proton gradient rather than the ATP pool size is a prerequisite for the normal processing of the energy-dependent degradation of ppGpp.Numerous functions in the bacterial cell are presently known to be regulated by guanosine 5'-diphosphate 3'-diphosphate (ppGpp) (for review see [I]). This compound which was also reported in chloroplasts [2] and mitochondria [3] appears as a general metabolic effector in procaryotes. If on one hand, ppGpp inhibits the synthesis of what might be called the 'structural' material of the cell, such as ribosomal RNA [4-71, ribosomal proteins [8-301, lipids [ l l , 121, peptidoglycan [13] etc., on the other hand it stimulates the expression of some inducible operons [4,The intracellular level of ppGpp may itself be greatly modified as a function of nutrient availability and other growth conditions. The mechanism of the relA gene product-dependent synthesis of ppGpp observed in the course of amino acid starvations, which constitutes a very early event of the stringent response, is now well understood [17--251. All deficiencies in the charge of tRNA also lead to an elevated level of guanosine 5'-triphosphate 3'-diphosphate (pppGpp), the physiological precursor of ppGpp 14-161.Abbreviations. ppGpp, guanosine 5'-diphosphate 3'-diphosphate; pppGpp, guanosine 5'-triphosphate 3'-diphosphate; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; (cHxN)zC, dicyclohexylcarbodiimide.[26-281. An accumulation of the single ppGpp can be elicited by carbon-source substitutions [29,30] energy poisons [3 I] or membrane-perturbating agents such as morphine analogs [32,33], i.e. treatments which do not affect the incorporation of amino acids but create a defined situation of unbalanced growth (downshifts). All these conditions, which do not change the basal level of ppGpp synthesis, have been found to reduce considerably the rate of ppGpp degradation [29,32].Mutations in the spoT gene were shown to have a similar effect [34,35] and recent experiments of ppGpp degradation in vitro ha...

A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydrophilic pores.

“…We can only speculate about the structure of the induced pore. Tetu et al (1980) have confirmed that the half life of ppGpp is increased 10 times when the protonmotive force is dissipated. These structures have, however, only been observed at much higher peptide to lipid molar ratios than used in this study.…”

SpoT-Triggered Stringent Response Controls usp Gene Expression in Pseudomonas aeruginosa

“…Despite the fact Wells et al (55) used acidic pH stress to induce the stringent response in H. pylori, they observed the same accumulation pattern for only ppGpp and not the precursor pppGpp, as we showed with alkaline stress in P. aeruginosa. The mechanism triggering the accumulation of ppGpp in response to pH stress might be linked to the pH-dependent perturbation of the proton gradient since treatment with the protonophor carbonyl cyanide m-chlorophenylhydrazone has also been shown to induce a SpoT-regulated stringent response by a reduction of the rate of ppGpp degradation in E. coli (50). Nevertheless, the capability of SpoT to detect and respond to a variety of apparently unrelated stress factors remains a highly complex and greatly unsolved network, which now can be additionally linked to environmental alkaline pH.…”

“…The second regulatory mechanism of the strin-gent response controls the balance between the synthetase and hydrolase activity of ppGpp by SpoT (8,59). In contrast to RelA, SpoT-controlled ppGpp accumulation is triggered by a variety of stimuli, e.g., inhibition of fatty acid metabolism (19,45), carbon deprivation (59), or membrane-perturbating agents (50).…”

Cellular localization of the Escherichia coli SpoT protein

“…In addition to detecting SpoT in high-speed pellets, we also found that the low-speed pellet (30,000 ϫ g) of crude extracts also contained substantial amounts of the SpoT protein. Lowspeed pellets consist primarily of crude membranes, and the localization of SpoT to this fraction could indicate membrane association, as suggested previously (29,30). To determine if SpoT is membrane associated, we fractionated crude membranes as described by Osborn and Munson (20).…”

“…Carbon source starvation was also shown to result in inhibition of SpoT-mediated (p)ppGppase and thereby account for (p)ppGpp accumulation under these conditions (16). In addition, (p)ppGppase activity is inhibited in vivo by osmotic shock (8), fatty acid synthesis inhibition (26), oxidative phosphorylation uncouplers (30), or long-chain alcohols (19). These effects can be dissociated from the effects of ATP levels (30), leading to the suggestion that the (p)ppGppase activity of SpoT is sensitive to membrane integrity in a manner that might involve membrane association of SpoT (29,30).…”