Abstract
Stringent response mediated by modified guanosine nucleotides is conserved across bacteria and is regulated through the Rel/Spo functions. In Escherichia coli, RelA and SpoT proteins synthesize the modified nucleotides ppGpp and pppGpp, together referred to as (p)ppGpp. SpoT is also the primary (p)ppGpp hydrolase. In this study, using hypomorphic relA alleles, we provide experimental evidence for SpoT-mediated negative regulation of the amplification of RelA-dependent stringent response. We investigated the kinetics of ppGpp degradation in cells recovering from stringent response in the complete absence of SpoT function. We found that, although greatly diminished, there was slow ppGpp degradation and growth resumption after a lag period, concomitant with decrease in ppGpp pool. We present evidence for reduction in the ppGpp degradation rate following an increase in pppGpp pool, during recovery from stringent response. From a genetic screen, the nudix hydrolases MutT and NudG were identified as over-expression suppressors of the growth defect of ΔspoT and ΔspoT ΔgppA strains. The effect of over-expression of these hydrolases on the stringent response to amino acid starvation and basal (p)ppGpp pool was studied. Over-expression of each hydrolase reduced the strength of the stringent response to amino acid starvation, and additionally, perturbed the ratio of ppGpp to pppGpp in strains with reduced SpoT hydrolase activity. In these strains that do not accumulate pppGpp during amino acid starvation, the expression of NudG or MutT supported pppGpp accumulation. This lends support to the idea that a reduction in the SpoT hydrolase activity is sufficient to cause the loss of pppGpp accumulation and therefore the phenomenon is independent of hydrolases that target pppGpp, such as GppA.
Highlights
Stringent response is a stress response ubiquitously found in microorganisms
In this study, using hypomorphic relA alleles or the overexpression of nudix hydrolases – conditions that support E. coli growth in the absence of SpoT function, we have examined the role of SpoT hydrolase function in (p)ppGpp metabolism during RelA mediated stringent response to amino acid starvation
We expected the relA:Tn10dTet transposon insertion after the 496th codon in the relA ORF to lead to synthesis of truncated RelA polypeptide, early stop codons were introduced in the relA ORF to allow synthesis of truncated RelA polypeptides with the N-terminal 455 amino acids or 496 amino acids. relA allele encoding the N-terminal 455 aa of RelA polypeptide has been expressed from plasmid, the truncated protein synthesized (p)ppGpp constitutively and did not respond to amino acid starvation (Schreiber et al, 1991; Svitil et al, 1993)
Summary
Stringent response is a stress response ubiquitously found in microorganisms. It is characterized by the accumulation of the signaling molecules ppGpp and pppGpp that are synthesized by the transfer of a pyrophosphate moiety from ATP to GDP or GTP, respectively, and collectively referred to as (p)ppGpp (Cashel et al, 1996). When pppGpp hydrolysis was prevented by the elimination of SpoT and GppA functions, RelA mediated (p)ppGpp synthesis was activated in the absence of starvation (Sanyal and Harinarayanan, 2020). SpoT hydrolase activity is essential for the growth of wild type E. coli, noninactivating suppressor mutations in the relA locus have been found to rescue the growth defect of spoT strain (Montero et al, 2014; Sanyal and Harinarayanan, 2020) These relA hypomorphs can be used to address (p)ppGpp turnover in the absence SpoT function. In this study, using hypomorphic relA alleles or the overexpression of nudix hydrolases – conditions that support E. coli growth in the absence of SpoT function, we have examined the role of SpoT hydrolase function in (p)ppGpp metabolism during RelA mediated stringent response to amino acid starvation. Our results show that the cellular pppGpp pool responds to the overall (p)ppGpp hydrolase activity of the cell in a counterintuitive manner, that is, when the cellular hydrolase activity was lowered its pool size decreased and when the hydrolase activity was increased using nudix hydrolases, its pool size increased
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