Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Abstract

A major bacterial alarmone, guanosine 3,5-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3,5-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.The stringent response, which is a pleiotropic adaptation to nutritional starvation and stress, is broadly conserved in bacteria and plant chloroplasts (1, 2). This physiological control is dominated by a rapid synthesis/degradation cycle of the stringent alarmones guanosine 3Ј-pyrophosphate 5Ј-triphosphate (pppGpp) 4 or guanosine 3Ј,5Ј-bispyrophosphate (ppGpp) ( Fig. 1) (3). In Escherichia coli grown under amino acid depletion, pppGpp is synthesized from ATP and GTP by ribosome-associated RelA activity and subsequently converted to ppGpp by the activity of 5Ј-nucleotidase (4). Besides this pathway, ppGpp is also directly synthesized from GDP by the activity of RelA. Thereafter, ppGpp is degraded to GDP and pyrophosphate by the activity of SpoT, which is a RelA homolog with reciprocal activities for ppGpp degradation and synthesis (5, 6). For several bacteria, SpoT physiologically acts as both the ppGpp synthetase and hydrolase and is known as the Rel/Spo homolog. The metabolism of these alarmone nucleotides is understood based on the function of these two enzymes, whereas recent studies have identified alternative players acting as ppGpp synthetase in bacteria (7,8). On the other hand, for the degradation of ppGpp, previous studies reported that SpoT-independent (p)ppGpp degradation activity exists in bacteria, whereas the biological meaning(s) and enzymatic mechanism(s) of these alternative pathways have not been characterized in any organisms (9 -12).The Nudix hydrolases constitute a large family of proteins that share a highly conserved amino acid sequence, the so-called "Nudix motif," which is distribut...

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

“…This result suggested that RelQ Ef prefers GDP over GTP as a substrate. However, there are other explanations for removal of the 5= ␥-phosphate from pppGpp, generating ppGpp, such as activities of nonspecific phosphohydrolases, including polyphosphate phosphatase, Nudix family hydrolases, or GTP-requiring enzymes for protein synthesis such as EF-G or EF-Tu (37)(38)(39)(40). A preference for GDP over GTP as an in vitro substrate has been observed for SASs from Mycobacterium smegmatis and S. aureus (24,41).…”

“…2B was possible because it involves separation of pure substrates and products from a defined synthesis reaction. The use of an HPLC system employing a column and mobilephase combination similar to those described by Ooga et al (38) overcame the hurdle of reliably separating pGpp standards from cell lysates. However, the selective loss of highly phosphorylated nucleotides (pGpp, ppGpp, and pppGpp) during sample processing and column separation, coupled with the reduced sensitivity of UV detection, greatly reduced our power to detect (pp)pGpp in E. faecalis.…”

Role of (p)ppGpp in biofilm formation and expression of filamentous structures in Bordetella pertussis

“…Thermus thermophilus is a Gram-negative, aerobic thermophile that has an orthologue of Rsh (RelA/SpoT); Rsh can both synthesize and degrade (p)ppGpp in response to nutritional starvation. In addition to Rsh, a ppGpp degradation pathway mediated by Ndx8 is present in T. thermophilus (Ooga et al, 2009). Additionally, Listeria monocytogenes strains that harbour a transposon insertion in pgpH, which encodes a metaldependent phosphohydrolase, are cold sensitive and contain increased levels of ppGpp (Liu et al, 2006 (Justo et al, 2004).…”

Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

“…The mutant gene obtained, hph5, was functional as a selection marker in T. thermophilus at up to 65 C. 6) We have also reported that mutant protein HPH5 was thermostabilized at the enzyme level at 17 C, 7) as well as crystallization and structural analysis of it. 8,9) Recently, another thermostable hph mutant was found to be functional at up to 82 C in T. thermophilus, 10) but no precise enzymatic characterization of this mutant protein has been reported.…”