Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis

Abstract

SummaryBacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelASpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins (~25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

“…S3D) (25,28). To understand this phenomenon in greater detail, we performed an in-depth kinetic analysis of SAS1 (Fig.…”

“…Both the synthetase and hydrolase domains of RelA are regulated in a reciprocal manner, ensuring inverse coupling of the opposing activities (19,22,23). The recently identified alarmone synthetases SAS1 (synonyms: YjbM and RelQ) and SAS2 (synonyms: YwaC and RelP) (24)(25)(26)(27) share high sequence identities of 20-30% with RSH proteins in their synthetase domains (18). However, the absence of the hydrolase and C-terminal domains found in RelA (Fig.…”

Streptococcus mutans: a new Gram-positive paradigm?

“…However, the serendipitous finding that the S. mutans DrelA mutant was still able to produce (p)ppGpp (Lemos et al, 2004) led to the discovery of two small monofunctional (p)ppGppsynthetase enzymes, named RelP and RelQ, in S. mutans (Lemos et al, 2007a). BLAST search analysis indicated that at least one of these enzymes was present in virtually every Gram-positive organism (Lemos et al, 2007a), and subsequent studies confirmed the functionality of these enzymes in other Gram-positive bacteria such as B. subtilis, S. pneumoniae and Enterococcus faecalis (Abranches et al, 2009;Battesti & Bouveret, 2009;Nanamiya et al, 2008). The identification of these small (p)ppGpp synthetase enzymes in Gram-positive bacteria reinvigorated the scientific interest in this fundamental stress response pathway.…”

Cytosolic ppGpp accumulation induces retarded plant growth and development

“…13 was synthesized in vitro with adapting its codon usage for expression in Arabidopsis. The DNA fragment was cloned into the estrogen-inducible expression vector pER8 at its SpeI and XhoI sites.…”

“…Specifically, we constructed a transgenic Arabidopsis expressing Bacillus subtilis ppGpp synthase gene (yjbM) under the control of the estrogen-induced promoter; the resulting line was designated as YJBMox. Because YjbM is a small protein (~25 kDa) consisting of a single ppGpp synthase domain, 13 YjbM expression could induce constitutive ppGpp synthesis in the cytosol.…”