Abstract
The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.
Highlights
The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses
We found that the strongest ppGpp-binding targets in B. anthracis can be categorized to three groups: (1) purine nucleotide synthesis proteins (Hpt[1], Xpt, Gmk, GuaC, PurA, and PurR); (2) ribosome and translation regulatory GTPases (HflX, Der, Obg, RbgA, TrmE, and Era); and (3) nucleotide hydrolytic enzymes, including Nucleoside Diphosphate linked to any moiety “X” (NuDiX) hydrolases and nucleotidases (Fig. 1b)
We have comprehensively characterized the interactomes of the related alarmones pppGpp and ppGpp and established the in vivo presence of pGpp as a closely related alarmone in Gram-positive Bacillus species
Summary
The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. We demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. We demonstrate pGpp as a third alarmone in Grampositive bacteria by establishing its presence in cells, systematically identifying its interacting targets, and revealing a key enzyme for pGpp production through hydrolyzing (p)ppGpp. We compare the targets of pGpp, ppGpp, and pppGpp through proteomic screens in Bacillus anthracis. Our work suggests a mechanism for the conversion and fine tuning of alarmone regulation and the physiological production of the alarmone pGpp
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