2 The abbreviations used are: c-di-AMP, cyclic di-AMP; CBS, cystathionine ␤-synthase; DRaCALA, differential radial capillary action of ligand assay; RCK, regulating conductance of K ϩ ; IPTG, isopropyl ␤-D-thiogalactopyranoside. cro ARTICLE

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.

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP. IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

Edited by Mike Shipston Many bacteria and some archaea produce the second messenger cyclic diadenosine monophosphate (c-di-AMP). c-di-AMP controls the uptake of osmolytes in Firmicutes, including the human pathogen Listeria monocytogenes, making it essential for growth. c-di-AMP is known to directly regulate several potassium channels involved in osmolyte transport in species such as Bacillus subtilis and Streptococcus pneumoniae, but whether this same mechanism is involved in L. monocytogenes, or even whether similar ion channels were present, was not known. Here, we have identified and characterized the putative L. monocytogenes' potassium transporters KimA, KtrCD, and KdpABC. We demonstrate that Escherichia coli expressing KimA and KtrCD, but not KdpABC, transport potassium into the cell, and both KimA and KtrCD are inhibited by c-di-AMP in vivo. For KimA, c-di-AMP-dependent regulation requires the C-terminal domain. In vitro assays demonstrated that the dinucleotide binds to the cytoplasmic regulatory subunit KtrC and to the KdpD sensor kinase of the KdpDE two-component system, which in Staphylococcus aureus regulates the corresponding KdpABC transporter. Finally, we also show that S. aureus contains a homolog of KimA, which mediates potassium transport. Thus, the c-di-AMP-dependent control of systems involved in potassium homeostasis seems to be conserved in phylogenetically related bacteria. Surprisingly, the growth of an L. monocytogenes mutant lacking the c-di-AMP-synthesizing enzyme cdaA is only weakly inhibited by potassium. Thus, the physiological impact of the c-di-AMP-dependent control of potassium uptake seems to be less pronounced in L. monocytogenes than in other Firmicutes. This work was supported by Grant CO 1139/2-1 from the Deutsche Forschungsgemeinschaft via Priority Program SPP1879, the Fonds der Chemischen Industrie, and the Max-Buchner-Forschungsstiftung (MBFSt-Kennziffer 3381) (to F. M. C.

Degradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-‘5’ exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness. However, the molecular basis of Orn’s unique cellular function remained unclear. Here, we show that Orn exhibits exquisite substrate preference for diribonucleotides. Crystal structures of substrate-bound Orn reveal an active site optimized for diribonucleotides. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, Orn is the one and only diribonucleotidase that completes the terminal step of RNA degradation. Together, our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of a specific intermediate pool, diribonucleotides, that affects cellular physiology and viability.