Microbial Primer: what is the stringent response and how does it allow bacteria to survive stress?

The stringent response is a conserved bacterial stress response that allows bacteria to alter their activity and survive under nutrient-limiting conditions. Activation of the stringent response is characterized by the production of intracellular signalling molecules, collectively termed (p)ppGpp, which interact with multiple targets inside bacterial cells. Together, these interactions induce a slow growth phenotype to aid bacterial survival by altering the transcriptomic profile of the cell, inhibiting ribosome biosynthesis and targeting enzymes involved in other key metabolic processes.

Adaptation to skin mycobiota promotes antibiotic tolerance in Staphylococcus aureus.

The microbiota can promote host health by inhibiting pathogen colonization, yet how host-resident fungi, or the mycobiota, contribute to this process remains unclear. The human skin mycobiota is uniquely stable compared to other body sites and dominated by yeasts of the genus Malassezia . We observe that colonization of human skin by Malassezia sympodialis significantly reduces subsequent colonization by the prominent bacterial pathogen Staphylococcus aureus . M. sympodialis secreted products possess potent bactericidal activity against S. aureus and are sufficient to impair S. aureus skin colonization. This bactericidal activity requires an acidic environment and is exacerbated by free fatty acids, demonstrating a unique synergy with host-derived epidermal defenses. Leveraging experimental evolution to pinpoint mechanisms of S. aureus adaptation in response to the skin mycobiota, we identified multiple mutations in the stringent response regulator Rel that promote survival against M. sympodialis . Similar Rel alleles have been reported in S. aureus clinical isolates, and natural Rel variants are sufficient for tolerance to M. sympodialis antagonism. Partial stringent response activation underlies tolerance to clinical antibiotics, with both laboratory-evolved and natural Rel variants conferring multidrug tolerance. These findings demonstrate the ability of the mycobiota to mediate pathogen colonization resistance, identify new mechanisms of bacterial adaptation in response to fungal antagonism, and reveal the potential for microbiota-driven evolution to shape pathogen antibiotic susceptibility. Highlights: - M. sympodialis reduces colonization of human skin by S. aureus - Bactericidal activity of M. sympodialis is exacerbated by features of the skin niche - S. aureus Rel variants are sufficient for tolerance to Malassezia antagonism - Evolved tolerance to yeast antagonism coincides with S. aureus multidrug tolerance.

Arginine-Functional Methacrylic Block Copolymer Nanoparticles: Synthesis, Characterization, and Adsorption onto a Model Planar Substrate.

Recently, we reported the synthesis of a hydrophilic aldehyde-functional methacrylic polymer (Angew. Chem., 2021, 60, 12032-12037). Herein we demonstrate that such polymers can be reacted with arginine in aqueous solution to produce arginine-functional methacrylic polymers without recourse to protecting group chemistry. Careful control of the solution pH is essential to ensure regioselective imine bond formation; subsequent reductive amination leads to a hydrolytically stable amide linkage. This new protocol was used to prepare a series of arginine-functionalized diblock copolymer nanoparticles of varying size via polymerization-induced self-assembly in aqueous media. Adsorption of these cationic nanoparticles onto silica was monitored using a quartz crystal microbalance. Strong electrostatic adsorption occurred at pH 7 (Γ = 14.7 mg m-2), whereas much weaker adsorption occurred at pH 3 (Γ = 1.9 mg m-2). These findings were corroborated by electron microscopy, which indicated a surface coverage of 42% at pH 7 but only 5% at pH 3.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Designing Effective Antimicrobial Nanostructured Surfaces: Highlighting the Lack of Consensus in the Literature.

Research into nanostructured materials, inspired by the topography of certain insect wings, has provided a potential pathway toward drug-free antibacterial surfaces, which may be vital in the ongoing battle against antimicrobial resistance. However, to produce viable antibacterial nanostructured surfaces, we must first understand the bactericidal mechanism of action and how to optimize them to kill the widest range of microorganisms. This review discusses the parameters of nanostructured surfaces that have been shown to influence their bactericidal efficiency and highlights the highly variable nature of many of the findings. A large-scale analysis of the literature is also presented, which further shows a lack of clarity in what is understood about the factors influencing bactericidal efficiency. The potential reasons for the ambiguity, including how the killing effect may be a result of multiple factors and issues with nonstandardized testing of the antibacterial properties of nanostructured surfaces, are then discussed. Finally, a standard method for testing of antimicrobial killing is proposed that will allow comparison between studies and enable a deeper understanding about nanostructured surfaces and how to optimize their bactericidal efficiency.

Stringent Response-Mediated Control of GTP Homeostasis Is Required for Long-Term Viability of Staphylococcus aureus.

Staphylococcus aureus is an opportunistic bacterial pathogen that often results in difficult-to-treat infections. One mechanism used by S. aureus to enhance survival during infection is the stringent response. This is a stress survival pathway that utilizes the nucleotides (p)ppGpp to reallocate bacterial resources, shutting down growth until conditions improve. Small colony variants (SCVs) of S. aureus are frequently associated with chronic infections, and this phenotype has previously been linked to a hyperactive stringent response. Here, we examine the role of (p)ppGpp in the long-term survival of S. aureus under nutrient-restricted conditions. When starved, a (p)ppGpp-null S. aureus mutant strain ((p)ppGpp0) initially had decreased viability. However, after 3 days we observed the presence and dominance of a population of small colonies. Similar to SCVs, these small colony isolates (p0-SCIs) had reduced growth but remained hemolytic and sensitive to gentamicin, phenotypes that have been tied to SCVs previously. Genomic analysis of the p0-SCIs revealed mutations arising within gmk, encoding an enzyme in the GTP synthesis pathway. We show that a (p)ppGpp0 strain has elevated levels of GTP, and that the mutations in the p0-SCIs all lower Gmk enzyme activity and consequently cellular GTP levels. We further show that in the absence of (p)ppGpp, cell viability can be rescued using the GuaA inhibitor decoyinine, which artificially lowers the intracellular GTP concentration. Our study highlights the role of (p)ppGpp in GTP homeostasis and underscores the importance of nucleotide signaling for long-term survival of S. aureus in nutrient-limiting conditions, such as those encountered during infections. IMPORTANCE Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp. These nucleotides function to shut down bacterial growth until conditions improve. Therefore, (p)ppGpp are important for bacterial survival and have been implicated in promoting chronic infections. Here, we investigate the importance of (p)ppGpp for long-term survival of bacteria in nutrient-limiting conditions similar to those in a human host. We discovered that in the absence of (p)ppGpp, bacterial viability decreases due to dysregulation of GTP homeostasis. However, the (p)ppGpp-null bacteria were able to compensate by introducing mutations in the GTP synthesis pathway that led to a reduction in GTP build-up and a rescue of viability. This study therefore highlights the importance of (p)ppGpp for the regulation of GTP levels and for long-term survival of S. aureus in restricted environments.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

The Stringent Response Inhibits 70S Ribosome Formation in Staphylococcus aureus by Impeding GTPase-Ribosome Interactions.

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control. IMPORTANCE The stringent response is a bacterial signaling network that utilizes the nucleotides pppGpp and ppGpp to reprogram cells in order to survive nutritional stresses. However, much about how these important nucleotides control cellular reprogramming is unknown. Our previous work revealed that (p)ppGpp can bind to and inhibit the enzymatic activity of four ribosome-associated GTPases (RA-GTPases), enzymes that facilitate maturation of the 50S and 30S ribosomal subunits. Here, we examine how this occurs mechanistically and demonstrate that this interaction prevents the accommodation of RA-GTPases on ribosomal subunits both in vitro and within bacterial cells, with the ppGpp-bound state structurally mimicking the inactive GDP-bound conformation of the enzyme. We additionally reveal that these GTPase enzymes have a greater affinity for OFF-state-inducing nucleotides, which is a mechanism likely to control ribosome assembly during growth. With this, we further our understanding of how ribosome function is controlled by (p)ppGpp, enabling bacterial survival during stress.

The stringent response and physiological roles of (pp)pGpp in bacteria.

The stringent response is a stress signalling system mediated by the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) in response to nutrient deprivation. Recent research highlights the complexity and broad range of functions that these alarmones control. This Review provides an update on our current understanding of the enzymes involved in ppGpp, pppGpp and guanosine 5'-monophosphate 3'-diphosphate (pGpp) (collectively (pp)pGpp) turnover, including those shown to produce pGpp and its analogue (pp)pApp. We describe the well-known interactions with RNA polymerase as well as a broader range of cellular target pathways controlled by (pp)pGpp, including DNA replication, transcription, nucleotide synthesis, ribosome biogenesis and function, as well as lipid metabolism. Finally, we review the role of ppGpp and pppGpp in bacterial pathogenesis, providing examples of how these nucleotides are involved in regulating many aspects of virulence and chronic infection.

The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly.

Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon induction of a nutrient stress-sensing signalling pathway termed the stringent response. This stress response is mediated by the alarmones guanosine tetra- and penta-phosphate ((p)ppGpp), the accumulation of which leads to a massive cellular response that slows growth and aids survival. The 70S bacterial ribosome is an intricate structure, with assembly both complex and highly modular. Presiding over the assembly process is a group of P-loop GTPases within the TRAFAC (Translation Factor Association) superclass that are crucial for correct positioning of both early and late stage ribosomal proteins (r-proteins) onto the rRNA. Often described as 'molecular switches', members of this GTPase superfamily readily bind and hydrolyse GTP to GDP in a cyclic manner that alters the propensity of the GTPase to carry out a function. TRAFAC GTPases are considered to act as checkpoints to ribosome assembly, involved in binding to immature sections in the GTP-bound state, preventing further r-protein association until maturation is complete. Here we review our current understanding of the impact of the stringent response and (p)ppGpp production on ribosome maturation in prokaryotic cells, focusing on the inhibition of (p)ppGpp on GTPase-mediated subunit assembly, but also touching upon the inhibition of rRNA transcription and protein translation.

The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus.

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.

Cyanophage MazG is a pyrophosphohydrolase but unable to hydrolyse magic spot nucleotides.

Bacteriophage possess a variety of auxiliary metabolic genes of bacterial origin. These proteins enable them to maximize infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein - a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate, however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine-tuned nature of viral metabolic processes.

Triggering the stringent response: signals responsible for activating (p)ppGpp synthesis in bacteria.

The stringent response is a conserved bacterial stress response mechanism that allows bacteria to respond to nutritional challenges. It is mediated by the alarmones pppGpp and ppGpp, nucleotides that are synthesized and hydrolyzed by members of the RSH superfamily. Whilst there are key differences in the binding targets for (p)ppGpp between Gram-negative and Gram-positive bacterial species, the transient accumulation of (p)ppGpp caused by nutritional stresses results in a global change in gene expression in all species. The RSH superfamily of enzymes is ubiquitous throughout the bacterial kingdom, and can be split into three main groups: the long-RSH enzymes; the small alarmone synthetases (SAS); and the small alarmone hydrolases (SAH). Despite the prevalence of these enzymes, there are important differences in the way in which they are regulated on a transcriptional and post-translational level. Here we provide an overview of the diverse regulatory mechanisms that are involved in governing this crucial signalling network. Understanding how the RSH superfamily members are regulated gives insights into the varied important biological roles for this signalling pathway across the bacteria.

The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus.

Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S aureus and found that the LAC*ΔgdpP strain of S aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-ß-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases--RsgA, RbgA, Era, HflX, and ObgE--as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

Cross-talk between two nucleotide-signaling pathways in Staphylococcus aureus.

Nucleotide-signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to external stimuli. The stringent response alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp) control a global response allowing cells to adapt to starvation conditions such as amino acid depletion. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that this signaling nucleotide is essential for the growth of Staphylococcus aureus, and its increased production during late growth phases indicates that c-di-AMP controls processes that are important for the survival of cells in stationary phase. By examining the transcriptional profile of cells with high levels of c-di-AMP, we reveal a significant overlap with a stringent response transcription signature. Examination of the intracellular nucleotide levels under stress conditions provides further evidence that high levels of c-di-AMP lead to an activation of the stringent response through a RelA/SpoT homologue (RSH) enzyme-dependent increase in the (p)ppGpp levels. This activation is shown to be indirect as c-di-AMP does not interact directly with the RSH protein. Our data extend this interconnection further by showing that the S. aureus c-di-AMP phosphodiesterase enzyme GdpP is inhibited in a dose-dependent manner by ppGpp, which itself is not a substrate for this enzyme. Altogether, these findings add a new layer of complexity to our understanding of nucleotide signaling in bacteria as they highlight intricate interconnections between different nucleotide-signaling networks.

Differential localization of LTA synthesis proteins and their interaction with the cell division machinery in Staphylococcus aureus.

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. In Staphylococcus aureus it consists of a polyglycerolphosphate-chain that is retained within the membrane via a glycolipid. Using an immunofluorescence approach, we show here that the LTA polymer is not surface exposed in S. aureus, as it can only be detected after digestion of the peptidoglycan layer. S. aureus mutants lacking LTA are enlarged and show aberrant positioning of septa, suggesting a link between LTA synthesis and the cell division process. Using a bacterial two-hybrid approach, we show that the three key LTA synthesis proteins, YpfP and LtaA, involved in glycolipid production, and LtaS, required for LTA backbone synthesis, interact with one another. All three proteins also interacted with numerous cell division and peptidoglycan synthesis proteins, suggesting the formation of a multi-enzyme complex and providing further evidence for the co-ordination of these processes. When assessed by fluorescence microscopy, YpfP and LtaA fluorescent protein fusions localized to the membrane while the LtaS enzyme accumulated at the cell division site. These data support a model whereby LTA backbone synthesis proceeds in S. aureus at the division site in co-ordination with cell division, while glycolipid synthesis takes place throughout the membrane.

Cyclic di-AMP: another second messenger enters the fray.

Nucleotide signalling molecules contribute to the regulation of cellular pathways in all forms of life. In recent years, the discovery of new signalling molecules in bacteria and archaea, as well as the elucidation of the pathways they regulate, has brought insights into signalling mechanisms not only in bacterial and archaeal cells but also in eukaryotic host cells. Here, we provide an overview of the synthesis and regulation of cyclic di-AMP (c-di-AMP), one of the latest cyclic nucleotide second messengers to be discovered in bacteria. We also discuss the currently known receptor proteins and pathways that are directly or indirectly controlled by c-di-AMP, the domain structure of the enzymes involved in its production and degradation, and the recognition of c-di-AMP by the eukaryotic host.

Systematic identification of conserved bacterial c-di-AMP receptor proteins.

Nucleotide signaling molecules are important messengers in key pathways that allow cellular responses to changing environments. Canonical secondary signaling molecules act through specific receptor proteins by direct binding to alter their activity. Cyclic diadenosine monophosphate (c-di-AMP) is an essential signaling molecule in bacteria that has only recently been discovered. Here we report on the identification of four Staphylococcus aureus c-di-AMP receptor proteins that are also widely distributed among other bacteria. Using an affinity pull-down assay we identified the potassium transporter-gating component KtrA as a c-di-AMP receptor protein, and it was further shown that this protein, together with c-di-AMP, enables S. aureus to grow in low potassium conditions. We defined the c-di-AMP binding activity within KtrA to the RCK_C (regulator of conductance of K(+)) domain. This domain is also found in a second S. aureus protein, a predicted cation/proton antiporter, CpaA, which as we show here also directly binds c-di-AMP. Because RCK_C domains are found in proteinaceous channels, transporters, and antiporters from all kingdoms of life, these findings have broad implications for the regulation of different pathways through nucleotide-dependent signaling. Using a genome-wide nucleotide protein interaction screen we further identified the histidine kinase protein KdpD that in many bacteria is also involved in the regulation of potassium transport and a PII-like signal transduction protein, which we renamed PstA, as c-di-AMP binding proteins. With the identification of these widely distributed c-di-AMP receptor proteins we link the c-di-AMP signaling network to a central metabolic process in bacteria.

The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid.

The Sbi protein of Staphylococcus aureus comprises two IgG-binding domains similar to those of protein A and a region that triggers the activation of complement C3. Sbi is expressed on the cell surface but its C-terminal domain lacks motifs associated with wall or membrane anchoring of proteins in Gram-positive bacteria. Cell-associated Sbi fractionates with the cytoplasmic membrane and is not solubilized during protoplast formation. S. aureus expressing Sbi truncates of the C-terminal Y domain allowed identification of residues that are required for association of Sbi with the membrane. Recombinant Sbi bound to purified cytoplasmic membrane material in vitro and to purified lipoteichoic acid. This explains how Sbi partitions with the membrane in fractionation experiments yet is partially exposed on the cell surface. An LTA-defective mutant of S. aureus had reduced levels of Sbi in the cytoplasmic membrane.