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Systematic Identification of Conserved Bacterial C-Di-AMP Receptor Proteins

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Systematic Identification of Conserved Bacterial C-Di-AMP Receptor Proteins Systematic identification of conserved bacterial c-di-AMP receptor proteins Rebecca M. Corrigana, Ivan Campeottoa, Tharshika Jeganathana, Kevin G. Roelofsb, Vincent T. Leeb,1, and Angelika Gründlinga,1 aSection of Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom; and bDepartment of Cell Biology and Molecular Genetics and Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742 Edited by Sankar Adhya, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved April 18, 2013 (received for review January 14, 2013) Nucleotide signaling molecules are important messengers in key diadenylate cyclases DacA, DisA, and YojJ and degraded by pathways that allow cellular responses to changing environ- the phosphodiesterase enzyme GdpP (4, 5, 11, 15–18). A va- ments. Canonical secondary signaling molecules act through riety of different phenotypes have been linked to altered c-di- specific receptor proteins by direct binding to alter their activity. AMP levels; an increase in c-di-AMP levels correlates with Cyclic diadenosine monophosphate (c-di-AMP) is an essential sig- increased acid resistance (16, 19) and altered antibiotic re- naling molecule in bacteria that has only recently been discovered. sistance, including an increase in methicillin resistance in S. fi Here we report on the identification of four Staphylococcus aureus aureus (11, 18, 20). Most notable, however, are the ndings that c-di-AMP receptor proteins that are also widely distributed among L. monocytogenes (5) and Bacillus subtilis (18) cannot grow in other bacteria. Using an affinity pull-down assay we identified the the absence of c-di-AMP, showing that in contrast to other sig- potassium transporter-gating component KtrA as a c-di-AMP recep- naling nucleotides, c-di-AMP controls essential cellular pathways. tor protein, and it was further shown that this protein, together with The molecular basis for this is currently not known, although it is assumed that, similar to other signaling molecules, c-di-AMP c-di-AMP, enables S. aureus to grow in low potassium conditions. We interacts with a specific set of target proteins and upon binding defined the c-di-AMP binding activity within KtrA to the RCK_C (reg- + alters their activity or function. Currently only one bacterial c-di- ulator of conductance of K ) domain. This domain is also found in MICROBIOLOGY S. aureus AMP receptor protein, the transcription factor DarR, has been a second protein, a predicted cation/proton antiporter, identified in Mycobacterium smegmatis (21). However, the absence CpaA, which as we show here also directly binds c-di-AMP. Because of close DarR homologs in many organisms that likely produce c-di- RCK_C domains are found in proteinaceous channels, transporters, AMP implies that additional c-di-AMP target proteins must exist. and antiporters from all kingdoms of life, these findings have broad In this study we have identified the potassium transporter-gating implications for the regulation of different pathways through nu- component KtrA as c-di-AMP target protein by using an affinity cleotide-dependent signaling. Using a genome-wide nucleotide pull-down assay. KtrA is a member of the widely distributed RCK + protein interaction screen we further identified the histidine kinase (regulator of conductance of K ) protein family, known to be in- protein KdpD that in many bacteria is also involved in the regulation volved in the gating of ion channels. Here we show that KtrA is of potassium transport and a PII-like signal transduction protein, required for the growth of S. aureus under potassium limiting which we renamed PstA, as c-di-AMP binding proteins. With the conditions. Through subsequent binding studies we show that c-di- identification of these widely distributed c-di-AMP receptor pro- AMP specifically interacts with the C-terminal RCK_C domain of teins we link the c-di-AMP signaling network to a central metabolic KtrA. A second S. aureus RCK_C domain-containing protein process in bacteria. CpaA, a predicted cation/proton antiporter, was subsequently identified bioinformatically and its interaction with c-di-AMP confirmed experimentally. Last, using a genome-wide in- ucleotide signaling molecules control fundamental processes fi in all forms of life. There is now a large body of evidence teraction screen, we identi ed the PII-like signal transduction N protein PstA and the histidine kinase KdpD as additional c-di-AMP linking nucleotides such as cAMP, cGMP, and guanosine tetra- fi (ppGpp) and pentaphosphate (pppGpp) to the control of fun- binding proteins. With the identi cation of these four widely damental metabolic pathways and stress response processes in distributed c-di-AMP binding proteins we provide a link between eukaryotic and prokaryotic cells (1–3). Cyclic dinucleotides in c-di-AMP and a fundamental cellular process in bacteria, namely particular have recently gained increased attention with the ion transport. identification of additional nucleotides such as cyclic dia- Results denosine monophosphate (c-di-AMP) and the hybrid c-AMP- GMP molecule in bacterial cells (4–6), as well as the discovery Identification of the c-di-AMP Target Protein KtrA. To identify c-di- fi that cyclic dinucleotides are also produced by eukaryotic cells AMP receptor proteins, we performed an af nity pull-down assay – (7–9). The dinucleotide cyclic diguanosine monophosphate using c-di-AMP coupled magnetic beads and protein extracts (c-di-GMP) and the molecular mechanisms by which it controls derived from the S. aureus strain LAC*. This strain is an eryth- cellular pathways has been well characterized, and it is now rec- romycin-sensitive derivative of the clinically relevant community- ognized as a central regulator in bacterial cells that controls the acquired methicillin-resistant USA300 strain LAC. One protein switch from free-living planktonic to a sessile biofilm-associated lifestyles. In pathogenic organisms this is often linked to coloni- zation of the human host and virulence (10). On the other hand, Author contributions: R.M.C., V.T.L., and A.G. designed research; R.M.C., I.C., T.J., and K.G.R. the function and the pathways controlled by the signaling nucle- performed research; V.T.L. contributed new reagents/analytic tools; R.M.C., I.C., K.G.R., V.T.L., otide c-di-AMP are less clear, largely owing to a gap in our and A.G. analyzed data; and R.M.C., V.T.L., and A.G. wrote the paper. knowledge of specific receptor proteins. The authors declare no conflict of interest. Many Gram-positive bacteria, including the important human This article is a PNAS Direct Submission. pathogens Staphylococcus aureus (11), Streptococcus pyogenes (12), Freely available online through the PNAS open access option. Listeria monocytogenes (5), and Mycobacterium tuberculosis (13), 1To whom correspondence may be addressed. E-mail: [email protected] or a.grundling@ produce c-di-AMP, and it is likely that c-di-AMP is also synthe- imperial.ac.uk. sized by several Gram-negative bacteria and a subset of archaea This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (14). c-di-AMP is synthesized by DisA_N domain-containing 1073/pnas.1300595110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1300595110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 systems are part of the RCK protein family and play an im- portant role in transporter gating (23, 24). KtrASA is a typical RCK protein, with an RCK_N domain and an RCK_C domain (Fig. 1C). On the basis of a structural model, it is likely that KtrASA assumes a similar two-lobed fold as the RCK domain in the potassium channel protein MthK of Methanobacterium ther- moautotrophicus (Fig. S1) (25, 26). Interestingly, a nucleotide- binding site for ATP and other nucleotides has been identified previously in the RCK_N domain of the B. subtilis protein KtrA (23) and according to a structural model the RCK_N domain of the S. aureus protein is likely to assume the same fold with the conserved GxGxxG motif forming part of a nucleotide-binding site and with aspartic acid residues D32 and D52 acting as crucial nucleotide-binding residues (Fig. S1) (23). c-di-AMP Binds to the RCK_C Domain of KtrA. To confirm the in- teraction between KtrASA andc-di-AMPandtodefine more pre- cisely the interaction domain, we adapted the differential radial capillary action of ligand assay (DRaCALA), which was previously used to study c-di-GMP-protein interactions (27). This assay is fi Fig. 1. Identi cation of S. aureus KtrASA as a potential c-di-AMP binding based on the principle that free nucleotides migrate outward when protein. (A) Silver-stained polyacrylamide gel of cytoplasmic S. aureus pro- spotted on nitrocellulose membranes, whereas bound ligand is se- teins retained on c-di-AMP-coupled (+) or uncoupled (-) beads. The protein questered to the protein and immobilized in a tight spot on the band enriched in the c-di-AMP lane (asterisk) was identified by mass spec- membrane (Fig. 2A). The distribution of free and bound ligand can trometry as S. aureus protein SAUSA300_0988 (KtrASA). (B) Illustration of be readily visualized and quantified using radiolabeled nucleotides. Ktr-type potassium transport systems, which are composed of a KtrB-type To determine whether c-di-AMP–protein interactions could be membrane component and a cytoplasmic KtrA-type gating component. (C) measured with this assay, we produced 32P-labeled c-di-AMP Schematic representation of the KtrASA domain structure with the RCK_N fi domain (amino acids 4–126) indicated in blue and RCK_C domain (amino acids (Fig. S2) and tested its interaction with puri ed S. aureus GdpP 135–219) shown in orange. The RCK_N domain of the B.
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