cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Abstract

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase. Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase. IntroductionCyclic AMP is used as a signal for eliciting diverse cellular responses in both bacteria and higher eukaryotes. Nucleotide cyclases utilize ATP and GTP to make cAMP or cGMP, respectively, and of the six classes of nucleotide cyclases that have been described (1Barzu O. Danchin A. Prog. Nucleic Acids Res. Mol. Biol. 1994; 49: 241-283Crossref PubMed Scopus (59) Google Scholar), the Class III adenylyl and guanylyl cyclases are encoded in many bacterial and almost all eukaryotic organisms (2Linder J.U. Schultz J.E. Cell Signal. 2003; 15: 1081-1089Crossref PubMed Scopus (143) Google Scholar). The genomes of pathogenic Mycobacterium tuberculosis, the causative agent of tuberculosis that accounts for almost 2 million deaths annually worldwide, harbor a number of genes that encode putative Class III nucleotide cyclases (3Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (43) Google Scholar) and a single cyclic nucleotide phosphodiesterase (4Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar), and many of these enzymes have been characterized biochemically as well as structurally (3Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (43) Google Scholar, 4Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar, 5Podobnik M. Tyagi R. Matange N. Dermol U. Gupta A.K. Mattoo R. Seshadri K. Visweswariah S.S. J. Biol. Chem. 2009; 284: 32846-32857Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar). Most recently, it has been reported that cAMP production by the Rv0386 adenylyl cyclase is critical for allowing M. tuberculosis to establish itself within the mammalian host and cause disease (7Agarwal N. Lamichhane G. Gupta R. Nolan S. Bishai W.R. Nature. 2009; 460: 98-102Crossref PubMed Scopus (168) Google Scholar). Thus, an understanding of the mechanisms underlying the production, utilization, and degradation of cAMP by M. tuberculosis may provide avenues for the identification of novel targets for drug development (8Shenoy A.R. Visweswariah S.S. Trends Microbiol. 2006; 14: 543-550Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).The genomes of non-pathogenic mycobacteria, such as Mycobacterium smegmatis, also encode multiple nucleotide cyclase genes, which could account for the high intracellular cAMP levels found in these bacteria (9Dass B.K. Sharma R. Shenoy A.R. Mattoo R. Visweswariah S.S. J. Bacteriol. 2008; 190: 3824-3834Crossref PubMed Scopus (31) Google Scholar). cAMP levels are modulated by various stress conditions in M. smegmatis (9Dass B.K. Sharma R. Shenoy A.R. Mattoo R. Visweswariah S.S. J. Bacteriol. 2008; 190: 3824-3834Crossref PubMed Scopus (31) Google Scholar), suggesting that cAMP participates in signaling within the bacterial cell, mediating its action by downstream effectors. The effectors of cAMP in mammalian cells have been well characterized and include protein kinase A (PKA), 4The abbreviations used are: PKAprotein kinase A2-ME2-mercaptoethanolCREBcAMP response element-bindingCBPCREB-binding proteincNMPcyclic nucleotide monophosphateCRPcatabolite repressor proteinDTTdithiothreitolGSTglutathione S-transferaseMALDI-TOFmatrix-assisted laser desorption/ionization, time of flightUSPuniversal stress proteinBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol(Sp)-cAMPSadenosine-3′,5′-cyclic monophosphorothioate, Sp isomer. exchange protein activated by cAMP (EPAC), and the hyperpolarization-activated cyclic nucleotide-gated channels (10Berman H.M. Ten Eyck L.F. Goodsell D.S. Haste N.M. Kornev A. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 45-50Crossref PubMed Scopus (164) Google Scholar, 11Rehmann H. Wittinghofer A. Bos J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 63-73Crossref PubMed Scopus (171) Google Scholar). The cyclic nucleotide (cNMP) binding domain in these proteins is conserved evolutionarily, and the best characterized protein with a cAMP binding domain in bacteria is the catabolite repressor protein, or CRP, that serves as a transcription factor, either enhancing or repressing transcription of a variety of genes in a cAMP-dependent manner (12Körner H. Sofia H.J. Zumft W.G. FEMS Microbiol. Rev. 2003; 27: 559-592Crossref PubMed Scopus (351) Google Scholar, 13Gallagher D.T. Smith N. Kim S.K. Robinson H. Reddy P.T. J. Biol. Chem. 2009; 284: 8228-8232Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 14Stapleton M. Haq I. Hunt D.M. Arnvig K.B. Artymiuk P.J. Buxton R.S. Green J. J. Biol. Chem. 2010; 285: 7016-7027Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Binding of cAMP allosterically regulates the activity of these effector proteins.Apart from CRP, no other downstream effector of cAMP in mycobacteria has been described. In this study we provide evidence for an entirely new signaling event mediated by cAMP and so far exclusive to mycobacteria. We show that the products of the Rv0998 gene and its ortholog in M. smegmatis, MSMEG_5458, are cNMP-binding proteins that share a significant similarity to the regulatory subunit of mammalian PKA. Remarkably, the cNMP domain in these proteins is fused to, and regulates the activity of, a GNAT-like protein acetyltransferase, which acetylates the epsilon amino group of a lysine residue in a Universal Stress Protein (USP, MSMEG_4207). This represents not only the first report of lysine acetylation in mycobacteria but also a novel effector function of cAMP in mycobacteria, which is perhaps reflective of the more complex cAMP/PKA/CREB/CBP pathway seen in eukaryotes (15Wang L. Tang Y. Cole P.A. Marmorstein R. Curr. Opin. Struct. Biol. 2008; 18: 741-747Crossref PubMed Scopus (131) Google Scholar).DISCUSSIONThe importance of cAMP in regulating biological processes has been conserved throughout evolution. What remains unique are downstream effectors coupled to cAMP. We now have identified a novel mechanism utilized by mycobacteria to potentially modify protein acetylation in a cAMP-responsive way.Our study represents the first biochemical characterization of proteins containing a fusion of a GNAT-like domain with a cNMP domain, and provides a novel downstream effector function of cyclic nucleotides in mycobacteria. Such a fusion was not identified in an earlier analysis of the evolution of cNMP binding modules in both eukaryotes, and prokaryotic genome sequences obtained from Global Ocean Sampling (34Kannan N. Wu J. Anand G.S. Yooseph S. Neuwald A.F. Venter J.C. Taylor S.S. Genome Biol. 2007; 8: R264Crossref PubMed Scopus (79) Google Scholar), perhaps indicating that this domain fusion may be unique to mycobacteria.In a phylogenetic analysis, MSMEG_5458 clusters with bacterial and fungal proteins, and based on a classification of the phosphate binding cassette, has residues that group it with fungal and alveolate regulatory subunits (35Canaves J.M. Taylor S.S. J. Mol. Evol. 2002; 54: 17-29Crossref PubMed Scopus (93) Google Scholar). The presence of a Ser/Thr residue at position 14 in the phosphate binding cassette (Ser-99 in Rv0998 and Thr-96 in MSMEG_5458) explains the observation that MSMEG_5458 binds to and is also regulated by cGMP (36Weber I.T. Shabb J.B. Corbin J.D. Biochemistry. 1989; 28: 6122-6127Crossref PubMed Scopus (90) Google Scholar). Whether cGMP has a physiological role in mycobacteria is uncertain, because the nucleotide cyclases encoded in mycobacterial genomes are predicted to be adenylyl cyclases, although some purified enzymes show low guanylyl cyclase activity (3Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (43) Google Scholar).The affinity of cAMP for MSMEG_5458 is in the range reported for the isolated A and B domains in the regulatory subunit of PKA (26Dostmann W.R. Taylor S.S. Genieser H.G. Jastorff B. Døskeland S.O. Ogreid D. J. Biol. Chem. 1990; 265: 10484-10491Abstract Full Text PDF PubMed Google Scholar). Given the high (∼100 μm to 1 mm) concentrations of intracellular cAMP in M. smegmatis (9Dass B.K. Sharma R. Shenoy A.R. Mattoo R. Visweswariah S.S. J. Bacteriol. 2008; 190: 3824-3834Crossref PubMed Scopus (31) Google Scholar), it is possible that MSMEG_5458 would almost always be completely saturated with cAMP. During evolution, this may have resulted in the production of a protein that shows only a 3-fold increase in substrate acetylation in the presence of cAMP. However, the very low acetyltransferase activity of Rv0998 in the absence of cAMP, and a dramatic increase in the presence, of cAMP, suggests that Rv0998 can be more tuned to the intracellular levels of cAMP and can modulate the levels of protein acetylation in the cell more precisely. It is conceivable that the actual rate of acetylation in vivo could be markedly different to that seen in vitro and could also be distinct for the two species.A number of proteins containing acetyltransferase domains are found encoded in mycobacteria (37Vagena E. Fakis G. Boukouvala S. Curr. Drug Metab. 2008; 9: 628-660Crossref PubMed Scopus (28) Google Scholar), and some have been characterized. For example, arylamine N-acetyltransferases, which catalyze the transfer of the acetyl group from acetyl-CoA to the free amino group of arylamines and hydrazines, can acetylate the antitubercular drug isoniazid, thus possibly preventing activation of the prodrug in the mycobacterial cell (28Sim E. Sandy J. Evangelopoulos D. Fullam E. Bhakta S. Westwood I. Krylova A. Lack N. Noble M. Curr. Drug Metab. 2008; 9: 510-519Crossref PubMed Scopus (41) Google Scholar). Rv0802c exhibits the GNAT fold, and co-crystallization with succinyl-CoA suggested its preferential use as a substrate (38Vetting M.W. Errey J.C. Blanchard J.S. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2008; 64: 978-985Crossref PubMed Scopus (19) Google Scholar). We have observed that MSMEG_5458 could utilize butyryl-CoA as the substrate for acetylation of USP while the activity with succinyl-CoA was much lower. Some GNAT-like enzymes can acetylate proteins (usually histones are used as model substrates) as well as low molecular aminoglycosides (39Vetting M.W. Magnet S. Nieves E. Roderick S.L. Blanchard J.S. Chem. Biol. 2004; 11: 565-573Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). It is conceivable that MSMEG_5458 and Rv0998 could have non-proteinaceous substrates in mycobacteria, although both these proteins were inactive against aminoglycoside antibiotics in our assays. It may also be worth testing if newly described mycobacterial aminoglycoside acetyltransferases such as eis could acetylate proteins in addition to kanamycin (40Zaunbrecher M.A. Sikes Jr., R.D. Metchock B. Shinnick T.M. Posey J.E. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 20004-20009Crossref PubMed Scopus (235) Google Scholar), especially because a number of proteins appear to be acetylated in a MSMEG_5458-independent manner in M. smegmatis.It is intriguing that the Glu-234 → Ala mutation in MSMEG_5458 reduced basal activity of the enzyme, but in the presence of cAMP, significant acetylation of USP was observed (Fig. 5C). In a recent study on the structure of the protein acetyltransferase from Sulfolobus, it was speculated that the process of inserting the lysine residue in a hydrophobic, substrate-binding pocket seen in protein acetyltransferase could significantly alter the pKa of the lysine residue, thereby allowing acetylation to proceed (41Brent M.M. Iwata A. Carten J. Zhao K. Marmorstein R. J. Biol. Chem. 2009; 284: 19412-19419Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Perhaps a similar mechanism is seen in MSMEG_5458 whereby cAMP binding could alter the hydrophobicity of the substrate binding site, thereby reducing the pKa of the lysine sufficiently to allow the acetylation reaction to occur, without the requirement of the Glu-234 residue to serve as a base (42Mehler E.L. Fuxreiter M. Simon I. Garcia-Moreno E.B. Proteins. 2002; 48: 283-292Crossref PubMed Scopus (118) Google Scholar).The tight association of USP with MSMEG_5458 is surprising, because enzyme-substrate complexes are rarely identified by pulldown assay approaches in the absence of an inhibitor. Perhaps this is indicative of USP being a good substrate for MSMEG_5458, with a high affinity for the enzyme (supplemental Fig. S4). Indeed, USP may be in a stable complex with MSMEG_5458 in cells under steady-state conditions. As a corollary, tight binding of USP would preclude the binding of other substrates to MSMEG_5458, and thus USP may act as a regulator of protein acetylation brought about by MSMEG_5458.In bacteria as well as eukaryotes, a number of enzymes involved in central metabolic pathways have recently been shown to be acetylated (43Wang Q. Zhang Y. Yang C. Xiong H. Lin Y. Yao J. Li H. Xie L. Zhao W. Yao Y. Ning Z.B. Zeng R. Xiong Y. Guan K.L. Zhao S. Zhao G.P. Science. 2010; 327: 1004-1007Crossref PubMed Scopus (777) Google Scholar, 44Zhao S. Xu W. Jiang W. Yu W. Lin Y. Zhang T. Yao J. Zhou L. Zeng Y. Li H. Li Y. Shi J. An W. Hancock S.M. He F. Qin L. Chin J. Yang P. Chen X. Lei Q. Xiong Y. Guan K.L. Science. 2010; 327: 1000-1004Crossref PubMed Scopus (1425) Google Scholar), suggesting a conserved means of regulation of intermediary metabolism. Protein acetylation in eukaryotes also regulates DNA-binding activity, protein-protein interaction, and protein stability via ubiquitination (45Yang X.J. Seto E. Mol. Cell. 2008; 31: 449-461Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar). In prokaryotes, there are reports of acetylation of the response regulator CheY (46Yan J. R. O. A. M. J. Mol. Biol. 2008; PubMed Scopus Google Scholar) and acetyl-CoA I. Cole J.D. J.C. Science. 2002; PubMed Scopus Google Scholar). Indeed, Sulfolobus, an for protein acetylation and has been described a the acetyltransferase protein acetyltransferase, and its substrate which is an DNA-binding protein J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Science. 2002; PubMed Scopus (235) Google Scholar). A analysis of acetylated proteins in E. that a number are metabolic and stress response proteins J. R. J. X. Kim S. H. Zhao Y. Mol. Cell 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). Such a study has not been in mycobacteria. reports on as a that on lysine residues in mycobacterial proteins and regulates stability J. J. Science. 2008; PubMed Scopus Google Scholar, F. J. 2010; PubMed Scopus Google Scholar), suggest that a between acetylation and may on lysine residues in proteins as a means of regulating stability within the This of would only if the of and acetylation and such would be of to is not an but are conserved in all mycobacteria, suggesting a unique requirement for this protein perhaps under conditions that are not in of M. and the are of cellular and the requirement of acetyl-CoA for acetyltransferases and for enzymes that protein lysine acetylation serves as a that cellular levels with protein activity J. J. Res. 2010; PubMed Scopus Google Scholar). In mycobacteria, a has a role to and that is cAMP. cAMP levels if under conditions could modulate the brought about by the of the cell and thereby protein The is to identify all acetylated proteins in mycobacteria and to many of are substrates for the Rv0998 family of to the of protein acetylation and in the of these important bacteria are also IntroductionCyclic AMP is used as a signal for eliciting diverse cellular responses in both bacteria and higher eukaryotes. Nucleotide cyclases utilize ATP and GTP to make cAMP or cGMP, respectively, and of the six classes of nucleotide cyclases that have been described (1Barzu O. Danchin A. Prog. Nucleic Acids Res. Mol. Biol. 1994; 49: 241-283Crossref PubMed Scopus (59) Google Scholar), the Class III adenylyl and guanylyl cyclases are encoded in many bacterial and almost all eukaryotic organisms (2Linder J.U. Schultz J.E. Cell Signal. 2003; 15: 1081-1089Crossref PubMed Scopus (143) Google Scholar). The genomes of pathogenic Mycobacterium tuberculosis, the causative agent of tuberculosis that accounts for almost 2 million deaths annually worldwide, harbor a number of genes that encode putative Class III nucleotide cyclases (3Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (43) Google Scholar) and a single cyclic nucleotide phosphodiesterase (4Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar), and many of these enzymes have been characterized biochemically as well as structurally (3Shenoy A.R. Visweswariah S.S. FEBS Lett. 2006; 580: 3344-3352Crossref PubMed Scopus (43) Google Scholar, 4Shenoy A.R. Sreenath N. Podobnik M. Kovacevic M. Visweswariah S.S. Biochemistry. 2005; 44: 15695-15704Crossref PubMed Scopus (59) Google Scholar, 5Podobnik M. Tyagi R. Matange N. Dermol U. Gupta A.K. Mattoo R. Seshadri K. Visweswariah S.S. J. Biol. Chem. 2009; 284: 32846-32857Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 6Shenoy A.R. Capuder M. Draskovic P. Lamba D. Visweswariah S.S. Podobnik M. J. Mol. Biol. 2007; 365: 211-225Crossref PubMed Scopus (53) Google Scholar). Most recently, it has been reported that cAMP production by the Rv0386 adenylyl cyclase is critical for allowing M. tuberculosis to establish itself within the mammalian host and cause disease (7Agarwal N. Lamichhane G. Gupta R. Nolan S. Bishai W.R. Nature. 2009; 460: 98-102Crossref PubMed Scopus (168) Google Scholar). Thus, an understanding of the mechanisms underlying the production, utilization, and degradation of cAMP by M. tuberculosis may provide avenues for the identification of novel targets for drug development (8Shenoy A.R. Visweswariah S.S. Trends Microbiol. 2006; 14: 543-550Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar).The genomes of non-pathogenic mycobacteria, such as Mycobacterium smegmatis, also encode multiple nucleotide cyclase genes, which could account for the high intracellular cAMP levels found in these bacteria (9Dass B.K. Sharma R. Shenoy A.R. Mattoo R. Visweswariah S.S. J. Bacteriol. 2008; 190: 3824-3834Crossref PubMed Scopus (31) Google Scholar). cAMP levels are modulated by various stress conditions in M. smegmatis (9Dass B.K. Sharma R. Shenoy A.R. Mattoo R. Visweswariah S.S. J. Bacteriol. 2008; 190: 3824-3834Crossref PubMed Scopus (31) Google Scholar), suggesting that cAMP participates in signaling within the bacterial cell, mediating its action by downstream effectors. The effectors of cAMP in mammalian cells have been well characterized and include protein kinase A (PKA), 4The abbreviations used are: PKAprotein kinase A2-ME2-mercaptoethanolCREBcAMP response element-bindingCBPCREB-binding proteincNMPcyclic nucleotide monophosphateCRPcatabolite repressor proteinDTTdithiothreitolGSTglutathione S-transferaseMALDI-TOFmatrix-assisted laser desorption/ionization, time of flightUSPuniversal stress proteinBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol(Sp)-cAMPSadenosine-3′,5′-cyclic monophosphorothioate, Sp isomer. exchange protein activated by cAMP (EPAC), and the hyperpolarization-activated cyclic nucleotide-gated channels (10Berman H.M. Ten Eyck L.F. Goodsell D.S. Haste N.M. Kornev A. Taylor S.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 45-50Crossref PubMed Scopus (164) Google Scholar, 11Rehmann H. Wittinghofer A. Bos J.L. Nat. Rev. Mol. Cell Biol. 2007; 8: 63-73Crossref PubMed Scopus (171) Google Scholar). The cyclic nucleotide (cNMP) binding domain in these proteins is conserved evolutionarily, and the best characterized protein with a cAMP binding domain in bacteria is the catabolite repressor protein, or CRP, that serves as a transcription factor, either enhancing or repressing transcription of a variety of genes in a cAMP-dependent manner (12Körner H. Sofia H.J. Zumft W.G. FEMS Microbiol. Rev. 2003; 27: 559-592Crossref PubMed Scopus (351) Google Scholar, 13Gallagher D.T. Smith N. Kim S.K. Robinson H. Reddy P.T. J. Biol. Chem. 2009; 284: 8228-8232Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 14Stapleton M. Haq I. Hunt D.M. Arnvig K.B. Artymiuk P.J. Buxton R.S. Green J. J. Biol. Chem. 2010; 285: 7016-7027Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Binding of cAMP allosterically regulates the activity of these effector proteins.Apart from CRP, no other downstream effector of cAMP in mycobacteria has been described. In this study we provide evidence for an entirely new signaling event mediated by cAMP and so far exclusive to mycobacteria. We show that the products of the Rv0998 gene and its ortholog in M. smegmatis, MSMEG_5458, are cNMP-binding proteins that share a significant similarity to the regulatory subunit of mammalian PKA. Remarkably, the cNMP domain in these proteins is fused to, and regulates the activity of, a GNAT-like protein acetyltransferase, which acetylates the epsilon amino group of a lysine residue in a Universal Stress Protein (USP, MSMEG_4207). This represents not only the first report of lysine acetylation in mycobacteria but also a novel effector function of cAMP in mycobacteria, which is perhaps reflective of the more complex cAMP/PKA/CREB/CBP pathway seen in eukaryotes (15Wang L. Tang Y. Cole P.A. Marmorstein R. Curr. Opin. Struct. Biol. 2008; 18: 741-747Crossref PubMed Scopus (131) Google Scholar).