The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 ？. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.
References
[1]
G？rke B, Stülke J (2008) Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol 6: 613–624.
[2]
Rojo F (2010) Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment. FEMS Microbiol Rev 34: 658–684.
[3]
Saier MH (1989) Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol Rev 53: 109–120.
[4]
Phillips AT, Mulfinger LM (1981) Cyclic adenosine 3′,5′-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis. J Bacteriol 145: 1286–1292.
[5]
Siegel LS, Hylemon PB, Phibbs PV (1977) Cyclic adenosine 3′,5′-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase in Pseudomonas and Bacteroides. J Bacteriol 129: 87–96.
[6]
Durham DR, Phibbs PV (1982) Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa. J Bacteriol 149: 534–541.
[7]
Morales G, Linares JF, Beloso A, Albar JP, Martínez JL, et al. (2004) The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds. J Bacteriol 186: 1337–1344.
[8]
Wolff JA, MacGregor CH, Eisenberg RC, Phibbs PV (1991) Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO. J Bacteriol 173: 4700–4706.
[9]
Hernández-Arranz S, Moreno R, Rojo F (2012) The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy. Environ Microbiol 15: 227–241.
[10]
O'Toole GA, Gibbs KA, Hager PW, Phibbs PV Jr, Kolter R (2000) The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa. J Bacteriol 182: 425–431.
[11]
Linares JF, Moreno R, Fajardo A, Martínez-Solano L, Escalante R, et al. (2010) The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Environ Microbiol 12: 3196–3212.
[12]
Huang J, Sonnleitner E, Ren B, Xu Y, Haas D (2012) Catabolite repression control of pyocyanin biosynthesis at an intersection of primary and secondary metabolism in Pseudomonas aeruginosa. Appl Environ Microbiol 78: 5016–5020.
[13]
Moreno R, Ruiz-Manzano A, Yuste L, Rojo F (2007) The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. Mol Microbiol 64: 665–675.
[14]
Moreno R, Marzi S, Romby P, Rojo F (2009) The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation. Nucleic Acids Res 37: 7678–7690.
[15]
Sonnleitner E, Abdou L, Haas D (2009) Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 106: 21866–21871.
[16]
Moreno R, Fonseca P, Rojo F (2012) Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression. Mol Microbiol 83: 24–40.
[17]
Collier DN, Hager PW, Phibbs PV (1996) Catabolite repression control in the Pseudomonads. Res Microbiol(147): 551–561.
[18]
MacGregor CH, Arora SK, Hager PW, Dail MB, Phibbs PV (1996) The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product. J Bacteriol 178: 5627–5635.
[19]
Wei Y, Zhang H, Gao ZQ, Xu JH, Liu QS, et al. (2013) Structure analysis of the global metabolic regulator Crc from Pseudomonas aeruginosa. IUBMB Life 65: 50–57.
[20]
Carpenter EP, Corbett A, Thomson H, Adacha J, Jensen K, et al. (2007) AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis. EMBO J 26: 1363–1372.
[21]
Mol CD, Izumi T, Mitra S, Tainer JA (2000) DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination. Nature 403: 451–456.
[22]
Vogel J, Luisi BF (2011) Hfq and its constellation of RNA. Nat Rev Microbiol 15: 578–589.
[23]
Beich-Frandsen M, Vecerek B, Konarev PV, Sj？blom B, Kloiber K, et al. (2011) Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq. Nucleic Acids Res 39: 4900–4915.
[24]
Link TM, Valentin-Hansen P, Brennan RG (2009) Structure of Escherichia coli Hfq bound to polyriboadenylate. Proc Natl Acad Sci U S A 106: 19292–19297.
[25]
Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326: 245–254.
[26]
Sauter C, Basquin J, Suck D (2003) Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res 31: 4091–4098.
[27]
Smyth PF, Clarke PH (1975) Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis. J Gen Microbiol 90: 81–90.
[28]
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006–2008.
[29]
Kabsch W (2010) Xds. Acta Cryst D 66: 125–132.
[30]
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, et al. (2011) Overview of the CCP4 suite and current developments. Acta Cryst D 67: 235–242.
[31]
McCoy AJ (2007) Solving structures of protein complexes by molecular replacement with Phaser. Acta Cryst D 63: 32–41.
[32]
Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Cryst D 53: 240–255.
[33]
Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Cryst D 66: 213–221.
[34]
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Cryst D 60: 2126–2132.
[35]
Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, et al. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Cryst D 66: 12–21.
[36]
DeLano WL (2002) The PyMol Molecular Graphics System. San Carlos: DeLano Scientific. http://www.pymol.org.
[37]
Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci 98: 10037–10041.
[38]
Sonnleitner E, Schuster M, Sorger-Domenigg T, Greenberg EP, Bl？si U (2006) Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa. Mol Microbiol 59: 1542–1558.
