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Volume 159, Issue Pt_8

Three functional β-carbonic anhydrases in PAO1: role in survival in ambient air Free

Abstract

Bacterial β-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO They maintain the intracellular balance of CO/bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative β-CAs that share 28–45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn and catalyse hydration of CO to bicarbonate. PAO102 (psCA1) was 19–26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II β-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of impaired PAO1 growth in ambient air and caused a minor defect at high CO. Thus, psCA1 contributes to the adaptation of to low CO conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.

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2013-08-01
2024-04-20
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References

  1. Aguilera J., Petit T., de Winde J. H., Pronk J. T.( 2005). Physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to high carbon dioxide concentrations. FEMS Yeast Res 5:579–593 [View Article][PubMed]
     [Google Scholar]
  2. Alber B. E., Ferry J. G.( 1994). A carbonic anhydrase from the archaeon Methanosarcina thermophila. Proc Natl Acad Sci U S A 91:6909–6913 [View Article][PubMed]
     [Google Scholar]
  3. Amoroso G., Morell-Avrahov L., Müller D., Klug K., Sültemeyer D.( 2005). The gene NCE103 (YNL036w) from Saccharomyces cerevisiae encodes a functional carbonic anhydrase and its transcription is regulated by the concentration of inorganic carbon in the medium. Mol Microbiol 56:549–558 [View Article][PubMed]
     [Google Scholar]
  4. Braus-Stromeyer S. A., Schnappauf G., Braus G. H., Gössner A. S., Drake H. L.( 1997). Carbonic anhydrase in Acetobacterium woodii and other acetogenic bacteria. J Bacteriol 179:7197–7200[PubMed]
     [Google Scholar]
  5. Brown O. R., Howitt H. F.( 1969). Growth inhibition and death of Escherichia coli from CO2 deprivation. Microbios 3:241–246
     [Google Scholar]
  6. ).Salmonella entericaenterica
  7. Burghout P., Cron L. E., Gradstedt H., Quintero B., Simonetti E., Bijlsma J. J. E., Bootsma H. J., Hermans P. W. M.( 2010). Carbonic anhydrase is essential for Streptococcus pneumoniae growth in environmental ambient air. J Bacteriol 192:4054–4062 [View Article][PubMed]
     [Google Scholar]
  8. Burghout P., Vullo D., Scozzafava A., Hermans P. W. M., Supuran C. T.( 2011). Inhibition of the β-carbonic anhydrase from Streptococcus pneumoniae by inorganic anions and small molecules: Toward innovative drug design of antiinfectives?. Bioorg Med Chem 19:243–248 [View Article][PubMed]
     [Google Scholar]
  9. Bury-Moné S., Mendz G. L., Ball G. E., Thibonnier M., Stingl K., Ecobichon C., Avé P., Huerre M., Labigne A.& other authors ( 2008). Roles of alpha and beta carbonic anhydrases of Helicobacter pylori in the urease-dependent response to acidity and in colonization of the murine gastric mucosa. Infect Immun 76:497–509 [View Article][PubMed]
     [Google Scholar]
  10. Covarrubias A., Larsson A. M., Högbom M., Lindberg J., Bergfors T., Björkelid C., Mowbray S. L., Unge T., Jones T. A.( 2005). Structure and function of carbonic anhydrases from Mycobacterium tuberculosis. J Biol Chem 280:18782–18789 [View Article][PubMed]
     [Google Scholar]
  11. Covarrubias A. S., Bergfors T., Jones T. A., Högbom M.( 2006). Structural mechanics of the pH-dependent activity of β-carbonic anhydrase from Mycobacterium tuberculosis. J Biol Chem 281:4993–4999 [View Article][PubMed]
     [Google Scholar]
  12. Cronk J. D., Endrizzi J. A., Cronk M. R., O’neill J. W., Zhang K. Y. J.( 2001). Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity. Protein Sci 10:911–922 [View Article][PubMed]
     [Google Scholar]
  13. Cronk J. D., Rowlett R. S., Zhang K. Y., Tu C., Endrizzi J. A., Lee J., Gareiss P. C., Preiss J. R.( 2006). Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase. Biochemistry 45:4351–4361 [View Article][PubMed]
     [Google Scholar]
  14. Dixon N. M., Kell D. B.( 1989). The inhibition by CO2 of the growth and metabolism of micro-organisms. J Appl Bacteriol 67:109–136 [View Article][PubMed]
     [Google Scholar]
  15. Dobrinski K. P., Boller A. J., Scott K. M.( 2010). Expression and function of four carbonic anhydrase homologs in the deep-sea chemolithoautotroph Thiomicrospira crunogena. Appl Environ Microbiol 76:3561–3567 [View Article][PubMed]
     [Google Scholar]
  16. Elleuche S., Pöggeler S.( 2010). Carbonic anhydrases in fungi. Microbiology 15:23–29 [View Article][PubMed]
     [Google Scholar]
  17. Enfors S. O., Molin G.( 1980). Effect of high concentrations of carbon dioxide on growth rate of Pseudomonas fragi, Bacillus cereus and Streptococcus cremoris. J Appl Bacteriol 48:409–416 [View Article][PubMed]
     [Google Scholar]
  18. Felce J., Saier M. H. Jr( 2004). Carbonic anhydrases fused to anion transporters of the SulP family: evidence for a novel type of bicarbonate transporter. J Mol Microbiol Biotechnol 8:169–176 [View Article][PubMed]
     [Google Scholar]
  19. Fukuzawa H., Suzuki E., Komukai Y., Miyachi S.( 1992). A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci U S A 89:4437–4441 [View Article][PubMed]
     [Google Scholar]
  20. Gill C. O., Tan K. H.( 1979). Effect of carbon dioxide on growth of Pseudomonas fluorescens. Appl Environ Microbiol 38:237–240[PubMed]
     [Google Scholar]
  21. Guilloton M. B., Korte J. J., Lamblin A. F., Fuchs J. A., Anderson P. M.( 1992). Carbonic anhydrase in Escherichia coli. A product of the cyn operon. J Biol Chem 267:3731–3734[PubMed]
     [Google Scholar]
  22. Guilloton M. B., Lamblin A. F., Kozliak E. I., Gerami-Nejad M., Tu C., Silverman D., Anderson P. M., Fuchs J. A.( 1993). A physiological role for cyanate-induced carbonic anhydrase in Escherichia coli. J Bacteriol 175:1443–1451[PubMed]
     [Google Scholar]
  23. Hashimoto M., Kato J.( 2003). Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure. Biosci Biotechnol Biochem 67:919–922 [View Article][PubMed]
     [Google Scholar]
  24. Hewett-Emmett D., Tashian R. E.( 1996). Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families. Mol Phylogenet Evol 5:50–77 [View Article][PubMed]
     [Google Scholar]
  25. Hoffmann K. M., Samardzic D., Heever K., Rowlett R. S.( 2011). Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion. Arch Biochem Biophys 511:80–87 [View Article][PubMed]
     [Google Scholar]
  26. Høiby N.( 2006). P. aeruginosa in cystic fibrosis patients resists host defenses, antibiotics. Microbe 1:571–577
     [Google Scholar]
  27. Innocenti A., Supuran C. T.( 2010). Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases. Bioorg Med Chem Lett 20:6208–6212 [View Article][PubMed]
     [Google Scholar]
  28. Iverson T. M., Alber B. E., Kisker C., Ferry J. G., Rees D. C.( 2000). A closer look at the active site of γ-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila. Biochemistry 39:9222–9231 [View Article][PubMed]
     [Google Scholar]
  29. Joseph P., Turtaut F., Ouahrani-Bettache S., Montero J. L., Nishimori I., Minakuchi T., Vullo D., Scozzafava A., Köhler S.& other authors ( 2010). Cloning, characterization, and inhibition studies of a beta-carbonic anhydrase from Brucella suis. J Med Chem 53:2277–2285 [View Article][PubMed]
     [Google Scholar]
  30. Joseph P., Ouahrani-Bettache S., Montero J. L., Nishimori I., Minakuchi T., Vullo D., Scozzafava A., Winum J. Y., Köhler S., Supuran C. T.( 2011). A new β-carbonic anhydrase from Brucella suis, its cloning, characterization, and inhibition with sulfonamides and sulfamates, leading to impaired pathogen growth. Bioorg Med Chem 19:1172–1178 [View Article][PubMed]
     [Google Scholar]
  31. Kalai S., Achour W., Abdeladhim A., Bejaoui M., Ben Hassen A.( 2005). [Pseudomonas aeruginosa isolated in immunocompromised patients: antimicrobial resistance, serotyping, and molecular typing]. Med Mal Infect 35:530–535 [View Article][PubMed]
     [Google Scholar]
  32. Kaur S., Mishra M. N., Tripathi A. K.( 2009). Regulation of expression and biochemical characterization of a β-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7. FEMS Microbiol Lett 299:149–158 [View Article][PubMed]
     [Google Scholar]
  33. Kempner W., Schlayer C.( 1942). Effect of CO2 on the growth rate of the Pneumococcus. J Bacteriol 43:387–396[PubMed]
     [Google Scholar]
  34. King A. D., Nagel C. W.( 1975). Influence of carbon dioxide upon the metabolism of Pseudomonas aeruginosa. J Food Sci 40:362–366 [View Article]
     [Google Scholar]
  35. Kusian B., Sültemeyer D., Bowien B.( 2002). Carbonic anhydrase is essential for growth of Ralstonia eutropha at ambient CO2 concentrations. J Bacteriol 184:5018–5026 [View Article][PubMed]
     [Google Scholar]
  36. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A.& other authors ( 2007). clustal w and clustal_x version 2.0. Bioinformatics 23:2947–2948 [View Article][PubMed]
     [Google Scholar]
  37. Mach H., Middaugh C. R., Lewis R. V.( 1992). Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem 200:74–80 [View Article][PubMed]
     [Google Scholar]
  38. Marchler-Bauer A., Lu S., Anderson J. B., Chitsaz F., Derbyshire M. K., DeWeese-Scott C., Fong J. H., Geer L. Y., Geer R. C.& other authors ( 2011). CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:Database issueD225–D229 [View Article][PubMed]
     [Google Scholar]
  39. Merlin C., Masters M., McAteer S., Coulson A.( 2003). Why is carbonic anhydrase essential to Escherichia coli?. J Bacteriol 185:6415–6424 [View Article][PubMed]
     [Google Scholar]
  40. Mesaros N., Nordmann P., Plésiat P., Roussel-Delvallez M., Van Eldere J., Glupczynski Y., Van Laethem Y., Jacobs F., Lebecque P.& other authors ( 2007). Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13:560–578 [View Article][PubMed]
     [Google Scholar]
  41. Mitsuhashi S., Ohnishi J., Hayashi M., Ikeda M.( 2004). A gene homologous to β-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions. Appl Microbiol Biotechnol 63:592–601 [View Article][PubMed]
     [Google Scholar]
  42. Mukherjee S., Saha B., Das A. K.( 2009). Differential chemical and thermal unfolding pattern of Rv3588c and Rv1284 of Mycobacterium tuberculosis – a comparison by fluorescence and circular dichroism spectroscopy. Biophys Chem 141:94–104 [View Article][PubMed]
     [Google Scholar]
  43. Nishimori I., Minakuchi T., Kohsaki T., Onishi S., Takeuchi H., Vullo D., Scozzafava A., Supuran C. T.( 2007). Carbonic anhydrase inhibitors: the beta-carbonic anhydrase from Helicobacter pylori is a new target for sulfonamide and sulfamate inhibitors. Bioorg Med Chem Lett 17:3585–3594 [View Article][PubMed]
     [Google Scholar]
  44. Nishimori I., Minakuchi T., Vullo D., Scozzafava A., Innocenti A., Supuran C. T.( 2009). Carbonic anhydrase inhibitors. Cloning, characterization, and inhibition studies of a new beta-carbonic anhydrase from Mycobacterium tuberculosis. J Med Chem 52:3116–3120 [View Article][PubMed]
     [Google Scholar]
  45. Nishimori I., Minakuchi T., Maresca A., Carta F., Scozzafava A., Supuran C. T.( 2010). The β-carbonic anhydrases from Mycobacterium tuberculosis as drug targets. Curr Pharm Des 16:3300–3309 [View Article][PubMed]
     [Google Scholar]
  46. Nishimori I., Minakuchi T., Vullo D., Scozzafava A., Supuran C. T.( 2011). Inhibition studies of the β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium with sulfonamides and sulfamates. Bioorg Med Chem 19:5023–5030 [View Article][PubMed]
     [Google Scholar]
  47. Ohri L. K., Plummer S.( 2004). Prevention and control of nosocomial infections, 4th edition. Ann Pharmacother 38:515–516 [View Article]
     [Google Scholar]
  48. Park H.-M., Park J. H., Choi J. W., Lee J., Kim B. Y., Jung C. H., Kim J. S.( 2012). Structures of the γ-class carbonic anhydrase homologue YrdA suggest a possible allosteric switch. Acta Crystallogr D Biol Crystallogr 68:920–926 [View Article][PubMed]
     [Google Scholar]
  49. Ramanan R., Kannan K., Vinayagamoorthy N., Ramkumar K., Sivanesan S., Chakrabarti T.( 2009). Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis. Biotechnol Bioprocess Eng 14:32–37 [View Article]
     [Google Scholar]
  50. Richard P., Le Floch R., Chamoux C., Pannier M., Espaze E., Richet H.( 1994). Pseudomonas aeruginosa outbreak in a burn unit: role of antimicrobials in the emergence of multiply resistant strains. J Infect Dis 170:377–383 [View Article][PubMed]
     [Google Scholar]
  51. Rowlett R. S.( 2010). Structure and catalytic mechanism of the β-carbonic anhydrases. Biochim Biophys Acta 1804:362–373 [View Article][PubMed]
     [Google Scholar]
  52. Sawaya M. R., Cannon G. C., Heinhorst S., Tanaka S., Williams E. B., Yeates T. O., Kerfeld C. A.( 2006). The structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two. J Biol Chem 281:7546–7555 [View Article][PubMed]
     [Google Scholar]
  53. Sears D. F., Eisenberg R. M.( 1961). A model representing a physiological role of CO2 at the cell membrane. J Gen Physiol 44:869–887 [View Article][PubMed]
     [Google Scholar]
  54. Şentürk M., Gülçin İ., Beydemir Ş., Küfrevioğlu Ö. İ., Supuran C. T.( 2011). In vitro inhibition of human carbonic anhydrase I and II isozymes with natural phenolic compounds. Chem Biol Drug Des 77:494–499 [View Article][PubMed]
     [Google Scholar]
  55. Smith K. S., Ferry J. G.( 1999). A plant-type (beta-class) carbonic anhydrase in the thermophilic methanoarchaeon Methanobacterium thermoautotrophicum. J Bacteriol 181:6247–6253[PubMed]
     [Google Scholar]
  56. Smith K. S., Ferry J. G.( 2000). Prokaryotic carbonic anhydrases. FEMS Microbiol Rev 24:335–366 [View Article][PubMed]
     [Google Scholar]
  57. Smith K. S., Jakubzick C., Whittam T. S., Ferry J. G.( 1999). Carbonic anhydrase is an ancient enzyme widespread in prokaryotes. Proc Natl Acad Sci U S A 96:15184–15189 [View Article][PubMed]
     [Google Scholar]
  58. Smith K. S., Cosper N. J., Stalhandske C., Scott R. A., Ferry J. G.( 2000). Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase. J Bacteriol 182:6605–6613 [View Article][PubMed]
     [Google Scholar]
  59. So A. K. C., Espie G. S.( 1998). Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803. Plant Mol Biol 37:205–215 [View Article][PubMed]
     [Google Scholar]
  60. Son M. S., Matthews W. J. Jr, Kang Y., Nguyen D. T., Hoang T. T.( 2007). In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect Immun 75:5313–5324 [View Article][PubMed]
     [Google Scholar]
  61. Supuran C. T.( 2007a). Novel targets against Helicoblacter pylori: a bioinformatic approach. Future Microbiol 2:111–114 [View Article]
     [Google Scholar]
  62. Supuran C. T.( 2007b). Carbonic anhydrases as drug targets–an overview. Curr Top Med Chem 7:825–833 [View Article][PubMed]
     [Google Scholar]
  63. Supuran C. T.( 2008). Carbonic anhydrases–an overview. Curr Pharm Des 14:603–614 [View Article][PubMed]
     [Google Scholar]
  64. Supuran C. T.( 2011). Bacterial carbonic anhydrases as drug targets: towards novel antibiotics?. Front Pharmacol 2:1–6 [View Article][PubMed]
     [Google Scholar]
  65. Supuran C. T., Scozzafava A.( 2007). Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem 15:4336–4350 [View Article][PubMed]
     [Google Scholar]
  66. Tripp B. C., Smith K., Ferry J. G.( 2001). Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem 276:48615–48618 [View Article][PubMed]
     [Google Scholar]
  67. Valdivia R. H., Falkow S.( 1997). Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277:2007–2011 [View Article][PubMed]
     [Google Scholar]
  68. Vullo D., Nishimori I., Minakuchi T., Scozzafava A., Supuran C. T.( 2011). Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium. Bioorg Med Chem Lett 21:3591–3595 [View Article][PubMed]
     [Google Scholar]
  69. Winum J. Y., Köhler S., Supuran C. T.( 2010). Brucella carbonic anhydrases: new targets for designing anti-infective agents. Curr Pharm Des 16:3310–3316 [View Article][PubMed]
     [Google Scholar]
  70. Xu Y., Feng L., Jeffrey P. D., Shi Y., Morel F. M. M.( 2008). Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms. Nature 452:56–61 [View Article][PubMed]
     [Google Scholar]
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Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air
Microbiology 159, 1748 (2013); https://doi.org/10.1099/mic.0.066357-0
/content/journal/micro/10.1099/mic.0.066357-0
/content/journal/micro/10.1099/mic.0.066357-0
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