Glycerol inhibition of melanin biosynthesis in the environmental Aeromonas salmonicida 34melT

Abstract

The environmental strain Aeromonas salmonicida subsp. pectinolytica 34melT produces abundant melanin through the homogentisate pathway in several culture media, but unexpectedly not when grown in a medium containing glycerol. Using this observation as a starting point, this study investigated the underlying causes of the inhibition of melanin synthesis by glycerol, to shed light on factors that affect melanin production in this microorganism. The effect of different carbon sources on melanin formation was related to the degree of oxidation of their C atoms, as the more reduced substrates delayed melanization more than the more oxidized ones, although only glycerol completely abolished melanin production. Glyphosate, an inhibitor of aromatic amino acid synthesis, did not affect melanization, while bicyclopyrone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (Hpd), the enzyme responsible for the synthesis of homogentisate, prevented melanin synthesis. These results showed that melanin production in 34melT depends on the degradation of aromatic amino acids from the growth medium and not on de novo aromatic amino acid synthesis. The presence of glycerol changed the secreted protein profile, but none of the proteins affected could be directly connected with melanin synthesis or transport. Transcription analysis of hpd, encoding the key enzyme for melanin synthesis, showed a clear inhibition caused by glycerol. The results obtained in this work indicate that a significant decrease in the transcription of hpd, together with a more reduced intracellular state, would lead to the abolishment of melanin synthesis observed. The effect of glycerol on melanization can thus be attributed to a combination of metabolic and regulatory effects.

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References

  1. Ahmad S, Lee SY, Kong HG, Jo EJ, Choi HK, Khan R, Lee SW (2016) Genetic determinants for pyomelanin production and its protective effect against oxidative stress in Ralstonia solanacearum. PLoS One 11:e0160845. https://doi.org/10.1371/journal.pone.0160845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Almeida-Paes R, Frases S, de Sousa Araújo G, de Oliveira MM, Gerfen GJ, Nosanchuk JD, Zancopé-Oliveira RM (2012) Biosynthesis and functions of a melanoid pigment produced by species of the Sporothrix complex in the presence of L-tyrosine. Appl Environ Microbiol 78:8623–8630. https://doi.org/10.1128/AEM.02414-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Arias-Barrau E, Olivera ER, Luengo JM, Fernández C, Galán B, García JL, Díaz E, Miñambres B (2004) The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. J Bacteriol 186:5062–5077. https://doi.org/10.1128/JB.186.15.5062-5077.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Arunasri K, Adil M, Khan PA, Shivaji S (2014) Global gene expression analysis of long-term stationary phase effects in E. coli K12 MG1655. PLoS One 9:e96701. https://doi.org/10.1371/journal.pone.0096701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Aubi O, Flydal MI, Zheng H, Skjærven L, Rekand I, Leiros HK, Haug BE, Cianciotto NP, Martinez A, Underhaug J (2015) Discovery of a specific inhibitor of pyomelanin synthesis in Legionella pneumophila. J Med Chem 58:8402–8412. https://doi.org/10.1021/acs.jmedchem.5b01589

    Article  CAS  PubMed  Google Scholar 

  6. Bongaerts J, Krämer M, Müller U, Raeven L, Wubbolts M (2001) Metabolic engineering for microbial production of aromatic amino acids and derived compounds. Metab Eng 3:289–300. https://doi.org/10.1006/mben.2001.0196

    Article  CAS  PubMed  Google Scholar 

  7. Chai B, Wang H, Chen X (2012) Draft genome sequence of high-melanin-yielding Aeromonas media strain WS. J Bacteriol 194:6693–6694. https://doi.org/10.1128/JB.01807-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chen LY (2013) Glycerol modulates water permeation through Escherichia coli aquaglyceroporin GlpF. Biochim Biophys Acta 1828:1786–1793. https://doi.org/10.1016/j.bbamem.2013.03.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Danilova LV, Gelfand MS, Lyubetsky VA, Laikova ON (2003) Computer-assisted analysis of regulation of the glycerol-3-phosphate metabolism in genomes of proteobacteria. Mol Biol 37:716–722. https://doi.org/10.1023/A:1026037027266

    Article  CAS  Google Scholar 

  10. de Almeida A, Giordano AM, Nikel PI, Pettinari MJ (2010) Effects of aeration on the synthesis of poly(3-hydroxybutyrate) from glycerol and glucose in recombinant Escherichia coli. Appl Environ Microbiol 76:2036–2040. https://doi.org/10.1128/AEM.02706-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ebanks RO, Goguen M, McKinnon S, Pinto DM, Ross NW (2005) Identification of the major outer membrane proteins of Aeromonas salmonicida. Dis Aquat Org 68:29–38. https://doi.org/10.3354/dao068029

    Article  CAS  PubMed  Google Scholar 

  12. Escapa IF, del Cerro C, García JL, Prieto MA (2013) The role of GlpR repressor in Pseudomonas putida KT2440 growth and PHA production from glycerol. Environ Microbiol 15:93–110. https://doi.org/10.1111/j.1462-2920.2012.02790.x

    Article  CAS  PubMed  Google Scholar 

  13. Fernández-Cañón JM, Granadino B, Beltrán-Valero de Bernabé D, Renedo M, Fernández-Ruiz E, Peñalva MA, Rodríguez de Córdoba S (1996) The molecular basis of alkaptonuria. Nat Genet 14:19–24. https://doi.org/10.1038/ng0996-19

    Article  PubMed  Google Scholar 

  14. Fuqua WC, Weiner RM (1993) The melA gene is essential for melanin biosynthesis in the marine bacterium Shewanella colwelliana. J Gen Microbiol 139:1105–1114. https://doi.org/10.1099/00221287-139-5-1105

    Article  CAS  PubMed  Google Scholar 

  15. Gonzalez R, Murarka A, Dharmadi Y, Yazdani SS (2008) A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli. Metab Eng 10:234–245. https://doi.org/10.1016/j.ymben.2008.05.001

    Article  CAS  PubMed  Google Scholar 

  16. Herrera MC, Krell T, Zhang X, Ramos JL (2009) PhhR binds to target sequences at different distances with respect to RNA polymerase in order to activate transcription. J Mol Biol 394:576–586. https://doi.org/10.1016/j.jmb.2009.09.045

    Article  CAS  PubMed  Google Scholar 

  17. Hunter RC, Newman DK (2010) A putative ABC transporter, HatABCDE, is among molecular determinants of pyomelanin production in Pseudomonas aeruginosa. J Bacteriol 192:5962–5971. https://doi.org/10.1128/JB.01021-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Keith KE, Killip L, He P, Moran GR, Valvano MA (2007) Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate. J Bacteriol 189:9057–9065. https://doi.org/10.1128/JB.00436-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Keller S, Macheleidt J, Scherlach K, Schmaler-Ripcke J, Jacobsen ID, Heinekamp T, Brakhage AA (2011) Pyomelanin formation in Aspergillus fumigatus requires HmgX and the transcriptional activator HmgR but is dispensable for virulence. PLoS One 6:e26604. https://doi.org/10.1371/journal.pone.0026604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kotob SI, Coon SL, Quintero EJ, Weiner RM (1995) Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Appl Environ Microbiol 61:1620–1622

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Larionov A, Krause A, Miller W (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics 6:62. https://doi.org/10.1186/1471-2105-6-62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Larson TJ, Cantwell JS, van Loo-Bhattacharya AT (1992) Interaction at a distance between multiple operators controls the adjacent, divergently transcribed glpTQ-glpACB operons of Escherichia coli K-12. J Biol Chem 267:6114–6121

    CAS  PubMed  Google Scholar 

  23. Loprasert S, Whangsuk W, Dubbs JM, Sallabhan R, Somsongkul K, Mongkolsuk S (2007) HpdR is a transcriptional activator of Sinorhizobium meliloti hpdA, which encodes a herbicide-targeted 4-hydroxyphenylpyruvate dioxygenase. J Bacteriol 189:3660–3664. https://doi.org/10.1128/JB.01662-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Martin JP, Batkoff B (1987) Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis. Free Radic Biol Med 3:241–250. https://doi.org/10.1016/S0891-5849(87)80031-X

    Article  CAS  PubMed  Google Scholar 

  25. McFall E, Newman EB (1996) Amino acids as carbon sources. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin EC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella: cellular and molecular biology, 2nd edn. ASM Press, Washington, DC, pp 358–379

    Google Scholar 

  26. Mistry JB, Bukhari M, Taylor AM (2013) Alkaptonuria. Rare Dis 1:e27475. https://doi.org/10.4161/rdis.27475

    Article  PubMed  PubMed Central  Google Scholar 

  27. Morales G, Linares JF, Beloso A, Albar JP, Martínez JL, Rojo F (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. https://doi.org/10.1128/JB.186.5.1337-1344.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nikel PI, Kim J, de Lorenzo V (2014) Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol 16:239–254. https://doi.org/10.1111/1462-2920.12224

    Article  CAS  PubMed  Google Scholar 

  29. Noorian P, Jie H, Chen Z, Kjelleberg S, Wilkins MR, Sun S, McDougald D (2017) Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii. FEMS Microbiol Ecol 93(12). https://doi.org/10.1093/femsec/fix147

  30. Nosanchuk JD, Casadevall A (2003) The contribution of melanin to microbial pathogenesis. Cell Microbiol 5:203–223. https://doi.org/10.1046/j.1462-5814.2003.00268.x

    Article  CAS  PubMed  Google Scholar 

  31. Palmer GC, Palmer KL, Jorth PA, Whiteley M (2010) Characterization of the Pseudomonas aeruginosa transcriptional response to phenylalanine and tyrosine. J Bacteriol 192:2722–2728. https://doi.org/10.1128/JB.00112-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pavan ME, Abbott SL, Zorzópulos J, Janda JM (2000) Aeromonas salmonicida subsp. pectinolytica subsp. nov., a new pectinase-positive subspecies isolated from a heavily polluted river. Int J Syst Evol Microbiol 50:1119–1124. https://doi.org/10.1099/00207713-50-3-1119

    Article  CAS  PubMed  Google Scholar 

  33. Pavan ME, Pavan EE, López NI, Levin L, Pettinari MJ (2015) Living in an extremely polluted environment: clues from the genome of melanin-producing Aeromonas salmonicida subsp. pectinolytica 34melT. Appl Environ Microbiol 81:5235–5248. https://doi.org/10.1128/AEM.00903-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Plonka PM, Grabacka M (2006) Melanin synthesis in microorganisms-biotechnological and medical aspects. Acta Biochim Pol 53:429–443

    CAS  PubMed  Google Scholar 

  35. Rocaboy-Faquet E, Noguer T, Romdhane S, Bertrand C, Dayan FE, Barthelmebs L (2014) Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors. Appl Microbiol Biotechnol 98:7243–7252. https://doi.org/10.1007/s00253-014-5793-5

    Article  CAS  PubMed  Google Scholar 

  36. Rodríguez-Rojas A, Mena A, Martín S, Borrell N, Oliver A, Blázquez J (2009) Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection. Microbiology 155:1050–1057. https://doi.org/10.1099/mic.0.024745-0

    Article  CAS  PubMed  Google Scholar 

  37. Rojo F (2010) Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment. FEMS Microbiol Rev 34:658–684. https://doi.org/10.1111/j.1574-6976.2010.00218.x

    Article  CAS  Google Scholar 

  38. Ryan A, Kaplan E, Nebel JC, Polycarpou E, Crescente V, Lowe E, Preston GM, Sim E (2014) Identification of NAD(P)H quinone oxidoreductase activity in azoreductases from P. aeruginosa: azoreductases and NAD(P)H quinone oxidoreductases belong to the same FMN-dependent superfamily of enzymes. PLoS One 9:e98551. https://doi.org/10.1371/journal.pone.0098551

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sanchez-Amat A, Ruzafa C, Solano F (1998) Comparative tyrosine degradation in Vibrio cholerae strains. The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell. Comp Biochem Physiol B Biochem Mol Biol 119:557–562. https://doi.org/10.1016/S0305-0491(98)00028-5

    Article  CAS  PubMed  Google Scholar 

  40. Santucci A, Bernardini G, Braconi D, Petricci E, Manetti F (2017) 4-Hydroxyphenylpyruvate dioxygenase and its inhibition in plants and animals: small molecules as herbicides and agents for the treatment of human inherited diseases. J Med Chem 60:4101–4125. https://doi.org/10.1021/acs.jmedchem.6b01395

    Article  CAS  PubMed  Google Scholar 

  41. Solano F (2014) Melanins: skin pigments and much more - types, structural models, biological functions, and formation routes. New J Sci 1:1–28. https://doi.org/10.1155/2014/498276

    Article  CAS  Google Scholar 

  42. Stepanova V, Rodionov DA (2011) Genomic analysis of transcriptional regulation of aromatic amino acid metabolism in gamma-proteobacteria. Department of Bioengineering and Bioinformatics of MV Lomonosov Moscow State University 352:186–188

    Google Scholar 

  43. Stuber K, Burr SE, Braun M, Wahli T, Frey J (2003) Type III secretion genes in Aeromonas salmonicida subsp salmonicida are located on a large thermolabile virulence plasmid. J Clin Microbiol 41:3854–3856. https://doi.org/10.1128/JCM.41.8.3854-3856.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tanaka KH, Vincent AT, Emond-Rheault JG, Adamczuk M, Frenette M, Charette SJ (2017) Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns. BMC Genomics 18:528. https://doi.org/10.1186/s12864-017-3921-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Turick CE, Caccavo F Jr, Tisa LS (2003) Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin. FEMS Microbiol Lett 220:99–104. https://doi.org/10.1016/S0378-1097(03)00096-X

    Article  CAS  PubMed  Google Scholar 

  46. Turick CE, Knox AS, Becnel JM, Ekechukwu AA, Milliken CE (2010) Properties and function of pyomelanin. In: Elnashar MM (ed) Biopolymers, 1st edn. Sciyo, Rijeka, pp 449–472. https://doi.org/10.5772/10273

    Chapter  Google Scholar 

  47. Upton C, Buckley JT (1995) A new family of lipolytic enzymes? Trends Biochem Sci 20:178–179. https://doi.org/10.1016/S0968-0004(00)89002-7

    Article  CAS  PubMed  Google Scholar 

  48. Vanden Bergh P, Heller M, Braga-Lagache S, Frey J (2013) The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors. Proteome Sci 11:42. https://doi.org/10.1186/1477-5956-11-42

    Article  CAS  Google Scholar 

  49. Vincent AT, Rouleau FD, Moineau S, Charette SJ (2017) Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species’ lifestyles and taxonomic dilemma. FEMS Microbiol Lett 364(23). https://doi.org/10.1093/femsle/fnx239

  50. Wang H, Qiao Y, Chai B, Qiu C, Chen X (2015) Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS. PLoS One 10:e0120923. https://doi.org/10.1371/journal.pone.0120923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wu L, Lin X, Peng X (2009) From proteome to genome for functional characterization of pH-dependent outer membrane proteins in Escherichia coli. J Proteome Res 8:1059–1070. https://doi.org/10.1021/pr800818r

    Article  CAS  PubMed  Google Scholar 

  52. Yu HB, Zhang YL, Lau YL, Yao F, Vilches S, Merino S, Tomas JM, Howard SP, Leung KY (2005) Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91. Appl Environ Microbiol 71:4469–4477. https://doi.org/10.1128/AEM.71.8.4469-4477.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zatkova A (2011) An update on molecular genetics of Alkaptonuria (AKU). J Inherit Metab Dis 34:1127–1136. https://doi.org/10.1007/s10545-011-9363-z

    Article  PubMed  Google Scholar 

  54. Zeng Z, Guo XP, Cai X, Wang P, Li B, Yang JL, Wang X (2017) Pyomelanin from Pseudoalteromonas lipolytica reduces biofouling. Microb Biotechnol 10:1718–1731. https://doi.org/10.1111/1751-7915.12773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

N.I.L. and M.J.P. are career investigators from CONICET. E.S.V. and D.E.E. hold doctoral fellowships from CONICET.

Funding

This work was partially supported by the University of Buenos Aires, CONICET, and Agencia Nacional de Promoción Científica y Tecnológica.

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M.E.P., N.I.L., and M.J.P. conceived the project and designed experiments. All authors carried out experiments. M.E.P., N.I.L., and M.J.P. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Nancy I. López or M. Julia Pettinari.

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Pavan, M.E., Venero, E.S., Egoburo, D.E. et al. Glycerol inhibition of melanin biosynthesis in the environmental Aeromonas salmonicida 34melT. Appl Microbiol Biotechnol 103, 1865–1876 (2019). https://doi.org/10.1007/s00253-018-9545-9

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Keywords

  • Aeromonas salmonicida subsp. pectinolytica
  • Glycerol
  • Melanin inhibition
  • Homogentisate pathway
  • 4-Hydroxyphenylpyruvate dioxygenase
  • hpd expression