Marine Biotechnology

, Volume 13, Issue 6, pp 1062–1073 | Cite as

Bioactivity, Chemical Profiling, and 16S rRNA-Based Phylogeny of Pseudoalteromonas Strains Collected on a Global Research Cruise

  • Nikolaj G. Vynne
  • Maria Månsson
  • Kristian F. Nielsen
  • Lone Gram
Original Article


One hundred one antibacterial Pseudoalteromonas strains that inhibited growth of a Vibrio anguillarum test strain were collected on a global research cruise (Galathea 3), and 51 of the strains repeatedly demonstrated antibacterial activity. Here, we profile secondary metabolites of these strains to determine if particular compounds serve as strain or species markers and to determine if the secondary metabolite profile of one strain represents the bioactivity of the entire species. 16S rRNA gene similarity divided the strains into two primary groups: One group (51 strains) consisted of bacteria which retained antibacterial activity, 48 of which were pigmented, and another group (50 strains) of bacteria which lost antibacterial activity upon sub-culturing, two of which were pigmented. The group that retained antibacterial activity consisted of six clusters in which strains were identified as Pseudoalteromonas luteoviolacea, Pseudoalteromonas aurantia, Pseudoalteromonas phenolica, Pseudoalteromonas ruthenica, Pseudoalteromonas rubra, and Pseudoalteromonas piscicida. HPLC-UV/VIS analyses identified key peaks, such as violacein in P. luteoviolacea. Some compounds, such as a novel bromoalterochromide, were detected in several species. HPLC-UV/VIS detected systematic intra-species differences for some groups, and testing several strains of a species was required to determine these differences. The majority of non-antibacterial, non-pigmented strains were identified as Pseudoalteromonas agarivorans, and HPLC-UV/VIS did not further differentiate this group. Pseudoalteromonas retaining antibacterial were more likely to originate from biotic or abiotic surfaces in contrast to planktonic strains. Hence, the pigmented, antibacterial Pseudoalteromonas have a niche specificity, and sampling from marine biofilm environments is a strategy for isolating novel marine bacteria that produce antibacterial compounds.


Pseudoalteromonas Antibacterial activity Secondary metabolites Bioprospecting Galathea 3 



We acknowledge Dr. Jesper B. Bruhn for valuable input during the early phase of this study. This study was supported by the Programme Commission on Health, Food and Welfare under the Danish Council for Strategic Research. The present work was carried out as part of the Galathea 3 expedition under the auspices of the Danish Expedition Foundation. This is Galathea 3 contribution no. p73.

Supplementary material

10126_2011_9369_MOESM1_ESM.doc (244 kb)
ESM 1 (DOC 244 kb)


  1. Armstrong E, Yan LM, Boyd KG, Wright PC, Burgess JG (2001) The symbiotic role of marine microbes on living surfaces. Hydrobiologia 461:37–40CrossRefGoogle Scholar
  2. Bewley CA, Faulkner DJ (1998) Lithistid sponges: star performers or hosts to the stars. Angew Chem Int Ed 37:2162–2178CrossRefGoogle Scholar
  3. Bowman JP (2007) Bioactive compound synthetic capacity and ecological significance of marine bacterial genus Pseudoalteromonas. Mar Drugs 5:220–241PubMedCrossRefGoogle Scholar
  4. Boyd KG, Adams DR, Burgess JG (1999) Antibacterial and repellent activities of marine bacteria associated with algal surfaces. Biofouling 14:227–236CrossRefGoogle Scholar
  5. Burgess JG, Jordan EM, Bregu M, Mearns-Spragg A, Boyd KG (1999) Microbial antagonism: a neglected avenue of natural products research. J Biotechnol 70:27–32PubMedCrossRefGoogle Scholar
  6. Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552PubMedGoogle Scholar
  7. Doull JL, Vining LC (1990) Nutritional control of actinorhodin production by Streptomyces coelicolor A3(2)—suppressive effects of nitrogen and phosphate. Appl Microbiol Biotechnol 32:449–454PubMedCrossRefGoogle Scholar
  8. Egan S, Holmström C, Kjelleberg S (2001) Pseudoalteromonas ulvae sp nov., a bacterium with antifouling activities isolated from the surface of a marine alga. Int J Syst Evol Microbiol 51:1499–1504PubMedGoogle Scholar
  9. Egan S, James S, Holmström C, Kjelleberg S (2002) Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata. Environ Microbiol 4:433–442PubMedCrossRefGoogle Scholar
  10. Egan S, Thomas T, Kjelleberg S (2008) Unlocking the diversity and biotechnological potential of marine surface associated microbial communities. Curr Opin Microbiol 11:219–225PubMedCrossRefGoogle Scholar
  11. Fisher RA (1958) Statistical methods for research workers. Hafner, New YorkGoogle Scholar
  12. Fox GE, Wisotzkey JD, Jurtshuk P (1992) How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Evol Microbiol 42:166Google Scholar
  13. Franks A, Egan S, Holmstrom C, James S, Lappin-Scott H, Kjelleberg S (2006) Inhibition of fungal colonization by Pseudoalteromonas tunicata provides a competitive advantage during surface colonization. Appl Environ Microbiol 72:6079–6087PubMedCrossRefGoogle Scholar
  14. Frisvad JC, Andersen B, Thrane U (2008) The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi. Mycol Res 112:231–240PubMedCrossRefGoogle Scholar
  15. Gauthier MJ (1976a) Morphological, physiological, and biochemical characteristics of some violet-pigmented bacteria isolated from seawater. Can J Microbiol 22:138–149PubMedCrossRefGoogle Scholar
  16. Gauthier MJ (1976b) Alteromonas rubra sp. nov., a new marine antibiotic-producing bacterium. Int J Syst Bacteriol 26:459–466CrossRefGoogle Scholar
  17. Gauthier MJ, Flatau GN (1976) Antibacterial activity of marine violet-pigmented Alteromonas with special reference to the production of brominated compounds. Can J Microbiol 22:1612–1619PubMedCrossRefGoogle Scholar
  18. Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA (2007) The current status of species recognition and identification in Aspergillus. Stud Mycol 59:1PubMedCrossRefGoogle Scholar
  19. Gram L, Melchiorsen J, Bruhn JB (2010) Antibacterial activity of marine culturable bacteria collected from a global sampling of ocean surface waters and surface swabs of marine organisms. Mar Biotechnol 12:439–451PubMedCrossRefGoogle Scholar
  20. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696PubMedCrossRefGoogle Scholar
  21. Hayashida-Soiza G, Uchida A, Mori N, Kuwahara Y, Ishida Y (2008) Purification and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain. J Appl Microbiol 105:1672–1677PubMedCrossRefGoogle Scholar
  22. Hjelm M, Bergh Ø, Riaza A, Nielsen J, Melchiorsen J, Jensen S, Duncan H, Ahrens P, Birkbeck H, Gram L (2004) Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units. Syst Appl Microbiol 27:360–371PubMedCrossRefGoogle Scholar
  23. Holmström C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30:285–293PubMedCrossRefGoogle Scholar
  24. Holmström C, James S, Egan S, Kjelleberg S (1996) Inhibition of common fouling organisms by marine bacterial isolates with special reference to the role of pigmented bacteria. Biofouling 10:251–259CrossRefGoogle Scholar
  25. Holmström C, Egan S, Franks A, McCloy S, Kjelleberg S (2002) Antifouling activities expressed by marine surface associated Pseudoalteromonas species. FEMS Microbiol Ecol 41:47–58PubMedCrossRefGoogle Scholar
  26. Hornemann U, Hurley LH, Speedie MK, Floss HG (1971) Biosynthesis of indolmycin. J Am Chem Soc 93:3028–3035PubMedCrossRefGoogle Scholar
  27. Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C (2001) Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol 67:1529–1535PubMedCrossRefGoogle Scholar
  28. Hurdle JG, O'Neill AJ, Chopra I (2004) Anti-staphylococcal activity of indolmycin, a potential topical agent for control of staphylococcal infections. J Antimicrob Chemothe 54:549–552CrossRefGoogle Scholar
  29. Isnansetyo A, Kamei Y (2003) MC21-A, a bactericidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenolica sp. nov. O-BC30T, against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 47:480–488PubMedCrossRefGoogle Scholar
  30. Ivanova EP, Flavier S, Christen R (2004) Phylogenetic relationships among marine Alteromonas-like proteobacteria: emended description of the family Alteromonadaceae and proposal of Pseudoalteromonadaceae fam. nov., Colwelliaceae fam. nov., Shewanellaceae fam. nov., Moritellaceae fam. nov., Ferrimonadaceae fam. nov., Idiomarinaceae fam. nov. and Psychromonadaceae fam. nov. Int J Syst Evol Microbiol 54:1773–1788PubMedCrossRefGoogle Scholar
  31. James SG, Holmström C, Kjelleberg S (1996) Purification and characterization of a novel antibacterial protein from the marine bacterium D2. Appl Environ Microbiol 62:2783–2788PubMedGoogle Scholar
  32. Jensen PR, Mafnas C (2006) Biogeography of the marine actinomycete Salinispora. Environ Microbiol 8:1881–1888PubMedCrossRefGoogle Scholar
  33. Jensen PR, Williams PG, Oh DC, Zeigler L, Fenical W (2007) Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 73:1146–1152PubMedCrossRefGoogle Scholar
  34. Jiang Z, Boyd KG, Mearns-Spragg A, Adams DR, Wright PC, Burgess JG (2000) Two diketopiperazines and one halogenated phenol from cultures of the marine bacterium, Pseudoalteromonas luteoviolacea. Nat Prod Let 14:435–440CrossRefGoogle Scholar
  35. Kalesperis GS, Prahlad KV, Lynch DL (1975) Toxigenic studies with the antibiotic pigments from Serratia marcescens. Can J Microbiol 21:213PubMedCrossRefGoogle Scholar
  36. Kalinovskaya NI, Ivanova EP, Alexeeva YV, Gorshkova NM, Kuznetsova TA, Dmitrenok AS, Nicolau DV (2004) Low-molecular-weight, biologically active compounds from marine Pseudoalteromonas species. Curr Microbiol 48:441–446PubMedCrossRefGoogle Scholar
  37. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066PubMedCrossRefGoogle Scholar
  38. Laatsch H, Pudleiner H (1989) Marine bakterien, I: synthese von pentabrompseudilin, einem phenylpyrrol aus Alteromonas luteoviolaceus. Liebigs Ann Chem 1989:863–881CrossRefGoogle Scholar
  39. Lichstein HC, Vandesand VF (1945) Violacein, an antibiotic pigment produced by Chromobacterium violaceum. J Infect Dis 76:47–51CrossRefGoogle Scholar
  40. Lovell FM (1966) Structure of a bromine-rich marine antibiotic. J Am Chem Soc 88:4510–4511CrossRefGoogle Scholar
  41. Månsson M, Phipps RK, Gram L, Munro MH, Larsen TO, Nielsen KF (2010) Explorative solid-phase extraction (E-SPE) for accelerated microbial natural product discovery. J Nat Prod 73:1126–1132PubMedCrossRefGoogle Scholar
  42. Matz C, Webb JS, Schupp PJ, Phang SY, Penesyan A, Egan S, Steinberg P, Kjelleberg S (2008) Marine biofilm bacteria evade eukaryotic predation by targeted chemical defense. PLoS ONE 3:e2744PubMedCrossRefGoogle Scholar
  43. McCarthy SA, Johnson RM, Kakimoto D (1994) Characterization of an antibiotic produced by Alteromonas luteoviolacea Gauthier 1982, 85 isolated from Kinko Bay, Japan. J Appl Microbiol 77:426–432CrossRefGoogle Scholar
  44. Mearns-Spragg A, Bregu M, Boyd KG, Burgess JG (1998) Cross-species induction and enhancement of antimicrobial activity produced by epibiotic bacteria from marine algae and invertebrates, after exposure to terrestrial bacteria. Lett Appl Microbiol 27:142–146PubMedCrossRefGoogle Scholar
  45. Newman DJ (2008) Natural products as leads to potential drugs: an old process or the new hope for drug discovery? J Med Chem 51:2589–2599PubMedCrossRefGoogle Scholar
  46. Nielsen KF, Smedsgaard J (2003) Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J Chromatogr A 1002:111–136PubMedCrossRefGoogle Scholar
  47. Nielsen KF, Sumarah MW, Frisvad JC, Miller JD (2006) Production of metabolites from the Penicillium roqueforti complex. J Agric Food Chem 54:3756–3763PubMedCrossRefGoogle Scholar
  48. Penesyan A, Marshall-Jones Z, Holmstrom C, Kjelleberg S, Egan S (2009) Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs. FEMS Microbiol Ecol 69:113–124PubMedCrossRefGoogle Scholar
  49. Rao D, Webb JS, Kjelleberg S (2005) Competitive interactions in mixed-species biofilms containing the marine bacterium Pseudoalteromonas tunicata. Appl Environ Microbiol 71:1729–1736PubMedCrossRefGoogle Scholar
  50. Sanchez S, Chavez A, Forero A, Garcia-Huante Y, Romero A, Sanchez M, Rocha D, Sanchez B, Avalos M, Guzman-Trampe S, Rodriguez-Sanoja R, Langley E, Ruiz B (2010) Carbon source regulation of antibiotic production. J Antibiot 63:442–459PubMedCrossRefGoogle Scholar
  51. Simmons TL, Coates RC, Clark BR, Engene N, Gonzalez D, Esquenazi E, Dorrestein PC, Gerwick WH (2008) Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages. P Natl A Sci USA 105:4587–4594CrossRefGoogle Scholar
  52. Skov MN, Pedersen K, Larsen JL (1995) Comparison of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling for typing of Vibrio anguillarum serovar O1. Appl Environ Microbiol 61:1540–1545PubMedGoogle Scholar
  53. Speitling M, Smetanina OE, Kuznetsova TA, Laatsch H (2007) Marine bacteria. XXXV. Bromoalterochromides A and A′, unprecedented chromopeptides from a marine Pseudoalteromonas maricaloris strain KMM 636. J Antibiot 60:36–42PubMedCrossRefGoogle Scholar
  54. Stackebrandt E, Frederiksen W, Garrity GM, Grimont PAD, Kampfer P, Maiden MCJ, Nesme X, Rossello-Mora R, Swings J, Truper HG, Vauterin L, Ward AC, Whitman WB (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52:1043–1047PubMedCrossRefGoogle Scholar
  55. Sudek S, Lopanik NB, Waggoner LE, Hildebrand M, Anderson C, Liu H, Patel A, Sherman DH, Haygood MG (2006) Identification of the putative bryostatin polyketide synthase gene cluster from “Candidatus Endobugula sertula”, the uncultivated microbial symbiont of the marine bryozoan Bugula neritina. J Nat Prod 70:67–74CrossRefGoogle Scholar
  56. Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56:564–577PubMedCrossRefGoogle Scholar
  57. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596PubMedCrossRefGoogle Scholar
  58. Violot S, Aghajari N, Czjzek M, Feller G, Sonan GK, Gouet P, Gerday C, Haser R, Receveur-Brechot V (2005) Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering. J Mol Biol 348:1211–1224PubMedCrossRefGoogle Scholar
  59. Werner RG (1980) Uptake of indolmycin in gram-positive bacteria. Antimicrob Agents Chemother 18:858PubMedGoogle Scholar
  60. Wietz M, Schramm A, Jørgensen B, Gram L (2010) Latitudinal patterns in the abundance of major marine bacterioplankton groups. Aquat Microb Ecol 61:179–189Google Scholar
  61. Wratten SJ, Wolfe MS, Andersen RJ, Faulkner DJ (1977) Antibiotic metabolites from a marine pseudomonad. Antimicrob Agents Chemother 11:411–414PubMedGoogle Scholar
  62. Zheng L, Yan XJ, Han XT, Chen HM, Lin W, Lee FSC, Wang XR (2006) Identification of norharman as the cytotoxic compound produced by the sponge (Hymeniacidon perleve)-associated marine bacterium Pseudoalteromonas piscicida and its apoptotic effect on cancer cells. Biotechnol Appl Biochem 44:135–142PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Nikolaj G. Vynne
    • 1
  • Maria Månsson
    • 2
  • Kristian F. Nielsen
    • 2
  • Lone Gram
    • 1
  1. 1.National Food InstituteTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Department of Systems BiologyTechnical University of DenmarkKgs. LyngbyDenmark

Personalised recommendations