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Applied and Environmental Microbiology, November 2005, p. 7263-7270, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7263-7270.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Ecology, Inhibitory Activity, and Morphogenesis of a Marine Antagonistic Bacterium Belonging to the Roseobacter Clade

Jesper Bartholin Bruhn,1* Kristian Fog Nielsen,2 Mette Hjelm,1 Michael Hansen,3 José Bresciani,3 Stefan Schulz,4 and Lone Gram1

Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, DTU Bldg. 221, DK-2800 Kongens, Lyngby, Denmark,1 Center for Microbial Biotechnology, BioCentrum, Technical University of Denmark, DK-2800 Kongens, Lyngby, Denmark,2 Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark,3 Institut für Organische Chemie, TU Braunschweig, Hagenring 30, D-38106 Braunschweig, Germany4

Received 3 January 2005/ Accepted 10 June 2005

Roseobacter strain 27-4 has been isolated from a turbot larval rearing unit and is capable of reducing mortality in turbot egg yolk sac larvae. Here, we demonstrate that the supernatant of Roseobacter 27-4 is lethal to the larval pathogens Vibrio anguillarum and Vibrio splendidus in a buffer system and inhibited their growth in marine broth. Liquid chromatography (LC) with both UV spectral detection and high-resolution mass spectrometry (HR-MS) identified the known antibacterial compound thiotropocin or its closely related precursor tropodithietic acid in the bioactive fractions. Antibacterial activity correlated with the appearance of a brownish pigment and was only formed in marine broth under static growth conditions. A thick biofilm of multicellular star-shaped aggregated cells formed at the air-liquid interface under static growth conditions. Here, the bioactive compound was the base peak in the LC-UV chromatograms of the extracts where it constituted 15% of the total peak area. Aerated conditions results in 10-fold-higher cell yield, however, cultures were nonpigmented, did not produce antibacterial activity, and grew as single cells. Production of antibacterial compounds may be quorum regulated, and we identified the acylated homoserine lactone (3-hydroxy-decanoyl homoserine lactone) from cultures of Roseobacter 27-4 using LC-HR-MS. The signal molecule was primarily detected in stagnant cultures. Roseobacter 27-4 grew between 10 and 30°C but died rapidly at 37°C. Also, the antibacterial compounds was sensitive to heat and was inactivated at 37°C in less than 2 days and at 25°C in 8 days. Using Roseobacter 27-4 as a probiotic culture will require that is be established in stagnant or adhered conditions and, due to the temperature sensitivity of the active compound, constant production must be ensured.

* Corresponding author. Mailing address: Danish Institute for Fisheries Research, Department of Seafood Research, Søltofts Plads, DTU Bldg. 221, DK-2800 Kgs. Lyngby, Denmark. Phone: 45-45-25-25-71. Fax: 45-45-88-47-74. E-mail: jbb{at}dfu.min.dk.

Applied and Environmental Microbiology, November 2005, p. 7263-7270, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7263-7270.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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