acid (TDA) that is an efficient inhibitor of V. anguillarum and other fish-pathogenic bacteria [22–25]. Phaeobacter spp. are commonly isolated from larval cultures of marine fish and shellfish [26–28], and do not appear to adversely affect fish larvae [22,29]. Ruegeria mobilis, a close relative to Phaeobacter also producing TDA, is a cosmopolitan marine bacterium that can be isolated from most ocean waters, apart from polar waters [30]. In a previous study, it was demonstrated that Phaeobacter and Ruegeria isolated from a turbot hatchery [26] could eliminate V. anguillarum in a seawater-based combined liquid-surface system [22]. It was demonstrated, using a TDA-negative mutant, that TDA production was likely a key factor in the pathogen inhibition. The purpose of the present study is to determine if P. gallaeciensis BS107 (DSM 17395) could antagonize V. anguillarum in fish larvae and cultures of their feed organisms. To the authors’ knowledge, no study on antagonistic probiotic bacteria has yet elucidated the mechanism of action in vivo. Therefore a non-antagonistic TDA- negative mutant of P. gallaeciensis BS107 (DSM 17395) was created to investigate the in vivo mechanism of action, as emphasized by Tinh et al. [15]. The type strain P. gallaeciensis BS107 (DSM17395) [28] was chosen, since its inhibition of V. anguillarum in Tetraselmis cultures was more pronounced than that of other Phaeobacter and Ruegeria strains, as assessed in a preliminary experiment (data not shown). Gnotobiotic algae and rotifers were used for studying probiotic and nutritional effects of the introduced organism, as recommended [15,31,32]. Materials and Methods Bacterial strains and media All strains and plasmids are listed in Table 1. Phaeobacter (Roseobacter) gallaeciensis BS107 (DSM17395) was isolated from seawater in scallop (Pecten maximus) cultures [28]. Vibrio anguillarum serotype O1 strain NB10 was used in algal and rotifer experiments. It was isolated from the Gulf of Bothnia and has caused disease in rainbow trout [33,34]. The strain has been tagged by insertion of plasmid pNQFlaC4-gfp27 (cat, gfp) into an intergenic region on the chromosome, and was kindly provided by D. Milton, University of Umea˚ [35]. V. anguillarum serotype O2a HI610 was used in challenge trials with cod larvae. The strain was isolated from diseased cod juveniles in the closed seawater basin Lake Parisvatn by the Institute of Marine Research (IMR), Norway, and has been used in challenge trials with cod [36–39]. It has been selected from a group of V. anguillarum strains of different serotypes, being the strain that caused the highest mortality in challenge trials with turbot, halibut and cod larvae [40]. Bacteria from frozen stock cultures (280uC) were streaked on half-strength Marine Agar (KMA; 27.6 g Difco 212185 Marine Agar, 15 g Instant Ocean Sea Salts, 7.5 g Agar, 1 l deionized water). KMA was also used for counting P. gallaeciensis. V. anguillarum was counted on Tryptone-Soy Agar (TSA; Oxoid CM0131) containing 6 mg/l chloramphenicol. The bacterial precultures for the algae and rotifer experiments were grown in 20 ml of KYTSS (2 g Bacto Yeast extract, 1.25 g Bacto Tryptone, 20 g Sigma Sea Salts, 1 l deionized water) [41] at 25uC with aeration (200 rpm) until OD600 = 1.0. The cells were harvested at 5,000 x g, washed twice, and used as inoculum for algae and rotifer experiments. Bacteria were diluted and washed in artificial seawater (ASW; 2% Sigma Sea Salts). Axenicity of algae and rotifer cultures was controlled by plating 100 ml on KMA and incubating for 7 days at 25uC. For the challenge trials, V. anguillarum HI610 was grown in tryptone-soy broth with additional 0.5% NaCl at 20uC with shaking at 60 rpm to an OD600 of about 0.5. The P. gallaeciensis Probiotic Phaeobacter for Marine Larviculture strains were grown in MB without shaking at 20uC until stationary phase was reached. All strains were harvested by centrifugation (1,825 x g), washed twice, and resuspended in aerated autoclaved 80% seawater. The bacterial concentrations in these suspensions were determined using a counting chamber for V. anguillarum, and for the P. gallaeciensis strains by measuring OD600 after centrifu- gation and dissolving in 0.1M NaOH. Generation of a TDA-negative Phaeobacter mutant A mutant library of P. gallaeciensis BS107 was created by random transposon insertion mutagenesis using the EZ-Tn5 ,R6Kcori/ KAN.Tnp Transposome Kit (Epicentre, Madison, WI), following the procedure of Geng et al. [42]. Ten non-pigmented mutants were selected, and absence of TDA production was confirmed by UHPLC-TOFMS and in an agar-diffusion test against V. anguillarum [27]. Growth rates of selected mutants were compared to the wild type in aerated (200 rpm) KYTSS cultures at 30uC, and one of the strains with a growth rate comparable to the wild type, BS107-Pda8, was chosen for further experiments. Using rescue cloning as described in the transposome kit manual, the mutated locus was identified as CDS104961, which encodes for a ‘‘periplasmic component of a TRAP-type C4-dicarboxylate transport system’’, as annotated on the BS107 genome on www. roseobase.org. Fluorescence tagging of Phaeobacter P. gallaeciensis BS107 was tagged chromosomally with a miniTn7(Gm)PA1/04/03DsRedExpress-a cassette, using a mini-Tn7 tagging system [43,44]. The delivery and helper plasmids were electroporated into P. gallaeciensis, followed by selection on KMA containing 75 mg/ml gentamicin. pPDA11, a transcriptional fusion of the tdaC promoter to a promoterless gfp gene ligated to the broad-host range plasmid pRK415, was constructed in an analogous manner to pHG1011 as described in Geng et al. 2010 [45]. Phaeobacter antagonism in algae Tetraselmis suecica CCAP 66/4 (Prasinophyceae) was obtained as axenic culture from the Culture Collection of Algae and Protozoa (Oban, UK). It was cultured in B-medium [46], a mineral algae medium, based on ASW. The 250-ml culture bottles were closed with cotton plugs and slowly aerated through a 0.2 mm syringe filter and a silicone tube, to prevent settling of particles. Light intensity on the bottles was 13,000 lux (daylight spectrum). Algal concentrations were assessed by measuring absorption at 665 nm, and calibrating with counts of axenic reference cultures in a Neubauer-improved counting chamber, using formaldehyde as fixative (0.5% final concentration). For each V. anguillarum inoculum level tested, eight bottles of 150 ml of B-medium were inoculated with 6.66104 cells/ml axenic T. suecica. Two bottles were inoculated with approximately 107 cfu/ml washed P. gallaeciensis BS107, two bottles with the same level of washed mutant P. gallaeciensis BS107-Pda8 cells, and four bottles were left axenic. The cultures were grown for 2 days and axenicity was checked. All cultures, except two axenic negative controls, were inoculated with V. anguillarum NB10 to concentrations of 10, 100, 1000, or 104 cfu/ml. Inoculum levels were verified by plate- counting. Concentrations of algae and both bacterial species were observed until day 5 after inoculation of the pathogen. Two independent experiments were performed for every initial concentration of V. anguillarum. Nannochloropsis oculata CCMP525 (Eustigmatophyceae) was obtained as axenic culture from the Center for Culture of Marine Phytoplankton (West Boothbay Harbor, ME). Since it did not grow in ASW-based B-medium, it PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43996