Strains and Culture conditions
Seawater S9905 was collected from Higashishina Kai 10 km from the coast of Yiriomote Island, Okinawa, Japan in May 1999 and seawater S0011 was collected from the coast of Miho, Shimizu City, Japan in November 2000. Seawater was treated by filtration with a 1.0 μm membrane filter to remove other microorganisms such as phytoplankton or flagellates before experimentation. All seawater samples were incubated in the dark. Bacteria were isolated from seawater sample after serial dilution in sterilized seawater (10-1 to 10-4), followed by spreading of 100 μl of each solution onto 1/10-diluted marine broth (Marine Broth 2216; Difco) or seawater based IDSM [17] agar plates. The plates were incubated at 30°C for up to 2 months, and marine bacteria were identified by growth on the plates containing 3% Nad in comparison with no growth on the agar plates containing 0.15% Nad for the same strain.
Seawater based IDSM medium was prepared using the following components (in grams/liter): NH4NO3, 1.0; NaCl, 30.0; MgSO4 • 7H2O, 0.5; KCl, 0.3; K2HPO4 1.5; C8H18N2O4S (HEPES), 2.38; CaCl2, 0.2. It also contained 10% glucose (10 ml) and 0.1 ml of 1 mM FeCl3.
Commercial siderophore desferroixamine (DEF, CIBA GEIGY) and synthetic 3OC6-HSL and C8-HSL [17] were filtered by 0.2 μm membrane before use. All containers were treated by 10%HCl more than 24 h before use.