PMC:59891 / 24601-30236 JSONTXT

{"project":"2_test","denotations":[{"id":"11716787-10877770-12178426","span":{"begin":711,"end":713},"obj":"10877770"},{"id":"11716787-10877770-12178427","span":{"begin":1299,"end":1301},"obj":"10877770"},{"id":"11716787-1987160-12178428","span":{"begin":3522,"end":3524},"obj":"1987160"},{"id":"11716787-10877770-12178429","span":{"begin":3771,"end":3773},"obj":"10877770"},{"id":"11716787-2952030-12178430","span":{"begin":3809,"end":3811},"obj":"2952030"}],"text":"Materials and Methods\n\nStrains and Culture conditions\nSeawater 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.\nSeawater 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.\nCommercial 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.\n\nMeasurement of bacterial numbers\nSeawater (30 L each) was incubated at 30°C for up to four weeks with the addition of DEF, plus 3OC6-HSL or plus C8-HSL; DEF only; HSL only; or without any addition. The concentration in seawater of each of the additions was adjusted to 0.1 nM.\nSeawater samples were collected after 24 h, 48 h, 72 h, 96 h, and 1 week of incubation. Total numbers of the microorganisms (cells/ml) in the seawater were determined by direct bacterial counting with 4', 6-diamino-2-phenylidole (DAPI) staining using epifluorescence microscopy [26]. Triplicate samples of seawater (1.35 ml) were collected and fixed with an equal volume of Mildform 10 NM (10% formalin neutral buffer). Three hundred microliters of 0.5 μg/ml DAPI solution was added and the solution was incubated for 5 min at room temperature. The DAPI reacted solution was filtered through a 0.2 μm GTBP membrane filter (MILLIPORE), after which the filter was fixed on a slide and was observed using an epifluorescence microscope (Nikon, EFD2). Total bacterial counts were obtained by calculation using the following formula: Bacterial number (cells/ml) = [Nave × Am] / [V × As]. Nave: Average number of cells counted from 20 views per sample; Am: Area of the filtration membrane (132.7 mm in this study); V: total filtered volume of seawater (3 ml); As: Area of counting view (64 × 10-6 mm in this study).\nCultivable species were enumerated by counting the number of colony forming units (CFU) on 1/10 Marine Broth or IDSM agar plates at 10-1 to 10-3 diluted seawater solutions.\n\nIdentification of bacteria by 16S rDNA sequences\nBacterial DNA were obtained by extracting pure cultivation cells with PureGene Kit (Gentra Systems). A 16S rDNA gene fragment (169 to 194 bp in length) was amplified by PCR using a universal primer complementary to position 517 to 534 (5'-ATTACCGCGGCTGCTGG-3') and a bacterial primer complementary to position 341 to 358 (5'-CCTACGGGAGGCAGCAG-3') [23]. The total 16S rDNA fragment was amplified by using two oligonucleotide primers, fD (5'-AGAGTTTGATCCTGGCTCAG-3') and rD (5'-AAGGAGGTGATCCAGCC-3') [31]. The PCR products were sequenced by using a 373 DNA sequencer (PE Biosystems) and analyzed in DDBJ databases.\n\nSiderophore production and cross-feeding assay\nSiderophore production and cross-feeding assays were performed as reported previously [17]. The chrome azurol S (CAS) assay [27] was used to detect siderophores. On CAS agar plates, the formation of colony and siderophore halos were evaluated following 7 days of colony incubation at 30°C. All isolated strains were inoculated on four different CAS plates: (i) without any addition, (ii) with the addition of 0.1 nM DEF, (iii) with the addition of 0.1 nM of 3OC6-HSL or C8-HSL, (iv) and with the addition of 0.1 nM DEF plus 0.1 nM of 3OC6-HSL or C8-HSL.\nThe induced siderophores of unknown strains were partially isolated and compared with desferroixamine by examination with the Csaky test [15] and the Arnow reaction [1] for their hydroxamate and catechol functionality respectively. In these assays, hydroxylamine and 2,3-dihydroxybenzoic acid, respectively, were used as the standards.\n\nDetermination of iron content in seawater\nCollected sea water 100 ml, was concentrated by lyophilization and resuspended in 2 ml of acid-treated distilled water. The salt component in seawater was removed by centrifugation. The concentration of iron in the seawater was measured with an inductively coupled plasma spectrometry (ICPS-1000IV) sequential plasma spectrometer (Shimazu) at the absorbance of iron atom (259.940 nm, 239.562 nm respectively). Iron standard solution (Fe100, WAKO Chemical Ltd) (Fe(NO3)3 in 0.1 mol/l • HNO3: 99 mg/L) was used for the determination of a calibration curve.\n\nNucleotide sequence accession number\nThe DDBJ GenBank accession numbers of the sequences for Erwinia nigrifluens, Shewanella putrefaciens, Pseudomonas doudoroffii., Caulobacter sp, Roseobacter sp., Cytophaga sp., Rhodobacter sp., Flavobacterim sp., Bartonella bacilliforms. Beta proteobacterium, Sphingomonas sp., and Gelidibacter sp. is Z96095, AF005255, AB019390, AB025196, Y15339, AB015545, U63949, U63955, M65249, AF026392, AB025720, AB001369, respectively. "}