Results

Analysis of total bacterial number in seawater
The total number of bacteria in S9905 seawater was analyzed by DAPI staining and counting during 7 days. The iron content in S9905 seawater was determined to be 7.8 nM by ICP analysis. Seawater was amended with trace amount of DEF (1 nM) plus 3OC6-HSL (1 nM) or C8-HSL (1 nM), DEF(1 nM) only, each of HSL(1 nM) only. Total bacterial numbers were shown to increase after the addition of DEF, HSL or DEF plus an HSL in comparison with those obtained from seawater without any addition (Fig. 1A). However, the total bacterial number in the seawater with the addition of DEF or HSL only was observed to reach to a maximum value and then began to decrease after the sixth day (Fig. 1A), after which low amounts were recorded during the remaining time of the four-week incubation period (data not shown). Total bacterial numbers in seawater treated with DEF plus an HSL increased during seven days and the value was found to start decreasing after day 14 during four-weeks incubation period (data not shown). Comparing the maximum values of the total bacterial numbers during the first week, the values for DEF, 3OC6-HSL, C8-HSL, DEF plus 3OC6-HSL, and DEF plus C8-HSL treated seawater were 2.50-fold, 2.69-fold, 1.60-fold 3.14-fold, and 2.62-fold respectively higher than plain seawater. The increase in total bacterial number in the seawater control was unclear but such bacterial changes in bacterial abundance have also been observed in several iron enrichment experiments [5,21].
To confirm the effects of such chemical compounds as a nutrient or not, high concentrations of DEF, HSL, DEF plus HSL were amended to the same seawater, and the total bacterial number was determined by DAPI counting (Fig. 1B). The total bacterial number was also stimulated by the addition of high concentrations of DEF, HSL, DEF plus HSL. The maximum value of S9905 seawater was 5.07 × 105 cells/ml, and 4.22 × 105 cells/ml for 1 μM of DEF plus 1 μM of 3OC6-HSL, and 1 μM of DEF plus 1 μM of C8-HSL amended seawater samples respectively, which was similar to 0.1 nM of DEF plus 0.1 nM of 3OC6-HSL (4.72 × 105 cells/ml), and 0.1 nM of DEF plus 0.1 nM of C8-HSL (3.95 × 105 cells/ml) amended seawater samples. Also, the trend of bacterial number increase was very similar for the seawater with the amended with HSL under high or low concentration. However, the maximum value of S9905 1 μM of DEF amended seawater was 1.42 × 105 cells/ml, which was lower than the control seawater (Fig. 1B).
Repeat experiments were performed with S0011 seawater, which was collected from a different location. The iron content in S0011 seawater was 11.2 nM and total bacterial number was shown in Fig. 2. S0011 seawater was observed to contain more bacteria (2.1 × 105 cells/ml) than that in S9905 seawater (2.07 × 104 cells/ml). The total bacteria number in 0.1 nM DEF plus 3OC6-HSL or C8-HSL (Fig 2A) showed a similar pattern with that in Fig 1A, where an increasing trend in 7 days incubation occurred except for a drop in values for 2 nd-day samples. Comparing the maximum values of the total bacterial numbers, the values for DEF, 3OC6-HSL, C8-HSL, DEF plus 3OC6-HSL, and DEF plus C8-HSL treated seawater were 3.64-fold, 2.73-fold, 2.73-fold, 4.84-fold, and 7.92-fold respectively higher than plain seawater. In the meantime, the bacterial counting were performed for high concentration amendments S0011 seawater (Fig. 2B), 3.80-fold, 3.66-fold 5.11-fold, and 8.18-fold respectively of bacterial number increase were observed in 1 μM of 3OC6-HSL, 1 μM of C8-HSL, 1 μM of DEF plus 1 μM of 3OC6-HSL, 1 μM of DEF plus 1 μM of C8-HSL treated seawater. There was not a large difference in the maximum values in the low (0.1 nM) and high concentration (1 μM) DEF and HSL amended seawater, but a slight change of bacterial number was also observed from 1 μM of DEF treated seawater.

Analysis of cultivable bacterial amount
To understand bacterial diversity, the cultivable species from the above seawater samples was further investigated. Samples were collected every 24 hr during one week and were evaluated by investigation of colony formation units (CFU) of different phenotypes on marine broth or IDSM agar plates. The phenotypes of observed strains obtained are summarized in table 1. Total numbers of cultivable bacteria in seawater samples as enumerated by CFU were found to occur as follows: without addition < with DEF < with 3OC6-HSL < with C8-HSL < with DEF plus 3OC6-HSL < with DEF plus C8-HSL. By comparison three phenotypes were obtained from the untreated seawater, seven, four and five phenotypes were obtained from the seawater with the addition of 0.1 nM DEF, 0.1 nM 3OC6-HSL or 0.1 nM C8-HSL respectively. Nine and eight phenotypes were isolated from seawater with the addition of 0.1 nM DEF plus 0.1 nM 3OC6-HSL, 0.1 nM DEF plus 0.1 nM C8-HSL respectively. CFU counting of each phenotype indicated that the relative contribution of each strain changed even though the same strains were detected in all samples. For example, as shown in Table 1: Strain GMO4-1 was detected in all six seawater samples; it was the preferentially cultivable strain in unamended seawater only. The relative percent contribution of other organisms increased (as calculated by CFU values) depending upon which amendment was used. The preferentially cultivable species from each amendment were: GMO4-4 and GMO4-5 (the same CFU value), GMO4-2, GMO4-12, GMO4-2, GMO4-8 from DEF, 3OC6-HSL, C8-HSL, DEF plus 3OC6-HSL, DEF plus C8-HSL amended sea water respectively (Table 1).

Identification of bacterial species by 16S rDNA sequences
To identify whether the eighteen total isolated phenotypes were different species or not, 16S rDNA analysis of all the strains was performed. Table 1 shows the 16S rDNA sequence homology of the strains. Each isolated strain obtained from the agar plates was identified to be unrelated.
Table 1 showed that the cultivable microorganism diversity obtained in the same seawater was affected by the addition of DEF and HSL. Erwinia nigrifluens GMO4-1 and Shewanella putrefaciens GMO4-2 were identified from all seawater samples, and indicates that these two species were not influenced by either DEF and HSL. Pseudomonas doudoroffii. GMO4-3 was isolated from the unamended seawater and seawater amended with DEF, or DEF plus C8-HSL. Some strains could only be isolated from the seawater after the additions. For example, Cytophaga sp. GMO4-6, Sphingomonas sp. GMO4-11, and Beta proteobacterium strain GMO 4-10 were isolated from the seawater with DEF plus 3OC6-HSL; Cytophaga sp. GMO4-6 was also obtained from the community that was treated with DEF plus C8-HSL. Furthermore, GMO4-13, GMO4-14, GMO4-15, GMO4-16, GMO4-17 and GMO4-18 were found to be unknown species cultivated only from seawater that was amended with DEF, DEF plus 3OC6-HSL, C8-HSL, or DEF plus C8-HSL respectively.

Relationship between bacterial growth and siderophore production under iron-limited conditions
To investigate whether the isolated strains grew under iron-limited conditions, all strains were inoculated on seawater-based IDSM agar plates containing 0.01 μM Fe(III) which is similar to the iron content in of seawater. Iron content in the seawater was detected to be 7.8 nM, which dissolved iron would be more less than 7.8 nM. Thus this seawater may be classified as an iron-limited environment for most microorganisms.
From eighteen isolates, only three strains were found to grow on iron-limited IDSM agar plates. Bacterial growth and siderophore production of all isolated strains were investigated with chrome azurol (CAS) assay [27] and the cross-feeding assay [17] with the addition of 0.1 nM each of DEF, 3OC6-HSL, C8-HSL and DEF plus 3OC6-HSL or C8-HSL respectively. Table 2 shows that the strains GMO4-11 Sphingomonas sp. and GMO4-14 did not grow on iron-limited IDSM agar medium and did not produce siderophores on CAS agar plates. However, their colonies and siderophore halo formation were observed on the IDSM and CAS agar plates after the addition of 0.1 nM DEF plus 0.1 nM 3OC6-HSL. Similarly, GMO4-16, and GMO4-18 only grew on the IDSM and CAS agar plates with the addition of DEF plus C8-HSL. Strains GMO4-11, GMO4-14, GMO4-16, GMO4-18 were further investigated in the A tumefaciens A136 reporter strain assay [34] using the synthetic HSLs as standards. However, 3OC6- and C8-HSLs were not detected in the supernatant extracts of these strains by this reporter strain assay. Also siderophore production by GMO4-11, GMO4-14, GMO4-16 and GMO4-18 in IDSM medium containing 0.01 μM Fe(III) with the addition of O.1 nM DEF plus 0.1 nM of 3OC6- or C8-HSL were partially extracted and investigated by the Csaky test and Arnow reaction (Table 3). These two assays are well known for the detection of hydroxamate (Csaky test) or catechol groups (Arnow reaction) which are typical functional groups that bind iron. The induced siderophores from the above four strains were most likely to be different types as shown by these assays. Furthermore, all of the siderophore components were observed to have different reactivities in the Csaky test and Arnow reaction with exogenous DEF. Siderophore components from GMO4-11, GMO4-14 were positive in the Arnow test and indicated the existence of catechol groups while those from GMO4-16 and GMO4-18 showed negative reactivities in both assays. Artificially added DEF is a hydroxamate and was positive in the Casky test. The added DEF (0.1 nM) to the cultivation medium of the four strains was at a lower concentration than the detection limit for both assays. Thus, the Arnow test positive components from GMO4-11 and GMO4-14, and siderophore components from GMO4-16 and GMO4-18 were siderophores produced by these strains only in response to the existence of DEF plus 3OC6-HSL or C8-HSL.