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.