Photolysis of iron-siderophore chelates promotes bacterial-algal mutualism

Abstract

Marine microalgae support world fisheries production and influence climate through various mechanisms. They are also responsible for harmful blooms that adversely impact coastal ecosystems and economies. Optimal growth and survival of many bloom-forming microalgae, including climatically important dinoflagellates and coccolithophores, requires the close association of specific bacterial species, but the reasons for these associations are unknown. Here, we report that several clades of Marinobacter ubiquitously found in close association with dinoflagellates and coccolithophores produce an unusual lower-affinity dicitrate siderophore, vibrioferrin (VF). Fe-VF chelates undergo photolysis at rates that are 10-20 times higher than siderophores produced by free-living marine bacteria, and unlike the latter, the VF photoproduct has no measurable affinity for iron. While both an algal-associated bacterium and a representative dinoflagellate partner, Scrippsiella trochoidea, used iron from Fe-VF chelates in the dark, in situ photolysis of the chelates in the presence of attenuated sunlight increased bacterial iron uptake by 70% and algal uptake by >20-fold. These results suggest that the bacteria promote algal assimilation of iron by facilitating photochemical redox cycling of this critical nutrient. Also, binary culture experiments and genomic evidence suggest that the algal cells release organic molecules that are used by the bacteria for growth. Such mutualistic sharing of iron and fixed carbon has important implications toward our understanding of the close beneficial interactions between marine bacteria and phytoplankton, and the effect of these interactions on algal blooms and climate.

Fig. 1.

The 16S rRNA gene phylogeny of the Marinobacter clade and VF production profile. (A) Maximum-likelihood neighbor-joining tree with bootstrap support (≥50%) of Marinobacter 16S rRNA genes. (B) Production and utilization of VF by Marinobacter for iron acquisition, as determined by LC-MS, NMR, PCR screening of VF biosynthetic genes, and siderophore growth-promotion assays. Dn, dinoflagellate; Co, coccolithophore; Dt, diatoms; ND, not determined. Orange shading indicates those strains capable of VF production and uptake; green shading indicates VF uptake only. Bar denotes nucleotide substitutions per site.*For Marinobacter sp. ELB17, production and uptake were presumed based on the presence of close homologs of VF biosynthetic and uptake genes.

Fig. 2.

VF-mediated iron uptake in Marinobacter sp. DG879 and the dinoflagellate S. trochoidea in the dark and in the presence of sunlight. (A) The photolysis of Fe(III)-VF in sunlight produces a photoproduct (VF*) and Fe(II)′, which is quickly oxidized in SW to Fe(III)′. (B) ⁵⁵Fe uptake rates by Marinobacter sp. DG879. Uptake was performed using 1 μM Fe(III) and 3 μM total VF either in the dark or in the presence of attenuated natural sunlight (450 μE·m⁻²·s⁻¹) at 20 °C. The uncoupler of oxidative phosphorylation, CCCP, and incubation of cells at 4 °C were used as metabolic inhibitors; incubations were carried out in the dark. The reductant ascorbate was used to prevent the oxidation of photochemically produced Fe(II) in bacteria exposed to sunlight. (C) ⁵⁵Fe uptake by axenic S. trochoidea cells. Uptake was performed in trace-metal- and EDTA-free Aquil using 50 nM Fe(III) and 500 nM VF either in the dark (filled circles) or in the presence of attenuated natural sunlight (open circles) as above. Error bars in B and C represent SD of triplicate cultures. Bars were not shown when smaller than the symbol.

Fig. 3.

Partial predicted translation of M. algicola DG893 FbpA homolog aligned with known FbpA proteins. M. algicola DG893 accession no. EDM49388. Protein PDB ID nos.: Mannheimia haemolytica (1SI0), Bordetella pertussis (1Y9U), Campylobacter jejuni (1Y4T), and Synechocystis sp. FutA1 (2PT2). Alignment was performed using Muscle as implemented in CLC Sequence Viewer (Version 6). Identical residues are depicted with blue shading and similar residues with green shading. Y195 and Y196 are universally conserved in FbpA proteins and are responsible for binding Fe³⁺. Y139 is only found in a subset of FbpA proteins and is present in the coordination sphere of Fe³⁺ as well.

Fig. 4.

Ferrireductase assay of axenic cultures of S. trochoidea. Formation of Fe(II) as measured as its BPDS complex in the presence (open circles) or absence (filled circles) of S. trochoidea cells.

Fig. 5.

Growth pattern of a binary culture of the bloom-forming S. trochoidea and an associated VF-producing Marinobacter strain (DG879) in f/2 medium. Growth of S. trochoidea (filled diamonds) in the presence of Marinobacter sp. DG879 (closed circles) is shown alongside Marinobacter sp. DG879 growing alone in f/2 medium (open circles). Error bars represent SD of cell counts from triplicate cultures.

Fig. 6.

Bacterial–algal mutualism based on the photoreductive dissociation of Fe(III)-VF chelates. On binding iron in the light, Fe(III)-VF photolyzes to ultimately produce Fe(III)′, which is then assimilated by both organisms. In the algae, the assimilated iron is needed in large amounts to support photosynthetic fixation of carbon. Some of this fixed carbon is excreted and used to support bacterial growth and VF production. Due to the thick boundary layer surrounding large algal cells such as dinoflagellates, diffusion of VF and excreted organic carbon away from the algal cell is highly diminished.