Synthetic aerobactin mediates bacterial proliferation of Vibrio fischeri relevant to the Vibrio-squid symbiosis.

Abstract

The ability to acquire iron drives microbial fitness in marine environments where iron solubility is extremely low. Iron uptake via the siderophore aerobactin plays an important role in Vibrio fischeri colonization in the light organ of the Hawaiian bobtail squid. To date, the contribution of aerobactin to this mutualistic relationship has not been demonstrated quantitatively. Here we report a revised total synthesis of aerobactin, characterize its stability and iron-binding properties, and demonstrate its importance to Vibrio-squid mutualism. Synthetic aerobactin was characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry and iron binding was measured via the Chrome Azurol S (CAS) assay. Stability experiments in artificial seawater showed that aerobactin degrades slowly, with a half-life on the order of weeks. The activity of synthetic aerobactin was then demonstrated by growth recovery experiments with wild-type V. fischeri ES114 and mutant strains deficient in aerobactin biosynthesis (ΔiucABCD), its outer-membrane aerobactin receptor (ΔiutA), and in the inner-membrane importer (ΔfhuCDB). Available iron was controlled in cell culture experiments by the addition of 2,2'-bipyridine (BPY) or citrate to experimental growth media, with bathocuproine disulfonic acid (BCDS) included as a membrane-impermeable, and poor iron-sequestering control. Exogenous aerobactin restored growth in iron-limited environments in a concentration-dependent manner, particularly in the biosynthesis-deficient mutant. Additionally, we report that BPY inhibits V. fischeri ES114 growth with a broad MIC range (128-256 μM) that highlights its capacity to disrupt the intracellular iron pool. Together, these findings support the use of AB-FeCit as the optimal media system for testing siderophore-dependent growth in V. fischeri and link synthetic aerobactin and iron availability to bacterial growth in a model marine mutualism.