Turnerbactin, a novel triscatecholate siderophore from the shipworm endosymbiont Teredinibacter turnerae T7901.

Andrew W Han,Moriah Sandy,Margo G Haygood,Amaro E Trindade-Silva,Daniel L Distel,Brian Fishman,Alison Butler,Carlos A G Soares

doi:10.1371/journal.pone.0076151

Abstract

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

Highlights

Iron is required by all but a few organisms and serves as an important cofactor for many essential enzymes
Due to its homology to the entD gene of enterobactin biosynthesis, the gene product of TERTU_1510 is proposed to be the P-pant transferase that posttranslationally attaches a P-pant moiety to a conserved Ser residue of the apo-peptidyl-carrier protein (PCP) domains of TnbF
We propose that turnerbactin is produced beginning with the production and activation of dihydroxybenzoic acid (DHB) by the protein products of tnbCEBA

Summary

Introduction

Iron is required by all but a few organisms and serves as an important cofactor for many essential enzymes. In the world’s oceans, 99% of the Fe(III) is bound by uncharacterized organic ligands [1], [2]. Iron is a limiting nutrient for bacteria in animals, where it is often tightly bound to proteins such as ferritin, transferrin, lactoferrin, or incorporated into heme containing proteins [3], [4]. In response to iron limitation, many bacteria and some fungi produce siderophores, low-molecular weight compounds with a high affinity for Fe(III). Hundreds of siderophores have been characterized, the majority being from terrestrial organisms [5], [6], [7]. Siderophores can be classified by their mode of biosynthesis, non-ribosomal peptide synthetase (NRPS)-dependent or NRPS-independent biosynthesis [8], as well as by their major type of functional binding groups, catechols, hydroxamic acids, and a-hydroxycarboxylic acids

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