Function-related replacement of bacterial siderophore pathways

Abstract

Bacterial genomes are rife with orphan biosynthetic gene clusters (BGCs) associated with secondary metabolism of unrealized natural product molecules. Often up to a tenth of the genome is predicted to code for the biosynthesis of diverse metabolites with mostly unknown structures and functions. This phenomenal diversity of BGCs coupled with their high rates of horizontal transfer raise questions about whether they are really active and beneficial, whether they are neutral and confer no advantage, or whether they are carried in genomes because they are parasitic or addictive. We previously reported that Salinispora bacteria broadly use the desferrioxamine family of siderophores for iron acquisition. Herein we describe a new and unrelated group of peptidic siderophores called salinichelins from a restricted number of Salinispora strains in which the desferrioxamine biosynthesis genes have been lost. We have reconstructed the evolutionary history of these two different siderophore families and show that the acquisition and retention of the new salinichelin siderophores co-occurs with the loss of the more ancient desferrioxamine pathway. This identical event occurred at least three times independently during the evolution of the genus. We surmise that certain BGCs may be extraneous because of their functional redundancy and demonstrate that the relative evolutionary pace of natural pathway replacement shows high selective pressure against retention of functionally superfluous gene clusters.

Figure 1

Deletion of a siderophore cluster. (a) Comparative genomic analysis showed that the majority of analyzed Salinispora genomes contain the full des cluster for the production of desferrioxamine under iron-limiting conditions (example S. pacifica CNY282). However, almost the whole biosynthesis cluster is deleted in 19 of the analyzed strains, whereas the surrounding genes (light gray) are highly conserved. Only the periplasmic binding protein and metal transporters (dark gray) remain. (b) Structure of desferrioxamine E.

Figure 2

Organization of the NRPS16/salinichelin BGC. Comparative genome mining revealed an orphan BGC only present in strains without the desferrioxamine gene cluster. The salinichelin (slc) gene cluster consists of 13 genes (slcA-slcM). Highlighted is the NRPS domain architecture of slcE. A, adenylation domain; C, condensation domain; E, epimerization domain; T, thiolation domain.

Figure 3

Production and structures of the salinichelins. (a) HPLC chromatograms of extracts of S. pacifica CNY-331 grown under Fe-limiting conditions leads to the production of a series of salinichelin siderophores (red trace) compared with the Fe-supplemented culture (blue trace). HPLC was monitored at 210 nm. (b) The structures of salinichelins A-C (2–4) is shown.

Figure 4

Siderophore evolution in Salinispora. (a) The character trace history analysis of slc (formerly NRPS16) was performed in Mesquite v2.75 (45). Each strain in the phylogenetic tree is depictured as circle, black marked strains contain the gene cluster, white strains do not. Arrows indicated where the gene cluster was likely acquired via horizontal gene transfer (HGT) independently in S. pacifica and twice in S. arenicola. (b) The phylogenetic gene tree of slc shows two main clades: one for S. pacifica and one for S. arenicola, indicating that the pathway was exchanged once between the two species and once within S. arenicola. (c) Evolutionary scenario of HGT of slc and the concurrent deletion of the more ancient des gene cluster as deviated from the comparison of the gene tree and species tree with Ranger-DTL (Supplementary Figures S17–S20; Bansal et al., 2012) and the character trace history.