Pseudobulbiferamides: Plasmid-Encoded Ureidopeptide Natural Products with Biosynthetic Gene Clusters Shared Among Marine Bacteria of Different Genera

Abstract

Ureidopeptidic natural products possess a wide variety of favorable pharmacological properties. In addition, they have been shown to mediate core physiological functions in producer bacteria. Here, we report that similar ureidopeptidic natural products with conserved biosynthetic gene clusters are produced by different bacterial genera that coinhabit marine invertebrate microbiomes. We demonstrate that a Microbulbifer strain isolated from a marine sponge can produce two different classes of ureidopeptide natural products encoded by two different biosynthetic gene clusters that are positioned on the bacterial chromosome and on a plasmid. The plasmid encoded ureidopeptide natural products, which we term the pseudobulbiferamides (5-8), resemble the ureidopeptide natural products produced by Pseudovibrio, a different marine bacterial genus that is likewise present in marine sponge commensal microbiomes. Using imaging mass spectrometry, we find that the two classes of Microbulbifer-derived ureidopeptides occupy different physical spaces relative to the bacterial colony, perhaps implying different roles for these two compound classes in Microbulbifer physiology and environmental interactions.

Figure 1

(A) Bulbiferamides isolated from marine Microbulbifer sp. bacteria. The Phe residue and the ureido linkage are highlighted in red. (B) MS/MS fragmentation spectra for bulbiferamide A (1, left) and pseudobulbiferamide A (5, right), demonstrating the conserved 165 and 26 Da neutral losses. Parent ions are marked by the blue diamonds. (C) Structures of pseudobulbiferamides A–D (5–8) and the shunt metabolite 9 described in this study. (D) COSY, HMBC, and selected ROESY correlations for 5–7, 9.

Figure 2

(A) Map of the 454 kbp Microbulbifer sp. MKSA007 plasmid bearing the mbp BGC. From outside in predicted open reading frames (ORFs) on the leading and lagging DNA strands with the position of the mbp BGC highlighted are the normalized plot of GC content (blue) and normalized plot of GC skew (purple/green). (B) mbp BGC. (Abbreviations: A, adenylation; CP, carrier protein; C, condensation; Ox, oxidase; TE, thioesterase; KS, ketosynthase; KR, ketoreductase; and AT, acyl transferase). Hybrid NRPS-PKS MbpD is rationalized to contain two NRPS modules with predicted A-domain specificities for Val and Pro and a single PKS module. (C) Inferred assembly line biosynthesis of pseudobulbiferamides mediated by NRPSs MbpA–C. Three C domains, color-coded, conceivably catalyze ureido bond formation, dehydration of a Thr side chain, and Cys to thiazoline oxidation.

Figure 3

(A) bulb BGCs detected in Microbulbifer strains, Microbulbifer sp. MLAF003, Microbulbifer sp. VAAF005, and Microbulbifer sp. MKSA007. The domain organization of the Microbulbifer sp. MKSA007 bulb NRPSs is denoted. The gene colored cyan encodes a putative transporter. C domains marked by * are split between two polypeptides. (B) Molecular network with nodes denoting bulbiferamides and pseudobulbiferamides detected in Microbulbifer sp. MKSA007 culture extracts. Nodes colored green, blue, and yellow are detected in liquid, solid, and both liquid and solid media culture extracts, respectively. The precursor ion m/z are labeled for each node. Nodes corresponding to different ionization states for both classes of molecules do not cluster. (C) Imaging mass spectrometry denoting differential localization of bulbiferamides and pseudobulbiferamides for bulbiferamide-only producing Microbulbifer sp. MLAF003 (left) and bulbiferamide and pseudobulbiferamides producing Microbulbifer sp. MKSA007 (right). Different bulbiferamide congeners detected in the two bacterial strains.