Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria

Abstract

Our understanding of secondary metabolite production in bacteria has been shaped primarily by studies of attached varieties such as symbionts, pathogens, and soil bacteria. Here we show that a strain of the single-celled, planktonic marine cyanobacterium Prochlorococcus-which conducts a sizable fraction of photosynthesis in the oceans-produces many cyclic, lanthionine-containing peptides (lantipeptides). Remarkably, in Prochlorococcus MIT9313 a single promiscuous enzyme transforms up to 29 different linear ribosomally synthesized peptides into a library of polycyclic, conformationally constrained products with highly diverse ring topologies. Genes encoding this system are found in variable abundances across the oceans-with a hot spot in a Galapagos hypersaline lagoon-suggesting they play a habitat- and/or community-specific role. The extraordinarily efficient pathway for generating structural diversity enables these cyanobacteria to produce as many secondary metabolites as model antibiotic-producing bacteria, but with much smaller genomes.

Fig. 1.

Enzymatic transformations and biosynthetic genes for lantipeptide synthesis in Prochlorococcus MIT9313. (A) Dehydration and cyclization reactions in lantibiotic/lantipeptide biosynthesis. (B) Genomic clusters in MIT9313 encoding ProcM and multiple ProcA peptides, their nomenclature, and locations on the genome. (C) High level of conservation in the N-terminal leader sequence and hypervariability of the C-terminal core peptide of ProcAs. The GG/GA cleavage site is marked by an arrow.

Fig. 2.

In vitro dehydration of ProcAs by ProcM. MALDI-TOF mass spectra of representative ProcAs modified by ProcM in vitro and subsequently proteolytically cleaved (LysC for ProcA1.2, GluC for all others) to remove part of the leader peptide (solid spectra). ProcM treatment was omitted in the dashed spectra. The sequence of each ProcA after protease cleavage is shown, and the number of dehydrations is determined by the mass difference between ProcM-treated and control samples (each dehydration = -18 Da). The control spectrum for ProcA1.7 does not include the parent peak because GluC cleavage of the unmodified core peptide results in two smaller peptides outside of the window shown.

Fig. 3.

In vitro cyclization of ProcAs by ProcM. (A) ESI-MS/MS analysis of ProcA1.1 G-1E (Upper) and ProcA1.7 (Lower) treated successively with ProcM and GluC. The labeled fragment ions support two nonoverlapping thioether rings for prochlorosin 1.1 (MIT9313) and show that, in prochlorosin 1.7 (MIT9313), Dhb1 and Dhb5 are not involved in rings. (B) Proposed structures of Pcn1.1, 1.7, 2.8, 3.3, 4.3, and 2.11 (MIT9313); see SI Appendix. Arrows illustrate the start of the putative core peptide. Asterisks indicate prochlorosins containing residues from the leader peptide (underlined).

Fig. 4.

In vivo production of prochlorosins. (A) Transcription of procM and procA genes by a log-phase culture of MIT9313. RT-reverse-transcriptase added (+) or omitted (−). rnpB is a control housekeeping gene. (B) Production of prochlorosin 2.1 (MIT9313) in vivo. The upper panel depicts ESI-MSMS analysis of ProcA2.1 modified in vitro by ProcM and digested with GluC; the lower panel shows ESI-MSMS analysis for prochlorosin 2.1 (MIT9313) from the spent media of a late-exponential stage culture of MIT9313. Fragment ions are indicated. The b2 and y′′28-y′′31 ions in the upper panel are derived from fragmentation in a stretch of residues (underlined) remaining from the leader peptide after GluC cleavage. Therefore, they are not observed in the product isolated from spent medium. Site directed mutagenesis studies suggest that Ser7 of Pcn2.1 (MIT9313) escapes dehydration (Fig. S18).

Fig. 5.

Prochlorococcus and Synechococcus lantipeptide genes in the Global Ocean Survey database. (A) Lantipeptide biosynthetic clusters in assembled metagenomic reads from the hypersaline lagoon site, GOS33. Blue—procM, red—procA, green—ABC transporters (red *—peptidase domain), light green—tolC, yellow—response regulator. (B) Distribution of procM- and procA-like genes in the GOS sites. Upper: Average number of core gene copies (normalized for gene length to 1,000 aa and to the database size at each GOS site in Mb), representing the relative abundance of Prochlorococcus and Synechococcus cells at each site. Lower: Ratio of procM and procA gene copies to core genes at each site. Wedges denote the location of the sampling sites in the bar graph. The sites of isolation of the Prochlorococcus MIT9313 and MIT9303 and Synechococcus RS9916 strains that contain lantipeptide genes are shown as yellow triangles.