Role of a Microcin-C–like biosynthetic gene cluster in allelopathic interactions in marine Synechococcus Javier Paz-Yepesa,b, Bianca Brahamshaa,1, and Brian Palenika,1 aMarine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093-0202; and bInstitut de Biologie de I’Ecole Normale Supérieure, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, 75230 Paris, France Edited* by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 6, 2013 (received for review April 2, 2013) Competition between phytoplankton species for nutrients and in coastal environments. Their discovery in 1979 (19, 20) soon light has been studied for many years, but allelopathic interactions led to a realization of their important contribution to primary between them have been more difficult to characterize. We used productivity (21, 22). It has also become clear that there is sig- liquid and plate assays to determine whether these interactions nificant diversity in the group (21), including distinct genetic occur between marine unicellular cyanobacteria of the genus Syn- clusters with distinct distributions in the water column (23–27), echococcus. We have found a clear growth impairment of Syne- implying the presence of distinct species with different ecological chococcus sp. CC9311 and Synechococcus sp. WH8102 when they niches. One might wonder whether allelopathic interactions are are cultured in the presence of Synechococcus sp. CC9605. The involved in this differentiation. In fact, antibiotic interactions genome of CC9605 contains a region showing homology to genes within microbial communities have been proposed as an effective of the Escherichia coli Microcin C (McC) biosynthetic pathway. McC way of maintaining bacterial diversity (28). is a ribosome-synthesized peptide that inhibits translation in sus- The complete genomic sequences of a number of these ceptible strains. We show that the CC9605 McC gene cluster is abundant photosynthetic microbes are available and have been expressed and that three genes (mccD, mccA, and mccB) are fur- analyzed in detail (29–33). Recently, metagenomic studies have ther induced by coculture with CC9311. CC9605 was resistant to also added to our picture of marine Synechococcus diversity McC purified from E. coli, whereas strains CC9311 and WH8102 (34). Collectively, genomic and metagenomic studies suggest were sensitive. Cloning the CC9605 McC biosynthetic gene cluster that these organisms have a core genome (shared by all Syn- into sensitive CC9311 led this strain to become resistant to both echococcus), potentially a clade-specific genome, and a vari- purified E. coli McC and Synechococcus sp. CC9605. A CC9605 mu- able, possibly strain-specific genome dominated by horizontal tant lacking mccA1, mccA2, and the N-terminal domain of mccB did gene transfer. not inhibit CC9311 growth, whereas the inhibition of WH8102 was Bioinformatic approaches have identified gene clusters with reduced. Our results suggest that an McC-like molecule is involved homology to the biosynthetic gene cluster of the antibiotic in the allelopathic interactions with CC9605. Microcin C (McC; also called Microcin C7, C51, or C7/C51) of Enterobacteriacea in two marine cyanobacteria: Synechococcus allelopathy | antibiotics | horizontal gene transfer strains CC9605 and RS9916 (35) (Fig. 1). Microcins are small (less than 10 kDa) bacteriocins produced by Escherichia coli and he production of secondary metabolites to inhibit either its close relatives (36, 37) that inhibit the growth of closely re- Tcompetitors or predators, known as allelopathy, is thought to lated bacterial species by targeting essential functions, such as play an important role in shaping microbial communities (1–3). DNA replication, transcription, and translation (35). Some The allelopathic potential of cyanobacteria was discovered in the microcins, such as the object of this study (McC), are initially ribosomally synthesized as a core peptide and heavily post- 1970s (4, 5), and subsequent studies have continued to focus fi largely on freshwater cyanobacteria (6–10). The cyanobacterial translationally modi ed by dedicated maturation enzymes (35). repertoire of secondary metabolites is large and synthesized The peptide moiety is required for the entry of unprocessed McC through diverse biochemical pathways (11). Recently, there has into sensitive cells, where it must be cleaved inside the target cell been increased interest in one class of compounds called bac- by peptidases to generate the inhibitory aminoacyl-nucleotide part of the drug (processed McC), which mimics the aspartyl teriocins, which are ribosomally synthesized peptides that be- adenylate and inhibits the aspartyl tRNA synthetase, thus come modified and are typically exported. Cyanobactins and blocking protein synthesis at the translation step (38). The McC- Prochlorosins are examples of these types of compounds pro- like compound from Synechococcus strains CC9605 and RS9916 duced by cyanobacteria (12–14). Bioinformatic analyses of cya- is structurally and functionally different from known ribosomally nobacterial genomes have shown the widespread occurrence of synthesized and posttranslationally modified peptides synthe- the potential to make bacteriocins of diverse types in almost sized by other cyanobacteria (39) [for example, the cyanobactins every cyanobacterium (15). produced by Microcystis and other cyanobacterial strains (13) or Despite the recognized ecological importance of marine cya- the lanthipeptides produced by Prochlorococcus MIT9313 (12)]. nobacteria, very little is known to date about their allelopathic McC was not found using comprehensive cyanobacterial genome interactions with each other or other marine bacteria (11). bioinformatic analyses for bacteriocins (15), possibly because Lanthipeptides called Prochlorosins have been discovered in the Prochlorococcus it is missing a double glycine processing site and a conserved marine cyanobacteria , although their func- leader peptide. tion is unclear, because antibiotic activity has not been shown (12, 14). Some marine intertidal cyanobacterial strains have been shown to produce substances with inhibitory effects on Gram- Author contributions: J.P.-Y., B.B., and B.P. designed research; J.P.-Y. performed research; positive bacteria or Artemia (16, 17), but the identity of these J.P.-Y., B.B., and B.P. analyzed data; and J.P.-Y., B.B., and B.P. wrote the paper. compounds is not known. Furthermore, certain marine bacteria The authors declare no conflict of interest. may produce compounds that positively or negatively affect the *This Direct Submission article had a prearranged editor. growth of marine cyanobacteria, which has been shown for 1To whom correspondence may be addressed. E-mail: [email protected] or bpalenik@ Prochlorococcus (18). ucsd.edu. Synechococcus Unicellular marine are cyanobacteria found This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. throughout the world’s oceans, and they are particularly abundant 1073/pnas.1306260110/-/DCSupplemental. 12030–12035 | PNAS | July 16, 2013 | vol. 110 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1306260110 Downloaded by guest on October 2, 2021 mccA mccB mccC mccD mccE mccF techniques, we characterized the allelopathic interactions be- E. coli tween different Synechococcus strains in liquid cocultures by AAY68494 AAY68495 AAY68496 AAY68497 AAY68498 AAY68499 measuring the relative abundance of each Synechococcus strain using the relative abundance of the rpoC1 marker gene and specific primers and conditions for each strain as described by Tai and Palenik (ref. 25; Table S1). Synechococcus sp. CC9605 always dominated when cocultured with CC9311 or WH8102 Core peptide N-acyl Aminopropyl Immunity (Fig. 2). Synechococcus sp. CC9311 dominated the coculture phosphoamide synthesis/ when growing with Synechococcus sp. WH8102 (Fig. 2). These bond formation attachment effects were also seen in solid medium. When a spot of CC9605 was plated on an existing lawn of CC9311 or WH8102, a zone of clearing developed, which was fivefold larger in the case of WH8102 (Fig. 3). The inhibition of CC9311 over WH8102 was also observed in solid medium. Both the liquid and plating approaches, thus, showed that CC9605 inhibits the growth of CC9311 and WH8102, likely through a bacteriocidal mechanism. Synechococcus CC9605 Is Resistant to E. coli McC. Because our fMet-Arg-Thr-Gly-Asn-Ala results could be explained by an McC-like compound potentially produced by CC9605, we characterized the toxic effect of McC purified from E. coli (38). Synechococcus strains WH8102 and CC9311 were very sensitive to E. coli McC, showing clear growth inhibition when McC was added up to 100 μM (CC9311) and Synechococcus mccD mccE1 mccE2 mccA1 mccA2 mccB 10 μM (WH8102) (Fig. S1). In contrast, Synechococcus strains E. coli sp. CC9605 CC9605 and RS9916 were resistant to McC (Fig. S1). ABB36235 ABB36236 ABB36237 NA NA ABB36238 mccE2 Synechococcus mccA1 mccA2 mccA3 mccB mccF Cloning from sp. CC9605 into an McC-Sensitive Synechococcus E. coli Strain Confers Resistance to McC. To determine whether the Synechococcus mccE2 sp. RS9916 EAU74189+89+90 EAU74191 EAU74192 sp. CC9605 gene could confer resistance to McC, we cloned this gene into an McC-sensitive E. coli strain, Fig. 1. Genetic organization of McC biosynthetic gene cluster in E. coli and TOP10. E. coli TOP10 carrying the plasmid pPY8 and containing predicted McC-like biosynthetic