Role of a Microcin-C–Like Biosynthetic Gene Cluster in Allelopathic Interactions in Marine Synechococcus

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 gene clusters in Synechococcus strains. Block the CC9605 mccE2 gene became fully resistant to the McC arrows denote genes, with arrows pointing in the direction of transcription. concentrations assayed (Fig. S2), whereas the controls, the Matching colors indicate homologous genes. Accession numbers for protein TOP10 strain alone, and TOP10 cells carrying plasmid pPY9, sequences from the National Center for Biotechnology Information Entrez which lacks the promoter and beginning of mccE2 (Table S2), database are indicated below each gene [Synechococcus sp. CC9605 ORFs were sensitive to McC (Fig. S2), showing a visible growth in- syncc9605_2504 (mccD), syncc9605_2505 (mccE1), syncc9605_2506 (mccE2), μ and syncc9605_2507 (mccB)]. NA, not annotated. At the top is a schematic hibition at McC concentrations ranging from 1 mM to 20 M. showing the McC biosynthesis operon along with the functions of individual mcc Synechococcus mcc gene products based on previously published information. Modified Expression Pattern of the Genes. In CC9605, from refs. 35 and 48. the mcc genes are in the order mccD, mccE1, mccE2, mccA1, mccA2, and mccB and were expressed under exponential growth as seen by RT-PCR of individual genes (Fig. S3). Cotranscription In E. coli, the McC biosynthetic cluster consists of the plasmid- of consecutive genes was also analyzed by RT-PCR, a common encoded mccABCDE operon (Fig. 1). mccA encodes the hep- method for analysis of cotranscription (42). RT-PCR amplifica- tapeptide McC precursor. In both Synechococcus strains, mccA tion products, indicating cotranscription of mccA2 and mccB and heptapeptide ORFs were not annotated in the gene cluster, but cotranscription of mccE2 and mccA1 (Fig. S4), were obtained two (sp. CC9605) and three (sp. RS9916) direct repeats, each potentially encoding 56- to 57-aa polypeptides, could be found (Fig. 1). It was suggested that the products of these ORFs might be subject to posttranslational modification and act as microcins (35). In E. coli, MccB adenylates the MccA heptapeptide, whereas MccD and the N-terminal domain of MccE are re- MICROBIOLOGY quired for phosphate modification with propylamine (40). McC in E. coli functions through a Trojan horse mechanism of ac- tion (38, 41). In this work, we describe an example of an allelopathic interaction occurring between marine Synechococcus strains. We focused on the inhibitory effect that strain CC9605 has on the growth of strains CC9311 and WH8102 and the hypothesis that it is because of a cyanobacterial version of an McC-like biosynthetic gene cluster encoded by Synechococcus sp. CC9605. Additionally, we studied the effect of purified E. coli McC on several marine Synechococcus strains. Results Fig. 2. Allelopathic interactions in liquid cocultures of Synechococcus Allelopathic Interactions Between Strains of Marine Synechococcus. strains. The proportion of each Synechococcus strain in liquid cocultures was Because it is not possible to distinguish Synechococcus strains determined by Q-PCR after 10 d and a subsequent 10 d (day 20) after from one another by microscopy or epifluorescence microscopy retransfer.

Paz-Yepes et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 12031 Downloaded by guest on October 2, 2021 Spot WH8102 CC9311 CC9605 of Synechococcus sp. CC9605 with Synechococcus sp. WH8102, Lawn E. coli strain TOP10, Vibrio cholerae T2123, and a mix of unknown heterotrophic marine bacteria isolated from a culture of the heterotrophic nanoﬂagellate Pteridomonas (Table S2). We did see a 2.83 ± 0.64-fold expression change with the mix of unknown marine heterotrophic bacteria (Fig. 4B), but we did not WH8102 see a signiﬁcant change in the mccA gene expression (Fig. 4B) when CC9605 was cocultured with the other organisms, including WH8102. In addition, 1.03-μm glass beads did not generate any change in the mccA gene expression (Fig. 4B).

Cloning the McC-Like Biosynthetic Gene Cluster from Synechococcus sp. CC9605 into Synechococcus sp. CC9311 Confers Resistance to CC9311 Synechococcus sp. CC9605 and Puriﬁed E. coli McC. We cloned the McC-like biosynthetic gene cluster from CC9605 into the comEC gene of CC9311, resulting in strain PY28 (Fig. S6 and Table S2). In liquid competition experiments between strain PY28 and Synechococcus sp. CC9605, PY28, unlike its WT parent CC9311, was able to grow in coculture with CC9605 (Fig. 5E). This ability

CC9605

Fig. 3. Study of allelopathic interactions between Synechococcus strains on A 14 solid medium. Plates containing lawns of each Synechococcus strain— WH8102, CC9311, and CC9605—with drops of the same Synechococcus 12 strains spotted in the middle of each plate.

10 with the primer pairs Mic-RT16/Mic-RT23 and Mic-RT5/Mic- RT8, respectively (Fig. S4). In contrast, no amplification product 8 was observed with Mic-RT1/Mic-RT10, Mic-RT1/Mic-RT20, Mic-RT3/Mic-RT6, andMic-RT14/Mic-RT8(Fig. S4), in- 6 dicating no cotranscription of the whole gene cluster or regions mccB-mccE2, mccE1-mccE2,ormccA1-mccA2, respectively. The Relative Expression (Fold Change) 4 results are summarized in the gene expression scheme shown in Fig. S4. 2 mccX:rpoC Synechococcus mcc sp. CC9311 Induces the Expression of Some Genes 0 in Synechococcus sp. CC9605. Using quantitative RT-PCR and in- 0 5 10 15 20 25 ternal primers specific for each ORF, we analyzed the expression Time (h) level of the ORFs associated with the biosynthesis of the McC- mccA mccB mccD mccE1 mccE2 like molecule in Synechococcus sp. CC9605. Because of the identical DNA sequence of the two mccA copies, we were unable B * to differentiate the expression level of each one separately; thus, 5 we determined the expression of both mccA ORFs together, and mccA will be referred to as a single gene. Among the ORFs 4 composing the McC-like biosynthetic gene cluster in CC9605, * the mccA and mccD genes were expressed in axenic exponen- tially growing cultures and strongly (more than threefold) up- 3 regulated in response to the addition of CC9311. Induction of mccA and mccD was observed after 6 h of mixed incubation, and 2 maximum expression occurred after 24 h for both mccA and mccD A ± ± Expression (Fold change) Relative genes (Fig. 4 ), increasing up to 8.82 0.93- and 13.11 1 rpoC 2.51-fold, respectively. In the case of the other three genes, only : mccB fi underwent a signi cant change in expression but only after mccA 24 h (2.91 ± 0.78-fold), whereas mccE1 and mccE2 did not ex- 0 s hibit any significant change in gene expression (Fig. 4A). E. coli Bead mccA induction was proportional to the concentration of CC9311 WH8102 Vibrio c.

CC9311 added to a coculture. When different initial concen- CC9605 control

trations of CC9311 were used in 24-h induction experiments, the CC9311 supernatant CC9311 broken cells 8 −1 xed unknown bacteria highest concentration (5 ± 1 × 10 cells mL )resultedinan Mi 8.82 ± 0.93-fold change of the mccA expression, whereas the mRNA level only changed 3.88 ± 2.8- and 1.01 ± 0.016-fold at Fig. 4. Expression of mcc-like genes in cocultures. (A) mccA and mccD genes ± × 7 ± × 6 −1 are induced in the presence of CC9311 to a greater extent than the other genes. lower cell concentrations (5 1 10 and 5 1 10 cells mL , (B) mccA gene expression is induced by Synechococcus CC9311 and a mixed respectively) (Fig. S5). population of heterotrophic marine bacteria of unknown types but not super- To determine whether expression of the mccA gene was in- natant from CC9311 cultures or some other bacteria, such as Synechococcus duced by other microorganisms, we carried out 14-h incubations WH8102, Vibrio,andE. coli, or glass beads. *Signiﬁcant expression changes.

12032 | www.pnas.org/cgi/doi/10.1073/pnas.1306260110 Paz-Yepes et al. Downloaded by guest on October 2, 2021 – was also evident in plating experiments, where after 14 20 d of WH8102 lawn WH8102 lawn solid coculture, an inhibition zone was not observed in the PY28 A B lawn (Fig. 5B), whereas CC9311 was still inhibited (Fig. 5A). We tested the sensitivity of strain PY28 to puriﬁed E. coli McC on plates, and as expected, it was fully resistant (Fig. 5D).

Partial Deletion of McC-like Biosynthesis Genes in Synechococcus sp. CC9605 Abolishes Growth Inhibition of Synechococcus sp. CC9311 and Reduces Growth Inhibition of Synechococcus sp. WH8102. The region of mccA1, mccA2, and the N-terminal domain of mccB in Syn- WT CC905 spot PY35 spot echococcus sp. CC9605 was substituted with a kanamycin resistance cassette, resulting in strain PY35 (Fig. S7 and Table S2). CC9311 lawn CC9311 lawn PY35 did not inhibit the growth of CC9311 (Fig. 6D) and still C D inhibited the growth of WH8102 (Fig. 6B), but it produced an

A WT lawn B PY28 lawn

WT CC905 spot PY35 spot

CC9605 spot CC9605 spot Fig. 6. Sensitivity of Synechococcus strains WH8102 and CC9311 to Syn- echococcus strains CC9605 and CC9605-PY35. Synechococcus strain WH8102 was inhibited by both (A) CC9605 and (B) the mutant strain CC9605-PY35, although the diameter of the zone of inhibition by the mutant was 56.7% smaller than the diameter produced by the WT strain. Unlike WH8102, the growth of CC9311 was only inhibited by (C) the WT strain CC9605, whereas C WT D PY28 (D) PY35 did not inhibit CC9311 growth. 0.2 mM 0.2 mM 0.1 mM 1 mM area of inhibition 57% smaller than the area produced by WT 0.1 mM A 1 mM CC9605 (Fig. 6 ). Discussion 10 M Most studies of allelopathic interactions between cyanobacteria have been performed in freshwater environments (11), and very 50 M 50 M little is known about this phenomenon among marine cyano- 10 M bacteria, especially Synechococcus. Using a quantitative PCR strategy to quantify marine Synechococcus clades within a liquid medium coculture (25), our results revealed that Synechococcus E sp. CC9605 clearly impairs the growth of strains CC9311 and 100 WH8102 under the conditions assayed (Figs. 2 and 3). WH8102 PY28 CC9605 90 seems to be ﬁvefold more sensitive than CC9311 to CC9605 in 80 solid medium. Interestingly, CC9605 and WH8102 co-occur in oligotrophic environments, where CC9605 is thought to be more 70 abundant than WH8102 (25, 43). We also found that Synecho- 60 coccus sp. CC9311, which does not encode an McC-like gene

Synechococcus MICROBIOLOGY 50 cluster, noticeably inhibits the growth of sp. WH8102 (Figs. 2 and 3), presumably by the production of a dif- 40 ferent allelochemical. These results are evidence of allelopathic 30 interactions between marine Synechococcus strains. Considering the zone of inhibition produced by CC9605 over 20 the other two strains on plates (Fig. 3), it is likely that the 10 CC9605 McC is secreted into the medium, like it is in E. coli, and

Strain Proportion (%) Synechococcus Strain Proportion 0 then taken up by the target cells. WH8102, CC9311, and CC9605 01020all encode genes homologous to the ATP-binding cassette E. coli Time (d) (ABC) transporter responsible for taking up McC in (44) that might be involved in the uptake of the CC9605 McC-like molecule. E. coli YejA, YejB, YejE, and YejF homologs are Fig. 5. Sensitivity of Synechococcus strain CC9311-PY28 to CC9605 and SYNW0709, SYNW0708, SYNW1683, and SYNW2325, re- purified E. coli McC. Synechococcus strain CC9311-PY28 resists CC9605 in (B) solid medium and (E) liquid conditions as well as (D) purified McC from spectively, in WH8102; sync_0939, sync_0938, sync_0676, and E. coli. WT CC9311 was sensitive to CC9605 in (A) solid medium and liquid sync_2703, respectively, in CC9311; and Syncc9605_1959, conditions (Fig. 2) as well as (C) purified McC from E. coli (concentrations of Syncc9605_1960, Syncc9605_800, and Syncc9605_2456, re- McC are indicated). spectively, in CC9605. However, unlike E. coli, CC9605 does

Paz-Yepes et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 12033 Downloaded by guest on October 2, 2021 not encode any homologs to the E. coli mccC efflux transporter growth could be ecologically costly but provide an important (Fig. 1). We hypothesize that the three ORFs upstream of the defensive role against competitors or even predators; having CC9605 McC biosynthetic gene cluster might be involved in the ability to further induce expression allows tuning of the de- CC9605 McC secretion. They are annotated as components of an fense to changing environmental conditions and co-occurring ABC efflux transporter and are also expressed under exponential microbes. growth in CC9605 (Fig. S3). Unlike CC9311, the growth of WH8102 was still inhibited by Allelopathic interactions involving antibiotics have been de- the mutant strain PY35, although to a lesser extent than the WT scribed for marine heterotrophic bacteria (1). Because we hy- strain CC9605 (Fig. 6 A and B), indicating that, in addition to an pothesized that CC9605 dominance over WH8102 and CC9311 McC-like compound, the strong growth inhibition of WH8102 by was due to an McC-like antibiotic, we tested the sensitivity of all CC9605 is also caused by at least another allelochemical that of these strains to McC purified from E. coli (38). McC inhibited CC9605 might produce. As has been shown by previous bio- the growth of CC9311 and WH8102 but not CC9605 or RS9916 informatic analyses of cyanobacterial genomes (15), CC9605 (Fig. S1), which are both known to carry an McC-like bio- bears two gene clusters that potentially encode for two different synthetic gene cluster (35). Therefore, E. coli McC can inhibit bacteriocins. We believe that at least one of these bacteriocins cyanobacteria. might also be involved in the allelopathic interaction observed Synechococcus sp. CC9605 has at least one resistance gene, the between CC9605 and WH8102. mccE2 gene, which we showed here can confer McC re- Interestingly, CC9605 exhibits a tendency to form aggregates sistance in E. coli (Fig. S2) and presumably provides self- that can constitute from 65% to 75% of total CC9605 abundance immunity against its own McC-like antibiotic. Synechococcus in some culture conditions (46). If aggregation occurs in the sp. RS9916 carries a putative mccF-like immunity gene instead of environment as well, it would result in higher McC-like con- an mccE-like gene (Fig. 1). centrations surrounding CC9605 cells. This behavior would be McC is produced by members of the family Enterobacteriaceae, significant in the oligotrophic environments that CC9605 and its biosynthetic pathway is thought to be horizontally inhabits (25, 43, 47), where the total microbial cell density is low transferred within the family (45). We believe that the two un- and the distance between single CC9605 cells and their nearest related marine Synechococcus strains, CC9605 and RS9916, po- neighbor would otherwise be high (∼200 cell lengths, which has tentially acquired the McC-like biosynthetic gene cluster by been suggested for Prochlorococcus) (12). It has also been pro- horizontal gene transfer. The nucleotide sequence also supports posed that antibiotic production may be a mechanism used by our hypothesis, because the G+C content determined for particle specialists to dominate the particle phase by deterring both McC-like gene clusters was 42.6% (CC9605) and 45.1% other potential colonizers (1). (RS9916), values significantly lower than the values found for We have shown that allelopathic interactions occur between their genomes: 59.2% (CC9605) and 59.8% (RS9916). Hori- marine Synechococcus strains. Our results suggest that the zontal gene transfer of the McC-like gene cluster into some product of an McC-like gene cluster is involved in these inter- Synechococcus strains would explain why it has not been found in actions when CC9605 is present, and provides an example of an other sequenced cyanobacterial genomes to date. active McC-like gene cluster outside of enteric bacteria. In ad- Surprisingly, when the competitor Synechococcus sp. CC9311 dition, we found that the growth of WH8102 was also inhibited was incubated with Synechococcus sp. CC9605 in a 24-h time by strain CC9311. Because this effect is not McC-dependent, course, the expression of some microcin-like genes, mccD and additional investigations are needed to identify this compound mccA, increased significantly after only 6 h of incubation (Fig. and other new compounds produced by Synechococcus strains 4A), whereas mccB was induced after 24 h. This result suggests that might be involved in such interactions. Given the significant that CC9605 can sense the presence of CC9311 and respond by abundance of Synechococcus in marine waters, such allelopathic inducing the expression of McC-like synthesis genes. Several interactions are likely to be relevant in shaping the composition molecular mechanisms could be responsible for the observed of the marine bacterial community as a whole. expression induction from physical contact without any kind of cell specificity to specific cell surface recognition. In the latter Materials and Methods case, it could be a response to a broad number of micro- The bacterial strains and plasmids used in this study are listed in Table S2. SI organisms, or alternatively, it could be a more specific response Materials and Methods discusses specific culture conditions, cyanobacterial to a few microorganisms. Our results suggest that the response genomic DNA and RNA extraction, quantitative real-time PCR, toxicity assay may occur through cell surface recognition of CC9311, because of E. coli McC on strains of Synechococcus, and genetic manipulation in we did not obtain significant induction of mccA expression when E. coli and Synechococcus strains. supernatant from a CC9311 culture, glass beads, or several other B Synechococcus Expression of mcc-Like Genes in Cocultures. We obtained the gene expression microorganisms was used (Fig. 4 ). However, sp. fi CC9605 does not exclusively recognize and respond to Synechococcus pro les of all of the genes contained in the McC-like biosynthetic gene cluster of Synechococcus sp. CC9605 after 1, 3, 6, 9, and 24 h of coculture with sp. CC9311. We found a 2.83 ± 0.64-fold induction when a mix- fi Synechococcus sp. CC9311. Batch cultures of CC9311 and CC9605 were ture of unidenti ed marine heterotrophic bacteria was used. grown up to late exponential/early stationary phase. Cells from both cul- − Additional studies are necessary to identify the molecular mecha- tures were mixed at the same initial cell density (∼5 ± 1 × 106 cells mL 1)and nism by which CC9605 recognizes CC9311 and induces the expres- grown at 25 °C under stirring conditions. A CC9605 culture was used as sion of some of the mcc-like genes. a control. At the indicated times, samples were taken, and RNA was The McC-like gene cluster from Synechococcus sp. CC9605 extracted and used in quantitative RT-PCR analysis. The gene expression of was cloned into the McC-sensitive Synechococcus sp. CC9311. As the different genes was analyzed using the oligonucleotides primer pairs expected, strain PY28 became resistant to Synechococcus sp. Mic-RT7/Mic-RT8 (mccA), Mic-RT9/Mic-RT21 (mccB), Mic-RT1/Mic-RT2 (mccD), CC9605 (Figs. 5 B and E), and PY28 also acquired immunity to Mic-RT12/Mic-RT20 (mccE1), and Mic-RT13/Mic-RT18 (mccE2) (Fig. 4A and Table S1). McC purified from E. coli (Fig. 5D). Furthermore, deletion of mccA1 mccA2 mccB The induction of mccA in response to different initial CC9311 cell con- , , and the N-terminal domain of in CC9605 centrations was determined. The starting cell concentration of CC9311 was (strain PY35) abolishes its inhibitory activity on strain CC9311 5 ± 1 × 108 cells mL−1. We did 24-h incubations of strains CC9311 and CC9605 (Fig. 6 C and D). Thus, our findings strongly support the idea and varied the initial cell density of CC9311 in a series of 10-fold dilutions. that allelopathic interactions between CC9605 and CC9311 are Induction of mccA after 14 h of coincubation with Synechococcus sp. because of an McC-like compound. The constitutive expression WH8102, E. coli strain TOP10, Vibrio cholerae T2123, a mix of unknown of these genes in Synechococcus sp. CC9605 under exponential heterotrophic marine bacteria (Table S2), or 1.03-μm glass beads (prewashed

12034 | www.pnas.org/cgi/doi/10.1073/pnas.1306260110 Paz-Yepes et al. Downloaded by guest on October 2, 2021 − two times with SN medium at a final bead concentration of 5.3 × 108 beads mL 1; and emission = 577 nM; Turner AU-10; Turner Designs). The specific growth Molecular Probes Inc.) was determined. rates of these cultures were determined using geometric mean linear re- gression of a plot of ln (culture fluorescence or cells per milliliter) vs. time Study of Allelopathic Interactions Between Marine Synechococcus Strains. To during midexponential phase. These growth conditions resulted in similar − study allelopathic interactions between different Synechococcus strains, growth rates for all of the cultures in the conditions assayed: 0.39 ± 0.04 d 1 − − duplicate 50-mL cultures consisting of two Synechococcus strains (CC9605 vs. (WH8102), 0.40 ± 0.07 d 1 (CC9311), 0.37 ± 0.06 d 1 (CC9605), and 0.40 ± − CC9311, CC9605 vs. WH8102, and CC9311 vs. WH8102) were inoculated into 0.06 d 1 (PY28). ∼ ± × 6 −1 SN medium at the same initial cell density ( 5 1 10 cells mL ) and Allelopathic interactions were also analyzed in solid medium using pour grown at 25 °C with shaking; 2 mL of each coculture were collected at the plates containing lawns of each Synechococcus strain (WH8102, CC9311, or − beginning of the experiment (zero time point) as well as after 10 d (10-d CC9605; 5 ± 1 × 106 cells mL 1). After incubation at 25 °C with a constant − − time point). After 10 d of growth, 1 mL each coculture was transferred to illumination of 10 microeinsteins m 2 s 1 for 1 wk, drops of each test Syn- 50 mL fresh SN medium, and 2 mL each coculture were collected after an- echococcus strain were spotted on the plate (Fig. 3). A circle of agarose was other 10 d of growth under the same conditions (20-d time point). DNA was − removed from the middle of the plate, and 100 μL cells (5 × 1010 cells mL 1) extracted as described above. To calculate the relative abundance of each premixed with SN containing 0.4% (wt/vol) agarose were spotted in the Synechococcus strain, quantitative PCR (Q-PCR) was carried out as described hole. As a control, each Synechococcus strain was plated separately without for Q-RT-PCR above but using genomic DNA instead of cDNA. Oligonucle- drops of other strains. otide primers and Q-PCR conditions specific for the rpoC1 gene of each Synechococcus strain were used as previously described (25) (Table S1). Each strain was also grown separately as a control; they were transferred using ACKNOWLEDGMENTS. We thank Dr. Doug Bartlett for sharing V. cholerae and Dr. Konstantin Severinov for sharing McC purified from E. coli. J.P.-Y. the same incubation periods as the experimental cocultures. When the was supported with a postdoctoral fellowship by the Fundación Alfonso control samples were collected, we mixed 1 mL each strain grown separately Martin Escudero (Spain) as well as by the Seventh Research Program of to recreate the combinations that we were assaying in the experiment. the European Union FP7/2007-2013 under Grant PIOF-GA-2011-301466. Re- Growth rates were calculated for each strain growing separately. Growth search was supported by National Science Foundation Grant IOS-1021421 (to was monitored by measuring phycoerythrin fluorescence (excitation = 544 nM B.B. and B.P.).

1. Long RA, Azam F (2001) Antagonistic interactions among marine pelagic bacteria. 25. Tai V, Palenik B (2009) Temporal variation of Synechococcus clades at a coastal Pacific Appl Environ Microbiol 67(11):4975–4983. Ocean monitoring site. ISME J 3(8):903–915. 2. Strom SL (2008) Microbial ecology of ocean biogeochemistry: A community perspec- 26. Tai V, Burton RS, Palenik B (2011) Temporal and spatial distributions of marine Syn- tive. Science 320(5879):1043–1045. echococcus in the Southern California Bight assessed by hybridization to bead-arrays. 3. Hibbing ME, Fuqua C, Parsek MR, Peterson SB (2010) Bacterial competition: Surviving Mar Ecol Prog Ser 426:133–147. and thriving in the microbial jungle. Nat Rev Microbiol 8(1):15–25. 27. Ahlgren NA, Rocap G (2012) Diversity and distribution of marine Synechococcus: 4. Keating KI (1977) Allelopathic influence on blue-green bloom sequence in a eutrophic Multiple gene phylogenies for consensus classification and development of qPCR lake. Science 196(4292):885–887. assays for sensitive measurement of clades in the ocean. Front Microbiol 3:213. 5. Keating KI (1978) Blue-green algal inhibition of diatom growth: Transition from 28. Czárán TL, Hoekstra RF, Pagie L (2002) Chemical warfare between microbes promotes mesotrophic to eutrophic community structure. Science 199(4332):971–973. biodiversity. Proc Natl Acad Sci USA 99(2):786–790. 6. Flores E, Wolk CP (1986) Production, by filamentous, nitrogen-fixing cyanobacteria, of 29. Palenik B, et al. (2003) The genome of a motile marine Synechococcus. Nature 424: a bacteriocin and of other antibiotics that kill related strains. Arch Microbiol 145(3):215–219. 1037–1042. 7. Legrand C, Rengefors K, Fistarol GO, Granéli E (2003) Allelopathy in phytoplankton- 30. Palenik B, et al. (2006) Genome sequence of Synechococcus CC9311: Insights into biochemical, ecological and evolutionary aspects. Phycologia 42(4):406–419. adaptation to a coastal environment. Proc Natl Acad Sci USA 103(36):13555–13559. 8. Leflaive J, Ten-Hage L (2007) Algal and cyanobacterial secondary metabolites in fresh- 31. Six C, et al. (2007) Diversity and evolution of phycobilisomes in marine Synechococcus waters: A comparison of allelopathic compounds and toxins. Freshw Biol 52(2):199–214. spp.: A comparative genomics study. Genome Biol 8(12):R259. 9. Leão PN, Vasconcelos MT, Vasconcelos VM (2009) Allelopathy in freshwater cyano- 32. Dufresne A, et al. (2008) Unraveling the genomic mosaic of a ubiquitous genus of bacteria. Crit Rev Microbiol 35(4):271–282. marine cyanobacteria. Genome Biol 9(5):R90. 10. Leão PN, Ramos V, Vale M, Machado JP, Vasconcelos VM (2012) Microbial community 33. Scanlan DJ, et al. (2009) Ecological genomics of marine picocyanobacteria. Microbiol changes elicited by exposure to cyanobacterial allelochemicals. Microb Ecol 63(1): Mol Biol Rev 73(2):249–299. 85–95. 34. Palenik B, Ren Q, Tai V, Paulsen IT (2009) Coastal Synechococcus metagenome reveals 11. Leão PN, Engene N, Antunes A, Gerwick WH, Vasconcelos V (2012) The chemical major roles for horizontal gene transfer and plasmids in population diversity. Environ ecology of cyanobacteria. Nat Prod Rep 29(3):372–391. Microbiol 11(2):349–359. 12. Li B, et al. (2010) Catalytic promiscuity in the biosynthesis of cyclic peptide secondary 35. Severinov K, Semenova E, Kazakov A, Kazakov T, Gelfand MS (2007) Low-molecular- metabolites in planktonic marine cyanobacteria. Proc Natl Acad Sci USA 107(23): weight post-translationally modified microcins. Mol Microbiol 65(6):1380–1394. 10430–10435. 36. Baquero F, Moreno F (1984) The microcins. FEMS Microbiol Lett 23:117–124. 13. Leikoski N, et al. (2012) Analysis of an inactive cyanobactin biosynthetic gene cluster 37. Baquero F, Bouanchaud D, Martinez-Perez MC, Fernandez C (1978) Microcin plasmids: leads to discovery of new natural products from strains of the genus Microcystis. PLoS A group of extrachromosomal elements coding for low-molecular-weight antibiotics One 7(8):e43002. in Escherichia coli. J Bacteriol 135(2):342–347. 14. Tang W, van der Donk WA (2012) Structural characterization of four prochlorosins: A 38. Metlitskaya A, et al. (2006) Aspartyl-tRNA synthetase is the target of peptide nucle- novel class of lantipeptides produced by planktonic marine cyanobacteria. Bio- otide antibiotic Microcin C. J Biol Chem 281(26):18033–18042. chemistry 51(21):4271–4279. 39. Arnison PG, et al. (2013) Ribosomally synthesized and post-translationally modified 15. Wang H, Fewer DP, Sivonen K (2011) Genome mining demonstrates the widespread oc- peptide natural products: Overview and recommendations for a universal nomen- currence of gene clusters encoding bacteriocins in cyanobacteria. PLoS ONE 6(7):e22384. clature. Nat Prod Rep 30(1):108–160.

16. Martins RF, et al. (2008) Antimicrobial and cytotoxic assessment of marine cyano- 40. González-Pastor JE, San Millán JL, Castilla MA, Moreno F (1995) Structure and orga- MICROBIOLOGY bacteria - Synechocystis and Synechococcus. Mar Drugs 6(1):1–11. nization of plasmid genes required to produce the translation inhibitor microcin C7. J 17. Frazão B, Martins R, Vasconcelos V (2010) Are known cyanotoxins involved in the Bacteriol 177(24):7131–7140. toxicity of picoplanktonic and filamentous North Atlantic marine cyanobacteria? Mar 41. Reader JS, et al. (2005) Major biocontrol of plant tumors targets tRNA synthetase. Drugs 8(6):1908–1919. Science 309(5740):1533. 18. Sher D, Thompson JW, Kashtan N, Croal L, Chisholm SW (2011) Response of Pro- 42. Paz-Yepes J, Merino-Puerto V, Herrero A, Flores E (2008) The amt gene cluster of chlorococcus ecotypes to co-culture with diverse marine bacteria. ISME J 5(7):1125–1132. the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 19. Waterbury JB, Watson SW, Guillard RRL, Brand LE (1979) Widespread occurrence of 190(19):6534–6539. a unicellular, marine, planktonic, cyanobacterium. Nature 277:293–294. 43. Zwirglmaier K, et al. (2008) Global phylogeography of marine Synechococcus and 20. Johnson PW, Sieburth JM (1979) Chroococcoid cyanobacteria in the sea: A ubiquitous Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes. and diverse phototrophic biomass. Limnol Oceanogr 24(5):928–935. Environ Microbiol 10(1):147–161. 21. Waterbury JB, Watson SW, Valois F, Franks D (1986) Biological and ecological char- 44. Novikova M, et al. (2007) The Escherichia coli Yej transporter is required for the up- acterization of the marine unicellular cyanobacterium Synechococcus. Can Bull Fish take of translation inhibitor microcin C. J Bacteriol 189(22):8361–8365. Aquat Sci 214:71–120. 45. Fomenko DE, et al. (2003) Microcin C51 plasmid genes: Possible source of horizontal 22. Olson RJ, Chisholm SW, Zettler ER, Armbrust EV (1990) Pigments, size, and distribution of gene transfer. Antimicrob Agents Chemother 47(9):2868–2874. Synechococcus in the North Atlantic and PacificOceans.Limnol Oceanogr 35(1):45–58. 46. Apple JK, Strom SL, Palenik B, Brahamsha B (2011) Variability in protist grazing and growth 23. Ferris MJ, Palenik B (1998) Niche adaptation in ocean cyanobacteria. Nature 396: on different marine Synechococcus isolates. Appl Environ Microbiol 77(9):3074–3084. 226–228. 47. Toledo G, Palenik B (2003) A Synechococcus serotype is found preferentially in surface 24. Fuller NJ, et al. (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the marine waters. Limnol Oceanogr 48(5):1744–1755. predominance of a single marine Synechococcus clade throughout a stratified water 48. Metlitskaya A, et al. (2009) Maturation of the translation inhibitor microcin C. column in the Red Sea. Appl Environ Microbiol 69(5):2430–2443. J Bacteriol 191(7):2380–2387.

Paz-Yepes et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 12035 Downloaded by guest on October 2, 2021