Abstract
An important factor dictating coral fitness is the quality of bacteria associated with corals and coral reefs. One way that bacteria benefit corals is by stimulating the larval to juvenile life cycle transition of settlement and metamorphosis. Tetrabromopyrrole (TBP) is a small molecule produced by bacteria that stimulates metamorphosis with and without attachment in a range of coral species. A standing debate remains, however, about whether TBP biosynthesis from live Pseudoalteromonas bacteria is the primary stimulant of coral metamorphosis. In this study, we create a Pseudoalteromonas sp. PS5 mutant lacking the TBP brominase gene, bmp2. Using this mutant, we confirm that the bmp2 gene is critical for TBP biosynthesis in Pseudoalteromonas sp. PS5. Mutation of this gene ablates the bacterium’s ability in live cultures to stimulate the metamorphosis of the stony coral Porites astreoides. We further demonstrate that expression of TBP biosynthesis genes is strongest in stationary and biofilm modes of growth, where Pseudoalteromonas sp. PS5 might exist within surface-attached biofilms on the sea floor. Finally, we create a modular transposon plasmid for genomic integration and fluorescent labeling of Pseudoalteromonas sp. PS5 cells. Our results functionally link a TBP biosynthesis gene from live bacteria to a morphogenic effect in corals. The genetic techniques established here provide new tools to explore coral-bacteria interactions and could help to inform future decisions about utilizing marine bacteria or their products for coral restoration.
Some marine bacteria stimulate the early life-cycle transition from larval to juvenile phases in corals (i.e., metamorphosis) [1,2,3]. These bacteria could be used to promote coral larval recruitment in the wild, cultivate corals for reseeding degraded reefs or the aquarium trade, or help test basic science questions in the laboratory. Single-species biofilms of Pseudoalteromonas bacteria, such as Pseudoalteromonas sp. strain PS5, have been shown to stimulate coral metamorphosis [2, 4, 5]. Moreover, the compound tetrabromopyrrole (TBP), extracted from Pseudoalteromonas bacteria or chemically synthesized, robustly promotes the metamorphosis of diverse coral species with and without attachment [2, 5,6,7] (Fig. 1A). However, an open question remains about whether live Pseudoalteromonas bacteria stimulate coral larval metamorphosis solely by producing TBP, or whether these Pseudoalteromonas strains produce yet unknown products that confer part or all of the stimulatory activity [6, 8].
In Pseudoalteromonas sp. PS5 and phylogenetically related strains, TBP biosynthesis from L-proline is performed by three enzymes and a carrier protein encoded by the brominated marine pyrroles/phenols (bmp) gene cluster [9], specifically genes bmp1-4 [10] (Fig. 1B). We thus sought to create a TBP deletion mutant in Pseudoalteromonas sp. PS5 to query coral larval metamorphosis by genetically inactivating the bmp2 gene, which codes for the brominase that installs all four bromine atoms in TBP [10]. We determined that Pseudoalteromonas sp. PS5 is amenable to genetic manipulation by conjugation using a broad-host-range gfp reporter plasmid from the Marine Modification Kit (MMK) plasmid system [11]. We generated a Pseudoalteromonas sp. PS5 mutant with an in-frame deletion of the bmp2 gene (∆bmp2) following previously established methods for double-homologous recombination [12] (Fig. 1B). The modified strains exhibited a similar growth rate to wild type; however, the plasmid containing and bmp2 mutant strains persisted longer in stationary phase (Fig. 1C). The mutation of bmp2, as anticipated, resulted in the complete loss of TBP production in the ∆bmp2 strain in stark contrast to the wild-type culture that readily produces TBP (1468 ± 401 µM TBP, P = 0.05, one-tailed Mann–Whitney test) (Fig. 1D). These results confirm that the bmp2 gene is required for TBP production in Pseudoalteromonas sp. PS5 under the conditions tested.
We next tested whether bacteria lacking the bmp2 gene were able to stimulate the metamorphosis of the Caribbean coral, Porites astreoides, which has been shown previously to undergo metamorphosis in response to Pseudoalteromonas sp. PS5 and TBP [2]. When exposed to biofilms of wild type Pseudoalteromonas sp. PS5, we observed the metamorphosis of coral larvae consistent with previous findings [2, 4,5,6], both attached to the substrate (11.6% ± 7.2, Adjusted P = 0.0002, Dunn’s multiple comparisons test) and unattached (55% ± 13.1, Adjusted P < 0.0001, Dunn’s multiple comparisons test) (Fig. 1E, Supplementary Table S1). In contrast, total morphogenesis of coral larvae was reduced from 63.5% in wild type biofilm treatments to 3.5% in the ∆bmp2 biofilm treatment (Adjusted P < 0.0001, Dunn’s multiple comparisons test) (Fig. 1E, Supplementary Table S1). We observed attached (0.6% ± 2.9) and unattached (5.1% ± 10.4) metamorphosis in the ∆bmp2 treatment at rates comparable to the background in the media disk and the seawater disk (placed directly in the Petri dishes with filtered seawater) controls. Our results demonstrate that the effect of Pseudoalteromonas sp. PS5 on coral metamorphosis is primarily due to the production of TBP.
We then questioned whether different growth conditions affect the expression of the bmp genes in Pseudoalteromonas sp. PS5. To this end, we cloned the bmp1 and bmp9 promoters, fused them with a NanoLuciferase (NLuc) reporter gene, and conjugated the resulting plasmids into Pseudoalteromonas sp. PS5 (Fig. 1B) [11]. Luminescence was measured during each growth phase with the highest activity measured in late stationary and biofilm phases (Fig. 1F). The bmp1 promoter displayed a 203-fold increase in expression between early stationary and biofilm (Fig. 1F). The bmp9 promoter followed similar expression profiles, suggesting that the gene cluster may be co-regulated. We also tested broad-host-range promoters, which displayed at least a 457-fold activity range compared to assay background across all tested conditions (Supplementary Fig. S1). Our results suggest that the expression of TBP biosynthesis genes is strongest when bacteria exist in a slow-growth state when Pseudoalteromonas sp. PS5 might occur within surface-attached biofilms on the sea floor.
Pseudoalteromonas species are known to associate with marine eukaryotes and produce interesting antimicrobial metabolites [13], yet the study of these host-microbe and microbe-microbe interactions remains challenging due to the limited genetic tools for their manipulation. We therefore developed an integrative Tn10 transposon for use in Pseudoalteromonas sp. PS5, which is compatible with existing modular genetic toolkit parts [11, 14, 15] (Supplementary Fig. S2). With the Tn10 transposon, we generated Pseudoalteromonas sp. PS5 with gfp-tags integrated into the genome (Fig. 1G, Supplementary Fig. S2), which were confirmed by whole genome sequencing to identify genomic insertion loci (Supplementary Table S2).
In this work, the genetic engineering of a marine microbe enabled us to test a standing question about the role of TBP in coral metamorphosis. Our results represent the first characterization of a gene in a marine bacterium conveying a morphogenic effect in corals. Previous studies suggest that Pseudoalteromonas species may not be present in ecologically relevant concentrations that would stimulate coral metamorphosis [6]. However, TBP could be used as a molecule to elucidate mechanisms of coral morphogenesis. The strains and methodological advancements developed in this study could be helpful for dissecting how TBP stimulates metamorphosis with and without attachment in corals [16] and more broadly for studying TBP’s effects on eukaryotic cell physiology [17,18,19,20]. Furthermore, these approaches could be applied to inform responsible use of coral probiotic strain candidates encoding the bmp gene cluster[21]. Beyond TBP, our results demonstrate how bacterial genetics can help characterize genes and gene products from bacteria in the context of non-model marine microbial interactions, providing new techniques to interrogate the microbial ecology of Pseudoalteromonas spp. We hope this work will represent a step towards elucidating function in bacteria-coral interactions and will inform the use of bacteria for coral reef restoration [22].
Data availability
Key plasmids generated for this study are available through Addgene (https://www.addgene.org/Nicholas_Shikuma/).
References
Tran C, Hadfield MG. Larvae of Pocillopora damicornis (Anthozoa) settle and metamorphose in response to surface-biofilm bacteria. Mar Ecol Prog Ser. 2011;433:85–96.
Sneed JM, Sharp KH, Ritchie KB, Paul VJ. The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals. Proc R Soc B Biol Sci. 2014;281:20133086.
Petersen LE, Kellermann MY, Nietzer S, Schupp PJ. Photosensitivity of the bacterial pigment cycloprodigiosin enables settlement in coral larvae—light as an understudied environmental factor. Front Mar Sci. 2021;8:1–13.
Negri AP, Webster NS, Hill RT, Heyward AJ. Metamorphosis of broadcast spawning corals in response to bacteria isolated from crustose algae. Mar Ecol Prog Ser. 2001;223:121–31.
Tebben J, Tapiolas DM, Motti CA, Abrego D, Negri AP, Blackall LL, et al. Induction of larval metamorphosis of the coral Acropora millepora by tetrabromopyrrole isolated from a Pseudoalteromonas bacterium. PLoS One. 2011;6:e19082.
Tebben J, Motti CA, Siboni N, Tapiolas DM, Negri AP, Schupp PJ, et al. Chemical mediation of coral larval settlement by crustose coralline algae. Sci Rep. 2015;5:10803.
Siboni N, Abrego D, Seneca F, Motti CA, Andreakis N, Tebben J, et al. Using bacterial extract along with differential gene expression in Acropora millepora Larvae to decouple the processes of attachment and metamorphosis. PLoS One. 2012;7:37774.
Petersen LE, Moeller M, Versluis D, Nietzer S, Kellermann MY, Schupp PJ. Mono- and multispecies biofilms from a crustose coralline alga induce settlement in the scleractinian coral Leptastrea purpurea. Coral Reefs. 2021;40:381–94.
Agarwal V, El Gamal AA, Yamanaka K, Poth D, Kersten RD, Schorn M, et al. Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nat Chem Biol. 2014;10:640–7.
El Gamal A, Agarwal V, Diethelm S, Rahman I, Schorn MA, Sneed JM, et al. Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair. Proc Natl Acad Sci USA. 2016;113:3797–802.
Alker AT, Farrell MV, Aspiras AE, Dunbar TL, Fedoriouk A, Jones JE, et al. A modular plasmid toolkit applied in marine Proteobacteria reveals functional insights during bacteria-stimulated metamorphosis. MBio. 2023;e0150223:1–17.
Alker AT, Delherbe N, Purdy TN, Moore BS, Shikuma NJ. Genetic examination of the marine bacterium Pseudoalteromonas luteoviolacea and effects of its metamorphosis-inducing factors. Environ Microbiol. 2020;22:4689–701.
Holmström C, Kjelleberg S. Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol. 1999;30:285–93.
Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol. 2015;4:975–86.
Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, et al. Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids. ACS Synth Biol. 2018;7:1279–90.
Siboni N, Abrego D, Motti CA, Tebben J, Harder T. Gene expression patterns during the early stages of chemically induced larval metamorphosis and settlement of the coral Acropora millepora. PLoS One. 2014;9:e91082.
Zheng J, Antrobus S, Feng W, Purdy TN, Moore BS, Pessah IN. Marine and anthropogenic bromopyrroles alter cellular Ca2+dynamics of murine cortical neuronal networks by targeting the ryanodine receptor and sarco/endoplasmic reticulum Ca2+-ATPase. Environ Sci Technol. 2021;55:16023–33.
Zheng J, Mckinnie SMK, El Gamal A, Feng W, Dong Y, Agarwal V, et al. Organohalogens naturally biosynthesized in marine environments and produced as disinfection byproducts alter sarco/endoplasmic reticulum Ca2+ dynamics. Env Sci Technol. 2018;52:5469–78.
Whalen KE, Kirby C, Nicholson RM, O’Reilly M, Moore BS, Harvey EL. The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton. Sci Rep. 2018;8:15498.
Akkipeddi SMK, Xu M, Chan KYK. Halogenated compound secreted by marine bacteria halts larval urchin development. J Exp Mar Bio Ecol. 2021;538:151540.
Ushijima B, Gunasekera SP, Meyer JL, Tittl J, Pitts KA, Thompson S, et al. Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease. Commun Biol. 2023;6:248.
Peixoto RS, Sweet M, Villela HDM, Cardoso P, Thomas T, Voolstra CR, et al. Coral probiotics: premise, promise, prospects. Ann. Rev. Animal Biosci. 2021;9:265–88.
Sullivan MJ, Petty NK, Beatson SA. Easyfig: a genome comparison visualizer. Bioinformatics. 2011;27:1009–10.
Acknowledgements
Corals were collected under FKNMS-2019-24 permit from the Florida Keys National Marine Sanctuary. We would like to thank SECORE International and Florida Department of Environmental Protection for support and personnel provided for coral larval collection. We would like to thank Erich Bartels, Cory Walter, Joe Kuehl, and Samantha Simpson, for collecting and returning the Porites astreoides colonies and Zach Ferris, Natalie Danek and Samantha Scheibler for helping with larval collection over the 2021-2022 larval collection seasons at the Mote Marine Laboratory’s Elizabeth Moore International Center for Coral Reef Research and Restoration in Summerland Key, Florida. We thank Tommy Demarco, Carle Dugan, Yesmarie de la Flor and Kelly Pitts for help setting up and scoring the coral metamorphosis assays. We would also like to thank Dr. Eric Allen, Dr. Kristen Marhaver, Dr. Raphael Ritson-Williams and Dr. Blake Ushijima for guidance and helpful discussions about coral metamorphosis and microbiology. Finally, thank you to the Shikuma Lab members, including Dr. Tiffany Dunbar, Dr. Kyle Malter, Dr. Kate Nesbit, Emily Darin and Andy Fedoriouk for their feedback and support with cloning, imaging, and editing the manuscript. Schematic figures were created with Biorender.com under agreement number PF2508LO46.
Funding
This work was supported by the National Science Foundation (2017232404, ATA; 1942251, NJS; OCE-1837116, BSM), the Gordon and Betty Moore Foundation (GBMF9344 to NJS.; https://doi.org/10.37807/GBMF9344), Office of Naval Research (N00014-20-1-2120 to NJS), the National Institutes of Health, (R35GM146722 to NJS; R01ES030316 to BSM) and the Alfred P. Sloan Foundation, Sloan Research Fellowship (NJS).
Author information
Authors and Affiliations
Contributions
ATA and NJS conceived the study. ATA and MVF performed cloning, bacterial experiments and analyses. ATA, AMD, JMS and VJP performed coral experiments and analyses. TNP, SA, BSM and ATA performed the chemical analyses. ATA, NJS, VJP, JMS and BSM acquired the research funds. ATA and NJS wrote the manuscript with contributions of writing and editing the manuscript by all co-authors.
Corresponding author
Ethics declarations
Competing interests
ATA and NJS are coinventors on provisional U.S. patent application Serial number 63/323,653, entitled “Genetic Engineering of Marine Bacteria for Biomaterial Production, Probiotic Use in Aquaculture and Marine Environmental Restoration” and assigned to San Diego State University Research Foundation.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Alker, A.T., Farrell, M.V., Demko, A.M. et al. Linking bacterial tetrabromopyrrole biosynthesis to coral metamorphosis. ISME COMMUN. 3, 98 (2023). https://doi.org/10.1038/s43705-023-00309-6
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s43705-023-00309-6