bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Phylogenetic analysis of the salinipostin g-butyrolactone gene cluster uncovers 2 new potential for bacterial signaling-molecule diversity 3 4 Kaitlin E. Creamera, Yuta Kudoa, Bradley S. Mooreb,c, Paul R. Jensena# 5 6 a Center for Marine Biotechnology and Biomedicine, Scripps Institution of 7 Oceanography, University of California San Diego, La Jolla, California, USA 8 b Center for Oceans and Human Health, Scripps Institution of Oceanography, University 9 of California San Diego, La Jolla, California, USA 10 c Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California 11 San Diego, La Jolla, California, USA 12 13 Running Head: Phylogenetic analysis of the salinipostin gene cluster 14 15 #Address correspondence to Paul R. Jensen, [email protected]. 16 17 Keywords salinipostin, g-butyrolactones, biosynthetic gene clusters, Salinispora, 18 bacterial signaling molecules, actinomycetes, HGT bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 19 Abstract 20 Bacteria communicate by small-molecule chemicals that facilitate intra- and inter- 21 species interactions. These extracellular signaling molecules mediate diverse processes 22 including virulence, bioluminescence, biofilm formation, motility, and specialized 23 metabolism. The signaling molecules produced by members of the phylum 24 Actinobacteria are generally comprised of g-butyrolactones, g-butenolides, and furans. 25 The best known actinomycete g-butyrolactone is A-factor, which triggers specialized 26 metabolism and morphological differentiation in the genus Streptomyces. Salinipostins 27 A-K are unique g-butyrolactone molecules with rare phosphotriester moieties that were 28 recently characterized from the marine actinomycete genus Salinispora. The production 29 of these compounds has been linked to the 9-gene biosynthetic gene cluster spt. Critical 30 to salinipostin assembly is the g-butyrolactone synthase encoded by spt9. Here, we 31 report the global distribution of spt9 among sequenced bacterial genomes, revealing a 32 surprising diversity of gene homologs across 12 bacterial phyla, the majority of which 33 are not known to produce g-butyrolactones. Further analyses uncovered a large group 34 of spt-like gene clusters outside of the genus Salinispora, suggesting the production of 35 new salinipostin-like diversity. These gene clusters show evidence of horizontal transfer 36 between many bacterial taxa and location specific homologous recombination exchange 37 among Salinispora strains. The results suggest that g-butyrolactone production may be 38 more widespread than previously recognized. The identification of new g-butyrolactone 39 biosynthetic gene clusters is the first step towards understanding the regulatory roles of 40 the encoded small molecules in Actinobacteria. 41 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 Importance 43 Signaling molecules orchestrate a wide variety of bacterial behaviors. Among 44 Actinobacteria, g-butyrolactones mediate morphological changes and regulate 45 specialized metabolism. Despite their importance, few g-butyrolactones have been 46 linked to their cognate biosynthetic gene clusters. A new series of g-butyrolactones 47 called the salinipostins was recently identified from the marine actinomycete genus 48 Salinispora and linked to the spt biosynthetic gene cluster. Here we report the detection 49 of spt-like gene clusters in diverse bacterial families not known for the production of this 50 class of compounds. This finding expands the taxonomic range of bacteria that may 51 employ this class of compounds and provides opportunities to discover new compounds 52 associated with chemical communication. 53 54 Introduction 55 Bacteria use chemical signaling molecules to regulate gene expression in a population 56 dependent manner. This process, known as quorum sensing, controls group behaviors 57 including swarming, bioluminescence, virulence, biofilm formation, cell competence, 58 DNA uptake, public good production, and specialized metabolism. In many Gram- 59 negative bacteria, quorum sensing is mediated by acyl-homoserine lactone (AHL) 60 autoinducers (AIs) and their cognate receptors (1). In some Gram-positive bacteria, 61 autoinducing peptides (AIPs) and their respective transmembrane two-component 62 histidine sensor kinases control similar group behaviors (2). Among Actinobacteria, g- 63 butyrolactone signaling molecules regulate morphological development and specialized 64 metabolite production. Given the importance of Actinobacteria for the production of bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 65 antibiotics and other useful compounds, the discovery of new signaling molecules could 66 facilitate the discovery of new natural products from the large number of “cryptic” gene 67 clusters detected in actinomycete genome sequences. 68 69 To date, the types of signaling molecules known to be produced by Actinobacteria 70 include g-butyrolactones (3–25), g-butenolides (26–30), furans (31, 32), PI factor (33), 71 and N-methylphenylalanyl-dehydrobutyrine diketopiperazine (34) (Figure 1). Most of 72 these were discovered from members of the genus Streptomyces. Sometimes referred 73 to as actinobacterial “hormones”, signaling molecules are commonly produced in low 74 amounts and have proven difficult to isolate and characterize. Many of these molecules 75 not only induce the production of specialized metabolites, but also regulate bacterial 76 morphogenesis and control complex regulatory systems (15). The first bacterial 77 signaling molecule discovered was A-factor (autoregulatory factor, 2-isocapryloyl-3R- 78 hydroxymethyl-g-butyrolactone) from the actinomycete Streptomyces griseus. It was 79 shown to trigger sporulation and the production of the antibiotic streptomycin (3). A- 80 factor biosynthesis requires a g-butyrolactone synthase and a reductase encoded by the 81 genes afsA and bprA, respectively (15), and its elucidation revitalized the search to link 82 signaling molecules to their biosynthetic genes (16, 32, 35–41). Most of the 83 biosynthetically characterized g-butyrolactones, g-butenolides, and furans have been 84 linked to afsA gene homologs via sequence similarity, biochemical verification, or A- 85 factor receptor binding assays. However, many afsA gene homologs observed in 86 Streptomyces, Kitasatospora, and Amycolatopsis genomes have yet to be linked to a bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 87 small molecule (42–51). Likewise, the Acl series of g-butyrolactones reported from S. 88 coelicolor has not been linked to an AfsA homolog (3, 14, 18) (Figure 1). 89 90 While most A-factor-like molecules have been identified from Streptomyces strains, it 91 remains possible that other actinobacterial taxa produce related signaling molecules. 92 This includes the obligate marine actinomycete genus Salinispora, which is comprised 93 of nine species: Salinispora tropica, S. arenicola, S. oceanensis, S. mooreana, S. 94 cortesiana, S. fenicalii, S. goodfellowii, S. vitiensis, and S. pacifica (52, 53) isolated from 95 marine sediments (54–57), seaweeds (55), and sponges (58, 59). The genus 96 Salinispora has proven to be a prolific source of specialized metabolites (60) including 97 the proteasome inhibitor salinosporamide A (61), which is currently in phase III clinical 98 trials as an anticancer agent. Whole-genome sequencing of 118 Salinispora strains 99 revealed 176 distinct biosynthetic gene clusters (BGCs), of which only 25 had been 100 linked to their products (62). In a subsequent study, a majority of Salinispora BGCs 101 were shown to be transcriptionally active under standard cultivation conditions, 102 suggesting that many of their small molecule products were being missed using 103 traditional detection and isolation techniques (63). Given that little is known about the 104 regulation of specialized metabolism in this genus, it remains possible that quorum 105 sensing signaling molecules may play a role in the regulation of BGC expression. 106 107 Recently, a series of compounds known as salinipostins A-K with rare bicyclic 108 phosphotriesters were identified from a Salinispora sp. RLUS08-036-SPS-B (64). While 109 these compounds were identified based on anti-malarial activity against Plasmodium bioRxiv preprint doi: https://doi.org/10.1101/2020.10.16.342204; this version posted October 16, 2020. The copyright holder for this preprint (which was not certified by peer review)