Genome analysis of two Pseudonocardia phylotypes associated with Acromyrmex leafcutter ants reveals their biosynthetic potential Neil A. Holmes2, Tabitha M. Innocent1, Daniel Heine3, Mahmoud Al Bassam2, Sarah F. Worsley2, Felix Trottmann3, Elaine H. Patrick2, Douglas W. Yu2, 4, J. Colin Murrell5, Morten Schiøtt1, Barrie Wilkinson3*, Jacobus J. Boomsma1*, Matthew I. Hutchings2* 1Centre for Social Evolution, University of Copenhagen, Denmark, 2School of Biological Sciences, University of East Anglia, United Kingdom, 3Department of Molecular Microbiology, John Innes Centre, United Kingdom, 4State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, China, 5School of Environmental Sciences, University of East Anglia, United Kingdom Submitted to Journal: Frontiers in Microbiology Specialty Section: Antimicrobials, Resistance and Chemotherapy ISSN: 1664-302X Article type: Original Research Article Received on: 15 Oct 2016 Accepted on: Provisional08 Dec 2016 Provisional PDF published on: 08 Dec 2016 Frontiers website link: www.frontiersin.org Citation: Holmes NA, Innocent TM, Heine D, Al_bassam M, Worsley SF, Trottmann F, Patrick EH, Yu DW, Murrell J, Schiøtt M, Wilkinson B, Boomsma JJ and Hutchings MI(2016) Genome analysis of two Pseudonocardia phylotypes associated with Acromyrmex leafcutter ants reveals their biosynthetic potential. Front. Microbiol. 7:2073. doi:10.3389/fmicb.2016.02073 Copyright statement: © 2016 Holmes, Innocent, Heine, Al_bassam, Worsley, Trottmann, Patrick, Yu, Murrell, Schiøtt, Wilkinson, Boomsma and Hutchings. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. This Provisional PDF corresponds to the article as it appeared upon acceptance, after peer-review. Fully formatted PDF and full text (HTML) versions will be made available soon. Frontiers in Microbiology | www.frontiersin.org Provisional 1 Genome analysis of two Pseudonocardia phylotypes associated with Acromyrmex 2 leafcutter ants reveals their biosynthetic potential 3 4 Neil A. Holmes1, Tabitha M. Innocent2, Daniel Heine3, Mahmoud Al Bassam1, Sarah F. 5 Worsley1, Felix Trottmann3, Elaine H. Patrick1, Douglas W. Yu1,4, J. Colin Murrell5, Morten 6 Schiøtt2, Barrie Wilkinson3, Jacobus J. Boomsma2, Matthew I. Hutchings1 7 8 1School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, 9 Norfolk, United Kingdom. 10 11 2Centre for Social Evolution, University of Copenhagen, Copenhagen, Denmark. 12 13 3Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, 14 Norwich, Norfolk, United Kingdom. 15 16 4State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, 17 Kunming, Yunnan,China. 18 19 5School of Environmental Sciences, University of East Anglia, Norwich Research Park, 20 Norwich, Norfolk, United Kingdom. 21 22 Correspondence: 23 Professor Jacobus Boomsma, Email: [email protected] 24 Professor Matt Hutchings, Email: [email protected] 25 Professor Barrie Wilkinson, Email: [email protected] 26 27 Manuscript Length: 6672 28 Number of Figures: 8 29 30 Keywords: Leafcutter ants, antibiotics, actinomycetes, Pseudonocardia, nystatin, 31 polyene,Provisional genome mining, Acromyrmex 32 Abstract. 33 34 The attine ants of South and Central America are ancient farmers, having evolved a 35 symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived 36 attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their 37 fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of 38 Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal 39 partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia 40 mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi 41 make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain 42 isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called 43 nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring 44 secondary metabolites of Pseudonocardia on a genome-wide scale. Here we report a genomic 45 analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with 46 Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely 47 on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 48 strains revealed that the phylotypes are distinct species and each encodes between 11-15 49 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 50 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding 51 nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. 52 Strains show variations in the arrangement of these BGCs that resemble those seen in 53 gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that 54 vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the 55 cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with 56 Acromyrmex ants and supports the hypothesis that Pseudonocardia species could provide a 57 valuable source of new antimicrobials. 58 59 1. Introduction. 60 61 Almost all antibiotics currently in clinical use are derived from the secondary 62 metabolites of a group of soil bacteria called actinomycetes, but the discovery of these strains 63 and their natural products (NPs) peaked in the 1950s. Since then, problems of rediscovery of 64 extant strains and compounds has led to a decline in the discovery of new classes of 65 metabolitesProvisional. As a result, few new antibiotics have made it to market in the last 50 years. More 66 recently, however, the field of NP discovery has been revitalised by large scale genome 67 sequencing, which has revealed that actinomycetes express less than 25% of their secondary 68 metabolite biosynthetic gene clusters (BGCs) in vitro (Doroghazi et al., 2014). Genome 69 mining for novel BGCs in soil actinomycetes isolated over the last 80 years, plus new 70 actinomycete strains isolated from under-explored environments, promises to yield thousands 71 of new NPs, including new anti-infective drugs (Katz and Baltz, 2016). One promising new 72 approach is to genome mine strains that have co-evolved with their eukaryotic hosts. Such 73 symbiotic relationships are known as protective mutualisms, because the plant or animal host 74 houses, feeds, and sometimes vertically transmits the bacteria in exchange for antibiotics that 75 protect them against infection (Clardy et al., 2009; Kaltenpoth, 2009; Seipke et al., 2011b). 76 Arguably, the best characterized examples are the protective mutualisms between the attine 77 ants of South and Central America and their vertically transmitted strains of Pseudonocardia 78 (Cafaro et al., 2011; Caldera and Currie, 2012). 79 The common ancestor of attine ants developed fungiculture 50-60 million years ago, 80 leading to the tribe Attini, which consists of ca. 15 genera and more than 230 species 81 (Nygaard et al., 2016; Schultz and Brady, 2008). The most evolutionary derived attines are 2 82 the genera Acromyrmex and Atta which are known as leafcutter ants because they actively cut 83 fresh leaves and feed them to their co-evolved symbiotic fungus Leucoagaricus 84 gongylophorus. In return for the maintenance and manuring services provided by the ant 85 farmers, the fungal cultivar produces gongylidia that are rich in fats and sugars and which the 86 ants harvest as the sole food source for their larvae and queen (De Fine Licht et al., 2014; 87 Schiøtt et al., 2010 ). Acromyrmex ants grow Pseudonocardia on their cuticles (Figure 1), 88 housed in specialized crypts that are connected to subcuticular glands through which the 89 hosts likely provide nutrients (Currie et al., 2006)(Andersen et al., 2013). The 90 Pseudonocardia strain provides the ants with at least one antifungal compound and the ants 91 use it to protect their fungus against disease, in particular from the specialized fungal 92 pathogen Escovopsis (Barke et al., 2010; Currie et al., 2003; Oh et al., 2009; Sit et al., 2015). 93 Pseudonocardia are considered rare actinomycetes because they are hard to isolate 94 from soil, and there are relatively few (<20) available genome sequences for this genus 95 (Barke et al., 2010; 2011; Sit et al., 2015). Like other filamentous actinomycetes, they grow 96 as multicellular hyphae and reproduce under nutrient stress conditions by erecting aerial 97 hyphae that undergo cell division to form spores. The spores provide an effective dispersal 98 mechanism for these non-motile bacteria. In the well-studied actinomycete genus 99 Streptomyces, antibiotic production occurs at the onset of sporulation, but little is known 100 about the developmental life-cycle of Pseudonocardia or the secondary metabolites they 101 encode. It was recently discovered that Pseudonocardia mutualists of the lower attine 102 Apterostigma dentigerum and