bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Bacterial secondary metabolite biosynthetic potential in soil varies with phylum, depth, and 2 vegetation type 3 4 Allison M. Sharrar1, Alexander Crits-Christoph2, Raphaël Méheust1,3, Spencer Diamond1, Evan 5 P. Starr2, and Jillian F. Banfield1,3* 6 7 *Corresponding author: [email protected] 8 9 1Department of Earth and Planetary Science, University of California, Berkeley, CA, USA 10 2Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA 11 3Innovative Genomics Institute, Berkeley, CA, USA 12 13 14 15 16 17 18 19 20 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Abstract 25 Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and 26 other compounds with clinical value that likely shape interactions among microbial community 27 members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of 28 all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies 29 across the phylogenetic diversity of bacterial species in soils, and whether the genetic potential for 30 production of these metabolites differs with soil type. We sampled soils and saprolite from three 31 sites in a northern California Critical Zone Observatory with varying vegetation and bedrock 32 characteristics and used metagenomic sequencing and assembly to reconstruct 1,334 microbial 33 genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite 34 production. We obtained genomes for prolific producers of secondary metabolites, including novel 35 groups within the Actinobacteria, Chloroflexi and candidate phylum Dormibactereota. 36 Surprisingly, one genome of a Candidate Phyla Radiation bacterium encoded for a ribosomally 37 synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly 38 available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were 39 enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by 40 taxonomy. 41 42 Introduction 43 Many soil microbes biosynthesize secondary metabolite molecules that play important 44 ecological roles in their complex and heterogeneous microenvironments. Secondary (or 45 “specialized”) metabolites are auxiliary compounds that microbes produce which are not required 46 for normal cell growth, but which benefit the cells in other ways. These compounds can have roles 2 bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 in nutrient acquisition, communication, inhibition, or other interactions with surrounding 48 organisms or the environment1. Examples of these molecules include antibiotics1, siderophores2, 49 quorum-sensing molecules3, immunosuppressants4, and degradative enzymes5. 50 Secondary metabolites are of interest for both their ecological and biogeochemical effects, 51 as well as their potential for use in medicine and biotechnology. Antibiotics are a class of 52 secondary metabolites with obvious human importance. Historically, antibiotic discovery relied 53 on being able to culture organisms from the environment; however, the vast majority of 54 environmental taxa cannot be cultured using current methods. Most known antibiotics are from 55 cultured members of Actinobacteria, Proteobacteria, and Firmicutes6. Because soil microbial 56 communities are so diverse and most microbial taxa in soil have not been well-described7, they 57 offer a wealth of potential for the discovery of new and important microbial products. 58 Secondary metabolites are produced by biosynthetic gene clusters (BGCs), groups of 59 colocated genes that function together to build a molecule. Nonribosomal peptide synthetases 60 (NRPSs) and polyketide synthases (PKSs) are two of the largest classes of BGCs, encompassing 61 most known antibiotics and antifungals6. NRPSs are characterized by condensation (CD) and 62 adenylation (AD) domains8 and PKSs contain ketosynthase (KS) domains and a variety of other 63 enzymatic domains9. These characteristic domains can be used to identify novel NRPS and PKS 64 gene clusters and their abundances can be used as a proxy for biosynthetic potential10. 65 Little is known about how environmental variables impact the distribution of secondary 66 metabolites in soil. Recent studies of microbial biosynthetic potential in soil have utilized amplicon 67 sequencing of NRPS and PKS domains11,12,13,14,15. One study involving soils from a variety of 68 environments demonstrated that NRPS and PKS domain richness was high in arid soils and low in 69 forested soils11. Others showed that the composition of these domains correlated with latitude12 3 bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 70 and vegetation13 at a continental scale and was distinct between urban and nonurban soils14. 71 Because these studies rely on degenerate PCR primers designed for known domains, only 72 sequences similar to known domains can be recovered. In contrast, genome-resolved 73 metagenomics is able to recover divergent sequences within their genomic and phylogenetic 74 context. Recently, this approach revealed abundant biosynthetic loci in Acidobacteria, 75 Verrucomicrobia, Gemmatimonadetes, and the candidate phylum Rokubacteria16. 76 Here, we reconstructed genomes from 129 metagenomes sampled from various soil types 77 and soil conditions in three northern California ecosystems and searched them for BGCs. The first 78 site is a meadow grassland, the second is a Douglas fir / Madrone forested hillslope, and the third 79 is an Oak grassland hillslope. A subset of the genomes from meadow grassland soils analyzed here 80 were previously reported in studies that both reported on novel BGCs16 and demonstrated that soil 81 depth and soil moisture affect microbial community structure and function17,18. We present a 82 comparative analysis of the biosynthetic potential of bacteria from many phyla and report how soil 83 microbiology and secondary metabolic potential vary with soil type and environmental conditions. 84 Metagenomic studies such as this have the ability to identify new environmental and taxonomic 85 targets key to the understanding of secondary metabolism ecology, and for the development of 86 microbial natural products of human interest. 87 88 Materials & Methods 89 Sampling sites 90 Soil and saprolite samples were taken in areas studied by the Eel River Critical Zone 91 Observatory (CZO). Samples were taken from soil depths of 20-200 cm over a four year period, 92 from 2013 to 2016. The Eel River CZO experiences a Mediterranean climate characterized by hot, 4 bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 93 dry summers and cool, wet winters. The first fall rain after the dry summer generally comes in 94 mid-late September and most rain falls between November and March. Average yearly rainfall 95 varies from about 1.8-2 m19. 96 Samples were collected at two sites within the Angelo Coast Range Reserve: Rivendell, a 97 forested hillslope20,21, and a nearby meadow17,18,22. The meadow and Rivendell are 1.5 km apart 98 (Fig. 1). Both are underlain by the Coastal Belt of the Franciscan Formation, which consists of 99 mostly argillite (shale), with some sandstone and conglomerate23. At Rivendell, the soil mostly 100 lacks distinct horizons24 and varies in depth from 30-75 cm with saprolite directly below19. The 101 northern slope of Rivendell is dominated by Douglas fir (Pseudotsuga menziesii) trees, while the 102 southern slope has more Pacific madrone (Arbutus menziesii) trees. In the Angelo meadow, grass 103 roots are confined to depths of <10 cm17. 104 A third study site, Sagehorn, is a hilly grassland located about 23 km to the southeast of 105 the other two sites (Fig. 1)25,26. Sagehorn is underlain by the Central Belt of the Franciscan 106 Formation, a mélange with a sheared argillaceous matrix containing blocks of sandstone and other 107 lithologies27. Sagehorn soils generally have a 30 cm thick organic-rich mineral A horizon underlain 108 by a 10-20 cm thick Bt horizon with a high clay content, directly above saprolite. The low porosity 109 mélange bedrock causes these layers to become entirely saturated in the winter wet season26. 110 Sagehorn is primarily a grassland with scattered Garry oak (Quercus garryana) trees. 111 112 113 114 115 5 bioRxiv preprint doi: https://doi.org/10.1101/818815; this version posted October 25, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 116 117 Figure 1 – Eel River CZO sampling scheme. Soil and saprolite samples were taken from depths of 20-200 cm 118 across three sites between 2013 and 2016.