bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055285; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC 4.0 International license. 1 Title: Symbiotic and non-symbiotic members of the genus Ensifer (syn. Sinorhizobium) are 2 separated into two clades based on comparative genomics and high-throughput phenotyping 3 4 Authors: Camilla Fagorzi1, Alexandru Ilie1, Francesca Decorosi2, Lisa Cangioli1, Carlo Viti2, 5 Alessio Mengoni1*, George C diCenzo1,3* 6 7 Affiliations: 1 Department of Biology, University of Florence, Sesto Fiorentino, Italy 8 2 Genexpress Laboratory, Department of Agriculture, Food, Environment and Forestry, 9 University of Florence, Sesto Fiorentino, Italy 10 3 Department of Biology, Queen’s University, Kingston, Ontario, Canada 11 12 * Corresponding Authors: Alessio Mengoni ([email protected]) and George diCenzo 13 ([email protected]). 14 15 Data Deposition: Genome sequences were deposited to the NCBI under the BioProject 16 accession PRJNA622509. As of the time of submission, the sequences are still being processed 17 by the NCBI and thus are not yet publicly available. 18 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055285; this version posted April 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC 4.0 International license. 19 ABSTRACT 20 Rhizobium – legume symbioses serve as a paradigmatic example for the study of mutualism 21 evolution. The genus Ensifer (syn. Sinorhizobium) contains diverse plant-associated bacteria, 22 a subset of which can fix nitrogen in symbiosis with legumes. To gain insights into the evolution 23 of symbiotic nitrogen fixation (SNF), and inter-kingdom mutualisms more generally, we 24 performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The 25 data suggest that SNF emerged several times within the genus Ensifer, likely through 26 independent horizontal gene transfer events. Yet, the majority (105 of 106) of the Ensifer strains 27 with the nodABC and nifHDK nodulation and nitrogen fixation genes were found within a 28 single, monophyletic clade. Comparative genomics highlighted several differences between the 29 “symbiotic” and “non-symbiotic” clades, including divergences in their pangenome content. 30 Additionally, strains of the symbiotic clade carried 325 fewer genes, on average, and appeared 31 to have fewer rRNA operons than strains of the non-symbiotic clade. Characterizing a subset 32 of ten Ensifer strains identified several phenotypic differences between the clades. Strains of 33 the non-symbiotic clade could catabolize 25% more carbon sources, on average, than strains 34 of the symbiotic clade, and they were better able to grow in LB medium and tolerate alkaline 35 conditions. On the other hand, strains of the symbiotic clade were better able to tolerate heat 36 stress and acidic conditions. We suggest that these data support the division of the genus Ensifer 37 into two main subgroups, as well as the hypothesis that pre-existing genetic features are 38 required to facilitate the evolution of SNF in bacteria. 39 40 Keywords: Mutualism, evolutionary biology, phenomics, comparative genomics, rhizobia, 41 Proteobacteria 42 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055285; this version posted April 25, 2020. 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-NC 4.0 International license. 43 INTRODUCTION 44 Symbioses are pervasive phenomena present in all Eukaryotic forms of life (López-García et 45 al. 2017). These includes the evolution of organelles, obligate symbioses, and facultative 46 symbiotic interactions (Douglas 2014), with symbiotic nitrogen fixation (SNF) being a 47 paradigmatic example of the latter (Masson-Boivin & Sachs 2018). SNF (the conversion of N2 48 to NH3) is performed by a polyphyletic group of bacteria from the Alphaproteobacteria and 49 Betaproteobacteria (whose nitrogen-fixing members are collectively called rhizobia) and 50 members of the genus Frankia (Wang & Young 2019; Masson-Boivin et al. 2009) while intra- 51 cellularly housed within specialized organs (nodules) of specific plants in the family Fabaceae 52 and the genus Parasponia, as well as the actinorhizal plants (Werner et al. 2014; Velzen et al. 53 2018; Griesmann et al. 2018). The advantages and evolutionary constraints to SNF have long 54 been investigated in the conceptual framework of mutualistic interactions and the exchange of 55 goods (see for instance (Sørensen et al. 2019; Werner et al. 2015; Heath & Tiffin 2007)), and 56 quantitative estimations with metabolic reconstructions have also been performed (Pfau et al. 57 2018; diCenzo, Tesi, et al. 2019). 58 The establishment of a symbiotic nitrogen-fixing interaction requires that the bacterium 59 encode several diverse molecular functions, including those related to signalling and metabolic 60 exchange with the host plant, nitrogenase and nitrogenase-related functions, and escaping or 61 resisting the plant immune system (Oldroyd et al. 2011; Haag et al. 2013; Poole et al. 2018). 62 In general, the core SNF genes (nod, nif, fix genes, among others) are located within mobile 63 genetic elements that include symbiotic islands and symbiotic (mega)-plasmids (Tian & Young 64 2019; Checcucci et al. 2019; Geddes et al. 2020), facilitating their spread through horizontal 65 gene transfer (HGT) (Sullivan et al. 1995; Barcellos et al. 2007; Pérez Carrascal et al. 2016). 66 Emphasizing the role of HGT in the evolution of rhizobia, rhizobia are dispersed across seven 67 families of the Alphaproteobacteria and one family of the Betaproteobacteria, and most genera 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055285; this version posted April 25, 2020. 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-NC 4.0 International license. 68 with rhizobia also contain non-rhizobia (Garrido-Oter et al. 2018; Wang 2019). 69 An interesting area of investigation is whether the evolution of mutualistic symbioses, 70 such as SNF, depends on metabolic/genetic requirements (“facilitators”, as in (Gerhart & 71 Kirschner 2007)) aside from the strict symbiotic genes (Zhao et al. 2017; Long 2001; Sanjuán 72 2016). In other words, i) is the acquisition of symbiotic genes present in genomic islands or 73 plasmids sufficient to become a symbiont or, ii) are metabolic pre-requirements or adaptation 74 successive to HGT required? A comparative genomics study of 1,314 Rhizobiales genomes 75 identified no functional difference between rhizobia and non-rhizobia based on Kyoto 76 Encyclopedia of Genes and Genomes (KEGG) annotations (Garrido-Oter et al. 2018), 77 suggestive of an absence of obvious facilitators. In contrast, experimental studies are generally 78 consistent with an important role of non-symbiotic genes in establishing or optimizing 79 rhizobium – legume symbioses. Several studies have shown that effective symbionts are not 80 produced following the transfer of symbiotic plasmids from rhizobia of the genera Rhizobium 81 or Ensifer (syn. Sinorhizobium) to closely related non-rhizobia from the genera Agrobacterium 82 or Ensifer (see for instance (Hooykaas et al. 1982; Finan et al. 1986; Rogel et al. 2001); 83 reviewed in (diCenzo, Zamani, et al. 2019)). Similarly, the same symbiotic island is associated 84 with vastly different symbiotic phenotypes depending on the Mesorhizobium genotype 85 (Nandasena et al. 2007; Haskett et al. 2016). Further supporting the need for additional 86 adaptations to support SNF, symbiosis plasmid transfer coupled to experimental evolution can 87 lead to the gain of more advanced symbiotic phenotypes (Doin de Moura et al. 2020). 88 The genus Ensifer provides an ideal model to further explore the differentiation, or lack 89 thereof, of symbiotic bacteria from non-symbionts. This genus comprises rhizobia such as E. 90 meliloti and E. fredii, as well as non-rhizobia like E. morelense and E. adhaerens, and many 91 members have been extensively studied producing an abundant set of experimental and 92 genomic data (for a recent review, see (diCenzo, Zamani, et al. 2019)). The genus Ensifer, as 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.23.055285; this version posted April 25, 2020. 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-NC 4.0 International license. 93 currently defined, resulted from the combination of the genera Sinorhizobium and Ensifer based 94 on similarities in the 16S rRNA and recA sequences of the type strains and the priority of the 95 name Ensifer (Young 2003; Willems et al.). Multilocus sequence analysis supported the 96 amalgamation of these genera (Martens et al. 2007), although it was subsequently noted that 97 E. adhaerens (the type strain) is an outgroup of this taxon based on whole genome 98 phylogenomics (Ormeño-Orrillo et al. 2015). A more recent taxonomy approach based on 99 genome phylogeny suggests that the genus Ensifer should again be split, with the initial type 100 strains of Ensifer and Sinorhizobium belonging to separate genera (Parks et al. 2018). 101 In this paper we report an extensive comparative genomic and phenotypic 102 characterization of legume symbionts and non-symbionts of the genus Ensifer. We identified 103 that SNF likely evolved multiple times through independent HGT events; even so, most 104 symbionts were found in a single clade, consistent with a requirement for pre-existing genetic 105 features to facilitate the evolution of SNF.