bioRxiv preprint doi: https://doi.org/10.1101/2021.04.09.439258; this version posted April 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 2 Genomic analysis of family UBA6911 (Group 18 3 Acidobacteria) expands the metabolic capacities of the 4 phylum and highlights adaptations to terrestrial habitats. 5 6 Archana Yadav1, Jenna C. Borrelli1, Mostafa S. Elshahed1, and Noha H. Youssef1* 7 8 1Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, 9 OK 10 *Correspondence: Noha H. Youssef: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.04.09.439258; this version posted April 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 11 Abstract 12 Approaches for recovering and analyzing genomes belonging to novel, hitherto unexplored 13 bacterial lineages have provided invaluable insights into the metabolic capabilities and 14 ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent 15 and ecologically successful lineages on earth yet, currently, multiple lineages within this phylum 16 remain unexplored. Here, we utilize genomes recovered from Zodletone spring, an anaerobic 17 sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil 18 and non-soil habitats, to examine the metabolic capabilities and ecological role of members of 19 the family UBA6911 (group18) Acidobacteria. The analyzed genomes clustered into five distinct 20 genera, with genera Gp18_AA60 and QHZH01 recovered from soils, genus Ga0209509 from 21 anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All 22 genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile 23 heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon 24 sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil- 25 dwelling genera were characterized by larger genome sizes, higher number of CRISPR loci, an 26 expanded carbohydrate active enzyme (CAZyme) machinery enabling de-branching of specific 27 sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, 28 and a sole dependence on aerobic respiration. In contrast, non-soil genomes encoded a more 29 versatile respiratory capacity for oxygen, nitrite, sulfate, trimethylamine N-oxide (TMAO) 30 respiration, as well as the potential for utilizing the Wood Ljungdahl (WL) pathway as an 31 electron sink during heterotrophic growth. Our results not only expand our knowledge of the 32 metabolism of a yet-uncultured bacterial lineage, but also provide interesting clues on how 33 terrestrialization and niche adaptation drives metabolic specialization within the Acidobacteria. bioRxiv preprint doi: https://doi.org/10.1101/2021.04.09.439258; this version posted April 10, 2021. 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. 34 Importance 35 Members of the Acidobacteria are important players in global biogeochemical cycles, especially 36 in soils. A wide range of Acidobacterial lineages remain currently unexplored. We present a 37 detailed genomic characterization of genomes belonging to the family UBA6911 (also known as 38 group 18) within the phylum Acidobacteria. The genomes belong to different genera and were 39 obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 40 and UBA6911), and anaerobic digestor (Genus Ga0209509). While all members of the family 41 shared common metabolic features, e.g. heterotrophic respiratory abilities, broad substrate 42 utilization capacities, and few auxotrophies; distinct differences between soil and non-soil genera 43 were observed. Soil genera were characterized by expanded genomes, higher numbers of 44 CRISPR loci, larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer 45 extractions from polymer side chains, and methylotrophic (methanol and methylamine) 46 degradation capacities. In contrast, non-soil genera encoded more versatile respiratory capacities 47 for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron 48 acceptors. Our results not only broaden our understanding of the metabolic capacities within the 49 Acidobacteria, but also, provide interesting clues on how terrestrialization shaped Acidobacteria 50 evolution and niche adaptation. 51 52 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.09.439258; this version posted April 10, 2021. 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. 53 Introduction 54 Our appreciation of the scope of phylogenetic and metabolic diversities within the microbial 55 world is rapidly expanding. Approaches enabling direct recovery of genomes from 56 environmental samples without the need for cultivation allow for deciphering the metabolic 57 capacities and putative physiological preferences of yet-uncultured taxa (1-9). Further, the 58 development of a genome-based taxonomic framework that incorporates environmentally- 59 sourced genomes (10) has opened the door for phylocentric (lineage-specific) studies. In such 60 investigations, comparative analysis of genomes belonging to a target lineage is conducted to 61 determine its common defining metabolic traits, the adaptive strategies of its members to various 62 environments, and evolutionary trajectories of and patterns of gene gain/loss across this lineage. 63 Members of the phylum Acidobacteria are one of the most dominant, diverse and 64 ecologically successful lineages within the bacterial domain (11-16). Originally proposed to 65 accommodate an eclectic group of acidophiles (17), aromatic compound degraders and 66 homoacetogens (18), and iron-reducers (19), it was subsequently identified as a soil-dwelling 67 bacterial lineage in early 16S rRNA gene-based diversity surveys (20-22). Subsequent 16S 68 rRNA studies have clearly shown its near-universal prevalence in a wide range of soils, where it 69 represents 5-50% of the overall community (23, 24). 70 Various taxonomic outlines have been proposed for the Acidobacteria. Genome-based 71 classification by the Genome Taxonomy Database (GTDB, (r95, October 2020) (10) splits the 72 phylum into 14 classes, 34 orders, 58 families, and 175 genera. This classification broadly, but 73 not always, corresponds to 16S rRNA gene-based taxonomic schemes in SILVA (25), the 26 74 groups (subdivisions) classification scheme (26), and the most recently proposed refined 75 class/order classification scheme (27) (Table S1). Regardless, a strong concordance between 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.09.439258; this version posted April 10, 2021. 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. 76 habitat and phylogeny was observed within most lineages in the Acidobacteria. Some lineages, 77 e.g. groups 1, 3 (both in Class Acidobacteriae in GTDB), and group 6 (Class Vicinamibacteria in 78 GTDB), have predominantly been encountered in soils, while others, e.g. groups 4 (Class 79 Blastocatellia in GTDB), 8 (Class Holophagae in GTDB), and 23 (Class Thermoanaerobaculia in 80 GTDB), are more prevalent in non-soil habitats (11). 81 Genomic analysis of cultured (11) and uncultured metagenome-assembled genomes 82 (MAGs) and single cell genomes (SAGs) (28-30) representatives of the phylum Acidobacteria 83 has provided valuable insights into their metabolic capacities and lifestyle. However, the 84 majority of genomic (and other –omics) approaches have mostly focused on cultured, and yet- 85 uncultured genomes of soil Acidobacteria (31-33). Genomic-based investigations of non-soil 86 Acidobacteria pure cultures (34-37), or MAGs (38, 39) are more limited and, consequently, 87 multiple lineages within the Acidobacteria remain unexplored. 88 We posit that genomic analysis of hitherto unexplored lineages of Acidobacteria would 89 not only expand our knowledge of their metabolic capacities, but also enable comparative 90 genomic investigation on how terrestrialization and niche adaptation shaped the evolutionary 91 trajectory and metabolic specialization within the phylum. To this end, we focus on a yet- 92 uncultured lineage in the Acidobacteria: Family UBA6911 (Subdivision 18 in (21), Class 1-2 in 93 (27), and group 18 in SILVA database release 138.1 (25)). We combine the analysis of genomes 94 recovered from Zodletone spring, an anaerobic sulfide and sulfur-rich spring in southwestern 95 Oklahoma, with available genomes from multiple disparate soil and non-soil habitats. Our goal 96 was to: understand the metabolic capacities, physiological preferences, and ecological