Abstract N-210 Contact Information The majority of the soil’s biosphere containins biodiveristy that remains yet to be discovered. The occurrence of novel bacterial phyla in soil, as well as the phylogenetic diversity within bacterial phyla with few cultured representatives (e.g. Acidobacteria, Anne Spain Dr. Mostafa S.Elshahed Verrucomicrobia, and Gemmatimonadetes) have been previously well documented. However, few studies have focused on the Composition, Diversity, and Novelty within Soil Proteobacteria Department of Botany and Microbiology Department of Microbiology and Molecular Genetics novel phylogenetic diversity within phyla containing numerous cultured representatives. Here, we present a detailed University of Oklahoma Oklahoma State University phylogenetic analysis of the Proteobacteria-affiliated clones identified in a 13,001 nearly full-length 16S rRNA gene clones 770 Van Vleet Oval 307 LSE derived from Oklahoma tall grass prairie soil. Proteobacteria was the most abundant phylum in the community, and comprised Norman, OK 73019 Stillwater, OK 74078 25% of total clones. The most abundant and diverse class within the Proteobacteria was Alphaproteobacteria, which comprised 405 325 5255 405 744 6790 39% of Proteobacteria clones, followed by the Deltaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, which made Anne M. Spain (1), Lee R. Krumholz (1), Mostafa S. Elshahed (2) up 37, 16, and 8% of Proteobacteria clones, respectively. Members of the Epsilonproteobacteria were not detected in the dataset. [email protected] [email protected] Detailed phylogenetic analysis indicated that 14% of the Proteobacteria clones belonged to 15 novel orders and 50% belonged (1) Dept. of Botany and Microbiology, University of Oklahoma, Norman, OK to orders with no described cultivated representatives or were unclassified. Within the Betaproteobacteria, one novel order and 13 novel families were detected. Also, 85% of Betaproteobacteria clones belonged to family-level lineages containing no (2) Dept. of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK described or characterized isolates and 69% belonged to orders containing no described representatives, MND1 and A21b. This work provides detailed phylogenetic evidence for novel lineages within the subdivisions of Proteobacteria and we can conclude Summary: Proteobacteria in Soil that although this phylum contains more cultivated microorganisms than any other phylum, the majority of soil Proteobacteria belong to lineages whose members remain uncultivated. Thus, the functions of soil microorganisms belonging to well-described Composition in Soil: Comparison of 4 Studies Novel Lineages of Soil phyla, such as Proteobacteria, are not yet understood. • Our study shows similar composition of Proteobacteria in soil to that found Proteobacteria in Minnesota farm soil. In this study, we found novel lineages in each • class of Proteobacteria % Proteobacteria estimates from pyrosequencing efforts are much higher than for clone libraries of near full-length sequences: possible that short • 15 Novel Orders Construction of a Large Clone Library from Dominant Phyla in the KFS Clone Library Alphaproteobacteria Deltaproteobacteria sequences lead to false taxonomic classifications. • 46 Novel Families Kessler Farm Soil (KFS): 13,001 Clones (>1%) General Composition and Novel Lineages General Composition and Novel Lineages • Kessler Farm Soil (KFS) collected from an undisturbed tall Undescribed Lineages of Soil grass prairie preserve in Central Oklahoma. No. Clones/OTUs 1043/168 No. Clones/OTUs 998/165 Proteobacteria No. Families: 45 (13 novel) No. Families: 33 (15 novel) • 16S rRNA genes: PCR-amplified (primers 27f and 1391r) and 25 8 The majority of soil Proteobacteria sequences 6 No. Orders 29 (8 novel) No. Orders 15 (6 novel) cloned into TOPO-TA cloning vector (Invitrogen). belong to uncharacterized lineages, containing no 4 % clones belonging to undescribed Families: 37% % clones belonging to undescribed Families: 85% • Clones sequenced at the DOE Joint Genome Institute. 20 named or described cultivated representatives. %2 Abundance % clones belonging to undescribed Orders: 28% % clones belonging to undescribed Orders: 64% • Sequences binned into different datasets based on % Abundance 0 • 50% of all Proteobacteria clones belong to 15 AlphaproteobacteriaDeltaproteobacteriaBetaproteobacteria taxonomic affiliations assigned using Ribosomal Database Gammaproteobacteria undescribed orders. Program’s Classifier program 10 Family-Level Lineages detected in KFS Soil Order-Level Family-Level Lineages • 65% Proteobacteria clones belong to % Abundance Order-Level Distribution • Sequences in each dataset binned into OTUs using 97% Betaproteobacteria AlphaproteobacteriaDeltaproteobacteria undescribed families. Gammaproteobacteria similarity cutoff (DOTUR) 5 of Clones Distribution detected in KFS Soil • Deltaproteobacteria contains the most clones Symbol Key • OTUs aligned using the Greengenes NAST alignment tool. of Clones Symbol Key belonging to undescribed lineages: [No. clones (no. OTUs)] Rhodoplanaceae [300 (15)] • NJ phylogenetic trees constructed using the ARB software 0 [No. clones (no. OTUs)] Families with named/ • 637 clones in orders containing no Bradyrhizobium [55 (2)] Bradyrhizobiales Symbol Key package from NAST-aligned sequences and closely related SPAM Symbol Key described cultivated Families with characterized cultivated representatives. Beijerckinaceae [3 (1)] representatives Solid slices: Order contains named/described sequences from the ARB May, 2007 database; taxonomic Firmicutes Solid slices: Order contains described, named • 848 clones in families containing no Chloroflexi Labrys [11 (1)] No named/described cultivated representatives Acidobacteria Bacteroidetes described, named Labrys assignments made based on Hugenholtz nomenclature. ProteobacteriaActinobacteria isolates cultivated representatives no cultivated Verrucomicrobia Planctomycetes A20 [2 (2)] characterized cultivated representatives. cultivated representatives A20 No cultivated No fill: Order-level lineage Myxococcales-KFS-1 [31 (17)] representatives Gemmatimonadetes Pattern slices: Order contains Devosia [9 (5)] representatives composed of clone Novel family undescribed, unnamed Novel family sequences only. cultivated representatives Rhizobiaceae [20 (4)] Rhizobiales JG37-AG-33 [27 (9)] Bootstrap value of branch to No fill: Order-level lineage Phyllobacteriaceae [11 (1)] Bootstrap value of branch to the right: >90 composed of clone the right: >90 5 Nannocystis [3 (2)] Bootstrap value: 70-89 Hyphomicrobiaceae [43 (10)] sequences only. Bootstrap value: 70-89 [1] Bootstrap value: 50-69 LO133 [32 (5)] Bradyrhizobiales Bootstrap value: 50-69 Myxococcales-KFS-2 Rhodomicrobium [8 (2)] Betaproteobacteria 11 Polyangiaceae [56 (29)] Gammaproteobacteria Alphaproteobacteria-KFS-1 [22 (6)] Alphaproteobacteria-KFS-1 Myxococcales-KFS-3 [4 (4)] Methylobacteraceae [22 (4)] Bradyrhizobiales Myxococcales General Composition and Novel Lineages SM1D08 [6 (5)] General Composition and Novel Lineages Rhodobacterales E48D05cD [1] Rhodobacterales−KFS−1 [5 (1)] No. Clones/OTUs 202/68 No. Clones/OTUs 432/78 Napoli-1B-37 [4 (3)] Caulobacter sp. A1 [14 (1)] No. Families: 19 (5 novel) No. Families: 23 (13 novel) Caulobacterales Brevundimonas [2 (2)] 9 Myxococcus [35 (8)] No. Orders 11 (0 novel) No. Orders 5 (1 novel) Nordella oligomobilis [8 (2)] % clones belonging to undescribed Families: 66% % clones belonging to undescribed Families: 85% 8 Anaeromyxobacter [7 (3)] Unclassified Nordella [6 (4)] Nordella % clones belonging to undescribed Orders: 58% FFCH5621 [3] % clones belonging to undescribed Orders: 69% Unclassified Nordella [39 (6)] Myxococcales-KFS-4 [1] KF−JG30−B3 [6 (3)] Ellin329 soil isolate Ellin329 10 Geobacteraceae [44 (2)] Desulfuromonadales H29 [1] Family-Level Lineages detected in KFS Soil H4 Order-Level Distribution isolate str. A0839 PB19 [3 (2)] PB19 Family-Level Lineages detected in KFS Soil Sphingomonadales−KFS−1 [89 (12)] Symbol Key Order-Level Distribution of Clones 9 Aquicella [20 (16)] Sphingomonas [25(11)] NB1-i [17 (2)] [No. clones (no. OTUs)] of Clones NB1-j Rickettsiella [2 (2)] MND1-KFS-1 [31 (3)] Symbol Key Porphyrobacter [15 (3)] Sphingomonadales Japan trench sediment clone NB1-j Symbol Key Families with FFCH15729 (2) Legionellales-KFS-1 [3 (3)] named/described [No. clones (no. OTUs)] H16 [13(3)] Solid slices: Order contains described, [38 (2)] MND1 FFCH5139 [1] cultivated representatives MND1-KFS-2 named cultivated representatives Symbol Key Families with FFCH11237 [13 (5)] 0 Legionella [8 (7)] no described or named Alphaproteobacteria-KFS-2 JG37−AG−128 [4 (2)] Pattern slices: Order contains undescribed, MND1-KFS-3 [1 (1)] named/described FFCH4696 [7 (3)] cultivated representatives Solid slices: Order contains EB1021 unnamed cultivated representatives Legionellales-KFS-2 [3 (3)] cultivated representatives no cultivated described, named Defluvicoccus [3 (1)] Defluvicoccus EB1021-KFS-1 [7 (5)] No fill: Order-level lineage composed of FFCH16310 [1]

no described or named Legionellales cultivated representatives MND1-KFS-4 [3 (1)] representatives cultivated representatives FFCH10111 [32 (1)] clone sequences only. 0 Coxiella [2 (2)] Pattern slices: Order contains Unclassified Alphaproteobacteria JG37-AG-2 [286 (10)] Novel family [36 (8)] no cultivated FFCH1465 [1] undescribed, unnamed Ellin6067 Burkholderiales representatives Rhodovibrio−KFS−1 [1] Legionellales-KFS-3 [1] Bootstrap value of branch to cultivated representatives FFCH2365 [3 (3)] mle1-7 [3 (2)] Rhodovibrio the right: >90 No fill: Order-level lineage Novel family Rhodovibrio sodomensi Legionellales-KFS-4 [2 (2)] Deltaproteobacteria-KFS-2 Bootstrap value: 70-89 composed of clone FFCH2097 [3 (3)] LO131 [120 (21)] MND1 Bootstrap value of branch to FFCH17422 [1] Beggiatoa Thiotrichaceae Bootstrap value: 50-69 sequences only. the right: >90 Unclassified Alphaproteobacteria FFCH18567 [2 (1)] Deltaproteobacteria-KFS-4 [1] FFCH11282 [1] Gammaproteobacteria MND1-KFS-5 [2 (2)] Bootstrap value: 70-89 Deltaproteobacteria-KFS-4 Unclassified Ellin314−KFS−1 [103 (4)] OM60 [1] OM60 Bootstrap value: 50-69 Ellin314 OM27 [3 (3)] Myxococcales Comamondadaceae [51 (10)] Ellin328 Arctic96B-1 [1] Arctic96B-1 Burkholderiales [33 (4)] Azospirillium [1] Azospirillales 2 Desulfovibrionaceae Desulfovibrionales Pseudomonadales -KFS-1 Burkholderiales Pseudomonadaceae FFCH11210 [11 (2)] Alcaligenaceae [1 (1)] Bdellovibrionales-KFS-1 [3 (3)] Enterobacteriaceae [23 (1)] Enterobacteriales FFCH8463 [2 (1)] Unclassified Alphaproteobacteria Bdellovibrionales Thauera/Azoarcus [2 (2)] FFCH1031 5 Bdellovibrio bacteriovorus [4 (3)] Shewanellaceae Shewanellaceae Rhodocyclales AKYG694 [13 (2)] AKYG694 mussel_symbiont_MAR1 [1] Azonexus/Dechloromonas [4 (1)] Deltaproteobacteria-KFS-5 [2 (2)] Deltaproteobacteria-KFS-5 SUP05 Alphaproteobacteria-KFS-3 [26 (5)] Alphaproteobacteria-KFS-3 Symbionts Sulfur-oxidizing symbionts Oxalobacteraceae [6 (3)] CrystalBog021E5 [12 (7)] Ellin361 [27 (3)] Ellin333 MIZ46 Burkholderiales sedimentary rock clone MIZ46 Methylobacter Methylobacter * Burkholderiaceae [1 (1)] Alphaproteobacteria-KFS-4 [8 (1)] Alphaproteobacteria-KFS-4 [3 (1)] Ellin339 [24 (5)] Alphaproteobacteria−KFS−5 [4 (2)] Alphaproteobacteria-KFS-5 Deltaproteobacteria-KFS-1 Deltaproteobacteria-KFS-1 Ellin339 A21b-KFS-1 [18 (4)] Spotted Fever Group SA29-B [75 (7)] soil isolate Ellin6095 FFCH5157 [50 (8)] Rickettsia−KFS−1 [1 (1)] Rickettsiales Deltaproteobacteria-KFS-3 AKYG940 [10 (3)] AKYG940 A21b-KFS-2 [40 (2)] Neorickettsia risticii FFCH15355 [14 (3)] MP1-2H [4 (2)] MP1-2H SMD12 [4 (3)] Oleomonas A21b-KFS-5 [4 (1)] 3 Syntrophobacteraceae Syntrophobacteraceae Alphaproteobacteria-KFS-6 [2 (2)] Alphaproteobacteria-KFS-6 Nitrosococcus Nitrosococcus rhizosphere clone Amb_16S_1908 A21b rhizosphere clone Amb_16S_715 BD7−3 [1] Oleomonas Entotheonella-KFS-1 [134 (10)] Acidithiobacillus-KFS-1 [4 (3)] Acidithiobacillus Caedibacteraceae [1] Candidatus Entotheonella A21b-KFS-3 [30 (3)] Consistiales 2 Entotheonella palauensis * Acidithiobacillus ferrooxidans Alphaproteobacteria-KFS-7 [1] Alphaproteobacteria-KFS-7 [3 (3)] Unclassified Xanthomonadales [3 (3)] A21b-KFS-4 Alphaproteobacteria-KFS-8 [2 (1)] Alphaproteobacteria-KFS-8 Deltaproteobacteria-KFS-6 [210 (9)] Deltaproteobacteria-KFS-6 * Xanthomonas Pelagibacter [4 (3)] A21b-KFS-6 [2 (1)] Consistiales mle1−8 [9 (4)] mle1-8 Xanthomonadales FFCH1925 [5 (1)] Unclassified Xanthomonadales [3 (1)] A21b-KFS-7 [2 (1)] Unclassified Alphaproteobacteria Acetobacteraceae [2 (2)] Lysobacter [9 (1)] Acetobacterales 0.10 Betaproteobacteria−KFS−1 [1 (1)] * Tree rooted with Betaproteobacteria-KFS-1 * Roseomonas Tree rooted with * Tree rooted with Frateuria * 16SrRNA gene sequence Candidatus Alysiosphaera [6 (4)] Candidatus Alysiosphaera 16SrRNA gene sequence 16SrRNA gene sequence from Chloroflexus Nevskiaceae [1] Nevskiaceae * Tree rooted with 16SrRNA gene sequence from Chloroflexus from Chloroflexus aurantiacus 0.10 from Chloroflexus aurantiacus 0.10 aurantiacus aurantiacus 0.10