Firmicutes

1. 96 Firmicutes LG Anth 17-2 (63-70) SE/R2A 100 AB110497 Dyella japonica 65 Hat Adj 7-14 (0-10) SE/R2A 40 56 100 AY741332 Burkholdaria cepacia Hat Anth 1-8 (0-30) R2A/SE 45 100 AF063219 Pseudamonas stutzeri Hat Anth 1-13 (0-30) SE 100 AY946283 Enterobacter sp. Hat Adj 7-22 (0-10) R2A/SE 100 57 58 Hat Anth 2-13 (43-69) R2A 86 AB033949 Sphingomonas sp. Proteobacteria Hat Anth 2-3 (43-69) SE/R2A Hat Anth 2-10 (43-69) SE/R2A 100 99 AF508207 Mesorhizobium septentrionale Acu Anth 27-9 (48-83) R2A 51 70 LG Anth 18-10 (0-16) R2A/SE 97 Hat Adj 7A-11R (0-10) SE/R2A 52 94 Hat Anth 2-5R (43-69) SE/R2A AF510586 Bradyrhizobium sp. 92 LG Anth 17-6 (63-70) SE/R2A 60 AB035490 alpha proteobacterium 29 50 LG Anth 18-11 (0-16) SE/R2A Hat Anth 2-24 (43-69) R2A/SE 74 98 DS Anth 16-11 (Buried) SE/R2A X95471 Actinomyces sp. 61 Hat Anth 2-6 (43-69) SE 100 X52921 Myobacterium fortuitum LG Anth 17-3 (63-70) SE 93 37 Y08853 Terrebacter sp. 100 Hat Anth 1-17R (0-30) SE/R2A 53 AJ717357 Microbacterium oxydans 99 LG Anth 17-8 (63-70) R2A/SE Actinobacteria AY572475 Anthrobacter sp. 76 Hat Anth 2-12c (43-69) R2A/SE 76 94 Hat Anth 2-15 (43-69) SE 100 AY651318 Anthrobacter sp. LG Anth 19-1 (16-43) SE/R2A 48 Hat Anth 3-9 (43-69) SE/R2A 96 Hat Anth 3-6 (78-100) SE/R2A Hat Anth 2-22(*9)R (0-30) R2A/SE 9 LG Anth 17-10 (63-70) R2A/SE 10 Hat Anth 2-7 (43-69) SE/R2A 36 LG Anth 18-1 (0-16) SE/R2A AY238335 Flexibacter sp. 100 LG Adj 22-7 (0-8) R2A/SE Bacteroidetes 99 Hat Adj 11-14 (80-120) SE/R2A 47 AF361187 Flexibacter sp. X64372 Isosphaera pallida 80 AJ862839 Methanoculleus thermophilus 0.05 Isolating Unique Bacteria from Terra Preta Systems: Using Culturing and Molecular Techniques as Tools for Characterizing Microbial Life in Amazonian Dark Earths Brendan O’Neill1, Julie Grossman1, Siu Mui Tsai2, Jose Elias Gomes2, Carlos Eduardo Garcia2, Dawit Solomon1, Biqing Liang1, Johannes Lehmann1 and Janice Thies1(1) Cornell University, Department of Crop and Soil Science, Ithaca, NY, (2) Centro de Energia Nuclear na Agricultura (CENA) Piracicaba, Brazil Results Introduction Phylogeny of 16S rDNA from Isolates Terra preta soils are anthropogenic soils created by pre-Colombian indigenous cultures through the incorporation of organic-rich material into existing highly weathered soil. The resulting anthrosols are highly fertile, due, in part, to high charcoal (black carbon) content, which also leads to their distinct, blackish color. These anthrosols likely harbor a unique microbial ecology which contribute to their sustained fertility. We examined anthrosols (Anth) and adjacent (Adj) background soils from four sites in the Amazon Basin: Hatahara (Hat), Dona Stella (DS), Lago Grande (LG) and Açutuba (Acu). We hypothesized that 1) bacterial populations were higher in Anth than Adj. soils 2) Using culturing and molecular techniques, bacterial populations would be more similar between anthrosols than within the same site on two soil types. MPN enumeration on liquid R2 medium Most probable number (log10) of bacteria colony forming units (CFU) g-1 ODW soil growing in R2 liquid medium by site, depth and soil (anthrosol = gray bar, adjacent soil = brown bar). MPN was calculated using MPNES software from Woomer et al. (1990), CI(0.95). Summary of bacterial isolates Methods Summary DNA sequencing – family level • Extracts from anthrosols and adjacent soils were used to inoculate: 1) 5 replicate tubes at 6 dilutions of liquid R2 media and incubated for 45 days at 30ºC. 2) R2A and soil extract (SE) solid media, incubated for 120 days at 30ºC. • Liquid media tubes were scored for positive growth and used to calculate most probable number (MPN). • Colonies were isolated from solid media plates, and cross-cultivated onto alternate media (R2A for colonies forming on SE and SE for colonies forming on R2A). • After media screening, 16S rDNA region was amplified using colony PCR, and resulting fragments digested with restriction endonucleases to determine unique isolates. • Select 16S rDNA regions were sequenced based on RFLP pattern and results from media screening. • From soil DNA extraction, a culture-independent DNA fingerprint, DGGE, was used to compare portions of 16S rDNA in soil types. • Select bands from DGGE were sequenced and compared. DGGE of community DNA by site and soil type • In spite of a smaller percentage of isolates forming initially and growing exclusively on SE agar from anthrosols (14,5%) compared to adjacent soils (34.1%), anthrosols have twice the number of unique RFLP types. • On a community level basis using DGGE, anthrosols are more similar to each other than different soil types form the same site. • Sequencing reveals much higher family-level diversity in anthrosols, and for screening purposes, culturing proved useful for identifying unique community members. Conclusions • MPN calculation shows that for every site, anthrosols have as higher or a culturable bacteria population than adjacent soils. • The majority of isolate diversoty in termsn of unique RFLP types was derived from anthrosol isolates (52.8%) and only a third of unique RFLP types came from adjacent soils.