Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with propos ed group specificity for planctomycetes, we examined anoxic bulk soil of fl ooded rice microcosms for the presence of novel planctomycete-like diversit y. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay w as assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence an alysis of cloned 16S rDNA, T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In cont rast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil co ntained 33 distinct T-RFs, a clearly higher level of complexity. A survey o f 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. Th e predominant T-RFs obtained from rice roots could be assigned to members o f the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descen t. Using a level of 16S rDNA sequence dissimilarity to cultured microorgani sms of approximately 20% as a threshold value, we detected 11 distinct bact erial lineages for which pure-culture representatives are not known. Four o f these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage w as affiliated with the spirochetes. The other five lineages either could no t be assigned to any of the main lines of bacterial descent or clearly expa nded the known diversity of division level lineages WS3 and OP3. Our result s indicate the presence of bacterial diversity at a subdivision and/or divi sion level that has not been detected previously by the so-called universal 16S rDNA PCR assays.