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

Abstract

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.

FIG. 1

16S rDNA-based T-RFLP fingerprint patterns obtained with the PV assay by using total DNA extracted from rice roots (A) and anoxic bulk soil (B) of a flooded rice microcosm. The numbers indicate the sizes of the T-RFs which could be correlated with phylogenetic information (Fig. 2 and 3). T-RFs for which no correspondence to cloned 16S rDNA could be found are indicated by arrows.

FIG. 2

Evolutionary distance dendrogram showing the positions of environmental 16S rDNA clone sequences recovered from rice roots (PRR clones) and anoxic bulk soil (PBS clones) of a flooded rice microcosm in relation to representative members of the order Planctomycetales and the division Verrucomicrobia. In addition to previously described taxa, the reference sequences included the Verrucomicrobia-like ultramicrobacteria VeGlc2, VeCb1, and VeSm13 (19) and environmental 16S rDNA sequences retrieved from marine aggregates (agg-27) (5), a trickling filter biofilm (Koll-18; GenBank accession no. AJ224943), a highly enriched consortium capable of anaerobic ammonia oxidation (anammox planctomycete) (44), and the anaerobic zone of a contaminated aquifer (WCHB sequences) (6). The individual lineages are designated as follows: Pir1 to Pir4 (Pir = Pirellula-like); Pla1 to Pla4 (Pla = planctomycete-like); and SD1, SD4, and SD5 (SD = recently proposed subdivisions of the division Verrucomicrobia [18]). The dendrogram was reconstructed by using the neighbor-joining method (40) in combination with a 50% consensus filter for the domain Bacteria. The numbers at the nodes indicate the percentages of recovery in 1,000 bootstrap resamplings. Only relevant bootstrap values of 70% or more are shown. The sizes of the T-RFs (in base pairs) indicated in brackets for the PRR and PBS clone sequences correspond to sizes in the T-RFLP diversity patterns shown in Fig. 1. The EMBL, GenBank, and DDBJ accession numbers are given in parentheses. Scale bar = 10% estimated difference in nucleotide sequence positions.

FIG. 3

Evolutionary distance dendrogram showing the positions of environmental 16S rDNA clone sequences recovered from rice roots (PRR clones) and anoxic bulk soil (PBS clones) of a flooded rice microcosm in relation to representative members of the Bacteria. In addition to previously described taxa, the reference sequences included environmental 16S rDNA sequences recovered from a hot spring in Yellowstone National Park (OPB-2) (17), a deep-sea sediment (BD4-9) (22), and the anaerobic zone of a contaminated aquifer (WCHA1-56) (6). The individual lineages are designated as follows: candidate divisions OP3 (17), WS3 (6), and BRC1 (= bacterial rice cluster); SP (= spirochete-like lineage); and NBL-UPA1 and NBL-UPA2 (NBL-UPA = novel bacterial lineage of uncertain phylogenetic affiliation). For WS3 and BRC1, the most divergent intralineage 16S rDNA clone sequences were used for the phylogenetic analysis. The dendrogram was reconstructed by using the neighbor-joining method (40) in combination with a 50% consensus filter for the domain Bacteria. The numbers at the nodes indicate the percentages of recovery of relevant branch points in 1,000 bootstrap resamplings. Branch points which were not supported by the confidence areas indicated around the nodes (bootstrap circle tool of the ARB program package) were at the division level collapsed back to the next significant branch point. For the most part such a lack of support corresponded to bootstrap values less than 50%. The root was determined by using archaeal 16S rRNA sequences as the outgroup reference. The sizes of the T-RFs (in base pairs) indicated in brackets for the PRR and PBS clone sequences correspond to the T-RFLP diversity patterns shown in Fig. 1. The EMBL, GenBank, and DDBJ accession numbers are given in parentheses. The only exception is the 16S rDNA sequence of Thermus thermarum LA3^T, for which the identity codes in the Ribosomal Database Project (30) and the ARB software package (http://www.biol.chemie.tu-muenchen.de/pub/ARB/) are indicated. Scale bar = 10% estimated difference in nucleotide sequence positions.