Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands)

Abstract

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass.

FIG. 1

Matching of clones with TGGE fingerprints of types A, F, and K generated from soil rRNA. The sequenced clones and their closest relatives are indicated. The 16S rRNA clusters are indicated in parentheses: HGC, high-G+C gram-positive bacteria; H/A, Holophaga/Acidobacterium cluster; Ver, Verrucomicrobium cluster; α and β, alpha and beta Proteobacteria. The column V6-hybridization summarizes the results of the V6 probe hybridization approach. Symbols: ++, positive identification by highly specific hybridization signal; +, positive identification by specific hybridization signal with minor cross-reactions; ∼, tentative identification by specific hybridization signal with major cross-reactions; ?, hybridization signals within the TGGE pattern too faint; K, A, or F, prominent sequence in type K, A or F; k, a or f, less abundant sequence in type K, A or F; −, not detected by the V6 probe.

FIG. 2

Phylogenetic tree of almost 8,000 SSU rRNA sequences within the ARB database. The clusters containing the DA sequences are highlighted. The numbers indicate the distribution of the DA sequences as compiled in Fig. 1. The alpha Proteobacteria and Cyanobacteria clusters also represent mitochondrial and chloroplast sequences, respectively. The Archaea and Eucarya branches are hidden. The bar in the lower right corner indicates the branch length and represents 0.1 base substitution per nucleotide.

FIG. 3

(A) Zoom into the cluster of the low-G+C gram-positive bacteria within the main tree in Fig. 2. The major clusters are represented by their best-known genera or species. The clusters containing the DA sequences are resolved, and the DA sequences are highlighted. One cluster is hidden but is presented in panel B. The bar in the lower right corner indicates the branch length. Abbreviations: B., Bacillus; P., Paenibacillus; B. sporoth., Bacillus sporothermodurans. (B) The hidden Bacillus cluster in panel A. The DA sequences found back in the TGGE fingerprints are highlighted, and two more DA sequences (DA026 and DA134) are also presented. One unnamed environmental sequence from Swedish groundwater is given by its accession number X91428 (38). The bar in the lower left corner indicates the branch length.

FIG. 3

(A) Zoom into the cluster of the low-G+C gram-positive bacteria within the main tree in Fig. 2. The major clusters are represented by their best-known genera or species. The clusters containing the DA sequences are resolved, and the DA sequences are highlighted. One cluster is hidden but is presented in panel B. The bar in the lower right corner indicates the branch length. Abbreviations: B., Bacillus; P., Paenibacillus; B. sporoth., Bacillus sporothermodurans. (B) The hidden Bacillus cluster in panel A. The DA sequences found back in the TGGE fingerprints are highlighted, and two more DA sequences (DA026 and DA134) are also presented. One unnamed environmental sequence from Swedish groundwater is given by its accession number X91428 (38). The bar in the lower left corner indicates the branch length.

FIG. 4

Zoom into the cluster of alpha Proteobacteria within the main tree in Fig. 2. The major clusters are represented by their best-known genera. The clusters containing the DA sequences are resolved, and the DA sequences found back in the TGGE fingerprints are highlighted. One more DA sequence (DA004) is also presented. The mitochondrial branch is hidden. Three unnamed environmental sequences are given by their accession numbers: those from Swedish groundwater (X91445) and Australian sludge (X84609 and X84612). Sequence “OS type O” originated from an Octopus Spring cyanobacterial mat (56). The bar in the lower right corner indicates the branch length.

FIG. 5

Zoom into the cluster of Holophaga and Acidobacterium within the main tree in Fig. 2. The DA sequences found back in the TGGE fingerprints are highlightened, and two more DA sequences (DA023, DA038) are also presented. Beyond the MC sequences from Australian forested soil (24) and the Japanese FIE and PAD sequences (53), other environmental sequences are marked with (A) from Amazonian forested soil (6), (H) from American mountain lakes (19), (B) from Australian sludge (4), and (L) from German agricultural soil (29). The 31 (L) sequences were assigned to (L) clusters where possible. The bar in the lower right corner indicates the branch-length.