Detection of Verrucomicrobia in a pasture soil by PCR-mediated amplification of 16S rRNA genes

Abstract

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.

FIG. 1

Alignments of oligonucleotide primers and target sequences. Mismatches are given in bold type. The nucleotide position numbering is taken from the work of Brosius et al. (5), the bacterial consensus sequence is from the review of Lane (18), and the GenBank accession numbers are given in square brackets. B, C or G or T; I, inosine; K, G or T; M, A or C; R, A or G; X, unspecified nucleotide; Y, C or T. Dots indicate identity with the Verrucomicrobia consensus. Mixed bases in the primers are equimolar.

FIG. 2

Phylogenetic dendrogram showing the relationships between cloned 16S rRNA gene fragments from the Ellinbank pasture soil (env. EV101 to env. EV155) and members of Verrucomicrobia. The 16S rRNA gene sequence from E. coli was used to root the dendrogram. Sequences prefixed with “env.” are cloned fragments from environmental samples. The GenBank accession numbers are given in square brackets. Nodes recovered in 90% or more of the 1,000 bootstrap dendrograms are indicated by filled circles, those recovered in 70 to 89% are indicated by open circles, and those recovered in less than 70% have no symbol. Bar, 10 inferred nucleotide substitutions per 100 positions.