Phylogenetic and metagenomic analysis of Verrucomicrobia in former agricultural grassland soil

The bacterial phylum Verrucomicrobia has a widespread distribution, and is known to be one of the most common and diverse phyla in soil habitats. However, members of this phylum have typically been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we examine the phylogenetic diversity of the Verrucomicrobia in a former agricultural field and gain access to genomic information via a metagenomic approach. We examined Verrucomicrobia -like 16S rRNA gene sequences recovered from general bacterial and phylum-specific libraries, revealing a dominance of subdivisions 1 and 2. A PCR-based screening method was developed to identify inserts containing verrucomicrobial 16S rRNA genes within a large-insert metagenomic library, and on screening of 28 800 clones, four fosmids were identified as containing verrucomicrobial genomic DNA. Full-length sequencing of fosmid inserts and gene annotation identified a total of 98 ORFs, representing a range of functions. No conservation of gene order was observed adjacent to the ribosomal operons. Fosmid inserts were further analyzed for tetranucleotide frequencies to identify remnants of past horizontal gene transfer events. The metagenomic approach utilized proved to be suitable for the recovery of verrucomicrobial genomic DNA, thereby providing a window into the genomes of members of this important, yet poorly characterized, bacterial phylum.