Laboratory cultivation of widespread and previously uncultured soil bacteria

Abstract

Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum ACTINOBACTERIA: In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.

FIG. 1.

Evolutionary distance tree of the bacterial domain showing an overview of culturable bacterial diversity for the Ellinbank soil samples at the family level. All family-level groupings (shown as wedges) were supported by bootstrap resampling (bootstrap proportion values, >75%). The phylogenetic depth of each of the phyla to which the families belong is shaded. Where the new isolates are the first isolated representatives of a proposed family, the family name is shown in boldface type. The 16S rRNA gene sequences of Sulfolobus acidocaldarius (GenBank accession no. D14876) and Methanococcus vannielii (accession no. M36507) and the hot spring 16S rRNA gene sequence pJP27 (accession no. L25852) were used as outgroups (data not shown) in the analysis. Bar, 0.05 change per nucleotide.