Detection and cultivation of soil verrucomicrobia - PubMed (original) (raw)

Detection and cultivation of soil verrucomicrobia

Parveen Sangwan et al. Appl Environ Microbiol. 2005 Dec.

Abstract

Only one isolate each of the class "Spartobacteria" (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class "Spartobacteria" and were related to "Chthoniobacter flavus." Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class "Spartobacteria" were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class "Spartobacteria" were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class "Spartobacteria" was inhibited by other culturable bacteria.

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Figures

FIG. 1.

Distribution of bacteria in the Ellinbank soil. Each point represents the results for a pooled 2-cm section (0 to 2, 2 to 4, 4 to 6, 6 to 8, and 8 to 10 cm). (A) The total cell count (▪), determined by microscopy, decreased with depth, whereas soil moisture (▵) was relatively constant. (B) The oligonucleotide probe VER1112 that targets 16S rRNA gene sequences of members of the class “_Spartobacteria_” (•) indicated that 16S rRNA genes from members of this class were more numerous than homologs from subdivision 3 (○) in PCR products generated by using a bacterium-specific primer pair. The errors bars represent SDs when they are larger than the symbol.

FIG. 2.

Evolutionary-distance dendrogram showing the relationships of newly isolated bacteria (in boldface) and other members of the class “_Spartobacteria_” and of subdivision 3 within the phylum Verrucomicrobia, based on comparisons of their 16S rRNA gene sequences. Also shown are two cultured members of the class Verrucomicrobiae. The dendrogram was constructed by using the Fitch-Margoliash (17) method from Jukes-Cantor (24) distances. The sequences of the 16S rRNA genes of Opitutus terrae (GenBank accession no. AJ229235) and Victivallis vadensis (GenBank accession AY049713) were used as outgroups and to root the tree; they are not shown in the figure. The GenBank accession numbers for the 16S rRNA gene sequences are given after each bacterium or clone name. Analyses using different algorithms resulted in very similar dendrograms. The number of times each branch point was recovered in dendrograms constructed from 1,000 bootstrapped datasets is indicated by symbols: •, recovered in >90%; ○, recovered in 75 to 89%. Nodes with no symbol were recovered in <75% of the bootstrapped datasets. The scale bar indicates 0.05 changes per nucleotide position.

FIG. 3.

Phase contrast photomicrographs, showing cells of isolate Ellin502, a member of the class “_Spartobacteria_” (A), and isolate Ellin514, a member of subdivision 3 (B). The scale bar represents 5 μm for both panels.

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Detection and cultivation of soil verrucomicrobia - PubMed (original) (raw)