New strategies for cultivation and detection of previously uncultured microbes

Abstract

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O(2) [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO(2) (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO(2). A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

FIG. 1.

PWPCR method to detect growth and monitor isolation of targeted bacteria. Of the three medium and incubation conditions shown in this diagram (conditions A, B, and C), growth of targeted bacteria (+) is represented only in condition C.

FIG. 2.

Detection of V. spinosum in a collection of diverse bacteria isolated from soil. (A) A single V. spinosum colony is shown among 94 other colonies growing on an agar plate. (B) PWPCR with Verrucomicrobia-specific primers using template in which V. spinosum colony material represented 1 part in 95 parts (i.e., plate in panel A; lane 1), 1 part in 189 parts (lane 2), 1 part in 471 parts (lane 3), 1 part in 941 parts (lane 4), and 1 part in 9,401 parts (lane 5). PWPCR of a control plate lacking V. spinosum (lane 6), negative control (no DNA; lane 7), V. spinosum DNA (50 ng; lane 8), and molecular size markers (lane M) is also shown. The positions of molecular size markers (in kilobases) are shown to the left of the gel.

FIG. 3.

Influence of medium additives and incubation conditions on CFU recovered from soil (A) and on the occurrence of Acidobacteria among the isolates (B). “Air” is actually the concentration of CO₂ (0.03% [vol/vol]) in normal air. Data in panel A represent the mean CFU recovered from soil among n samples. Error bars represent sample standard deviations. Data in panel B represent the frequency with which Acidobacteria were detected among the plates in panel A using PWPCR. These data were subjected to chi-square analyses with Bonferroni's error rate adjustment. Statistical significance (α = 0.10, df = 1) is indicated by an asterisk.

FIG. 4.

(Left) Maximum-likelihood tree of subdivisions 1 to 4 of the phylum Acidobacteria based on 16S rRNA gene sequences from organisms in culture and PCR-generated clones from soil. Isolate designations and accession numbers are given. Isolates obtained in this study are shown in boldface type. Bootstrap values for branch points of the major subdivisions are given. Branch points conserved in all analyses with bootstrap values of >75% (closed circles) and bootstrap values of 50 to 74% (open circles) are indicated. Subdivisions 2 to 4 are labeled, bracketed, and condensed as shaded trapezia with the number of sequences represented in parentheses. 16S rRNA gene sequences of members of subdivisions 6 to 8 were used as outgroups (not shown). The scale bar represents 0.10 change per nucleotide. (Right) The scanning electron micrograph shows KBS89 cells trapped in an extracellular matrix.

FIG. 5.

(A) Maximum-likelihood tree of subdivisions 1 to 4 and 6 of the phylum Verrucomicrobia based on 16S rRNA gene sequences from organisms in culture and PCR-generated clones from environmental samples. Isolate designations and accession numbers are given. Isolates obtained in this study are shown in boldface type. Bootstrap values for branch points of the major subdivisions are given. Branch points conserved in all analyses with bootstrap values of >75% (closed circles) and bootstrap values of 50 to 74% (open circles) are indicated. Subdivisions 1 to 3 and 6 are labeled, bracketed, and condensed as shaded trapezia with the number of represented sequences in parentheses to simplify presentation of the tree. 16S rRNA gene sequences of members of the phylum Planctomycetes were used as an outgroup (not shown). The scale bar represents 0.10 change per nucleotide. (B and C) The scanning electron micrograph of TAV2 (B) shows the doublet cell morphology shared by all TAV isolates, and that of TAV1 (C) shows the encapsulation of the cells in an extracellular matrix, a morphological feature not shared by TAV2, TAV3, or TAV4.