Article Figures & Data
Figures
- 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.
Tables
- TABLE 1.
PCR primers used in this study
Primer Positionsa Sequence (5′→3′) Annealing tempb Reference or source F2 CAG TCA CGA CGT TGT AAA ACG ACG GC 62.0 28 R4*c CAG GAA ACA GCT ATG ACC ATG 28 Acd31F 15-31 GAT CCT GGC TCA GAA TC 56.7 3 Ver53F 37-53 TGG CGG CGT GGW TAA GA 61.0 D. Buckleyd 1492R 1492-1510 GGT TAC CTT GTT ACG ACT T 30 ↵ a The positions of the target region are given using E. coli numbering system of the 16S rRNA gene. Primers F2 and R4* complement regions of the multiple cloning site of pCR2.1 or pCR4.0.
↵ b Annealing temperatures optimized for the conditions used in this study are given for the forward primer of each primer pair.
↵ c Primer R4* was modified from the primer in reference 28 by omission of five deoxynucleotides from the 5′ end.
↵ d Personal communication.
