Culture-independent isolation of environmental bacterial species for genomic analysis

Fluorescence in situ hybridization (FISH) has been a commonly used method in molecular ecology studies. In many cases, one can visualize bacterial populations based upon the hybridization of FISH probes with bacterial 16S rRNA sequences in whole cell hybridizations. This process has been termed the full cycle rRNA approach. Several research groups have posited that the full cycle rRNA approach can be utilized to identify uncultured bacterial populations despite the limited ability to validate the accuracy of FISH probes for these groups. To account for these limitations, it has been suggested that the simultaneous hybridization of two or more probes on different loci within the same 16S rRNA molecule (i.e. 'the multiple-probes concept') can be used to evaluate the accuracy of FISH for uncultured populations in the absence of culture based validation methods.; In this dissertation we experimentally tested the accuracy of this hypothesis. Specifically, we utilized the multiple probes concept, as described by Ludwig et al., to design a pair of FISH probes for the visualization and identification of an uncultured Acidobacterium population within the planktonic community of an artesian spring in rural north-central Idaho (Ludwig et al. 1997). Upon recovery of a population that hybridized both probes and analysis of 1.4 kb 16S rRNA gene sequences obtained from their amplified genomic DNA, it was found that at least four different non-Acidobacterium species hybridized the probes. Additionally, our results indicated that probe length and the tertiary structure of the 30S ribosomal small subunit may be important criteria to consider in the de novo design of FISH probes, since in the recovered population contained several mismatches for both probes. Based upon these results the null hypothesis, that the multiple probes concept could accurately identify the uncultured species of interest, was rejected in favor of an alternative hypothesis that the multiple-probes concept could not be used to accurately identify an uncultured bacterial species in the groundwater community investigated. In conclusion, the findings suggested that the multiple-probes approach cannot be reliably used as the sole validation procedure to determine the accuracy of FISH probes designed for uncultured bacterial populations.