Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model
- 11 August 2004
- journal article
- research article
- Published by Wiley in Environmental Microbiology
- Vol. 6 (9), 928-937
- https://doi.org/10.1111/j.1462-2920.2004.00575.x
Abstract
Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.Keywords
This publication has 49 references indexed in Scilit:
- Quantification of cell‐specific substrate uptake by probe‐defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridizationEnvironmental Microbiology, 2003
- Identification of Differences in Genome Content among phlD -Positive Pseudomonas fluorescens Strains by Using PCR-Based Subtractive HybridizationApplied and Environmental Microbiology, 2002
- Suppressive Subtractive Hybridization Detects Extensive Genomic Diversity in Thermotoga maritimaJournal of Bacteriology, 2002
- Genomic Analysis of the F3031 Brazilian Purpuric Fever Clone of Haemophilus influenzae Biogroup Aegyptius by PCR-Based Subtractive HybridizationInfection and Immunity, 2002
- Microbial communities in different soil types do not converge after diesel contaminationJournal of Applied Microbiology, 2002
- Screening of Environmental DNA Libraries for the Presence of Genes Conferring Na + (Li + )/H + Antiporter Activity on Escherichia coli : Characterization of the Recovered Genes and the Corresponding Gene ProductsJournal of Bacteriology, 2001
- Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyses using rRNA-targeted oligonucleotide probesFEMS Microbiology Ecology, 1997
- A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicumBiochemical and Biophysical Research Communications, 1992
- Microbial Ecology and Evolution: A Ribosomal RNA ApproachAnnual Review of Microbiology, 1986
- Evidence for the reductive pathway of deoxyribonucleotide synthesis in an archaebacteriumFEBS Letters, 1981