Phylogenetic Screening of Ribosomal RNA Gene-Containing Clones in Bacterial Artificial Chromosome (BAC) Libraries from Different Depths in Monterey Bay

• M.T. Suzuki ^[1] ; C.M. Preston ^[2] ; O. Béjà ^{[2] [3]} ; J.R. de la Torre ^[2] ; G.F. Steward ^{[2] [5]} ; E.F. DeLong ^{[2] [4]}
  1. [1] University of Maryland, College Park

     University of Maryland, College Park

     Estados Unidos

     
  2. [2] Monterey Bay Aquarium Research Institute

     Monterey Bay Aquarium Research Institute

     Estados Unidos

     
  3. [3] Technion – Israel Institute of Technology

     Technion – Israel Institute of Technology

     Israel

     
  4. [4] Massachusetts Institute of Technology

     Massachusetts Institute of Technology

     City of Cambridge, Estados Unidos

     
  5. [5] Department of Oceanography, University of Hawaii, Honolulu, USA

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• Localización: Microbial ecology, ISSN-e 1432-184X, ISSN 0095-3628, Vol. 48, Nº. 4, 2004, págs. 473-488
• Idioma: inglés
• Texto completo no disponible (Saber más ...)
• Resumen

  • Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.