Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage

Cultivation-independent surveys of ribosomal RNA genes have revealed the ex istence of novel microbial lineages, many with no known cultivated represen tatives. Ribosomal RNA-based analyses, however, often do not provide signif icant information beyond phylogenetic affiliation. Analysis of large genome fragments recovered directly from microbial communities represents one pro mising approach for characterizing uncultivated microbial species better. T o assess further the utility of this approach, we constructed large-insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of pla nktonic marine microbial assemblages. The BAC libraries we prepared had ave rage insert sizes of approximate to 80 kb, with maximal insert sizes >150 k b. A rapid screening method assessing the phylogenetic diversity and repres entation in the library was developed and applied. In general, representati on in the libraries agreed well with previous culture-independent surveys b ased on polymerase chain reaction (PCR)-amplified rRNA fragments. A signifi cant fraction of the genome fragments in the BAC libraries originated from as yet uncultivated microbial species, thought to be abundant and widely di stributed in the marine environment. One entire BAC insert, derived from an uncultivated, surface-dwelling euryarchaeote, was sequenced completely. Th e planktonic euryarchaeal genome fragment contained some typical archaeal g enes, as well as unique open reading frames (ORFs) suggesting novel functio n. In total, our results verify the utility of BAC libraries for providing access to the genomes of as yet uncultivated microbial species, Further ana lysis of these BAC libraries has the potential to provide significant insig ht into the genomic potential and ecological roles of many indigenous micro bial species, cultivated or not.