CHARACTERIZATION OF UNCULTIVATED PROKARYOTES - ISOLATION AND ANALYSISOF A 40-KILOBASE-PAIR GENOME FRAGMENT FRONT A PLANKTONIC MARINE ARCHAEON

One potential approach for characterizing uncultivated prokaryotes fro m natural assemblages involves genomic analysis of DNA fragments retri eved directly from naturally occurring microbial biomass. In this stud y, we sought to isolate large genomic fragments from a widely distribu ted and relatively abundant but as yet uncultivated group of prokaryot es, the planktonic marine Archaea. A fosmid DNA library was prepared f rom a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. P hylogenetic analyses of the small subunit rRNA sequence demonstrated i ts close relationship to that of previously described planktonic archa ea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bo re highest similarity to archaeal homologs, including large subunit ri bosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its aff iliation with the Crenarchaeote subdivision of Archaea. Two gene fragm ents encoding proteins not previously found in Archaea were also ident ified: RNA helicase, responsible for the ATP-dependent alteration of R NA secondary structure, and glutamate semialdehyde aminotransferase, a n enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retriev ed from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokar yotes.