Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon

Abstract

One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from 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. Phylogenetic analyses of the small subunit rRNA sequence demonstrated its close relationship to that of previously described planktonic archaea, 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 bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.Characterization of complex microbial communities by isolation and analysis of phylogenetically informative gene sequences has been an exciting development in microbiology (27,32). Studies using molecular phylogenetic approaches based on small subunit (ssu) rRNA sequence analyses have resulted in new estimates of the phylogenetic diversity contained within naturally occurring microbial assemblages. Entirely new phylogenetic lineages, which may sometimes represent major constituents of natural microbial communities, have been revealed by using such approaches (3,4,7,13,16,22,29). Although results of these studies have contributed substantially to assessments of naturally occurring microbial diversity, their utility as predictors of the physiological attributes of newly described phylotypes has been more limited. This is partly because many phylogenetically coherent prokaryote lineages, for example, the proteobacteria, often encompass a bewildering array of physiological and metabolic diversity. The problem is further complicated by the fact that many of the newly discovered phylotypes revealed by molecular phylogenetic analysis are proving difficult to enrich and isolate in pure culture. These limitations suggest the need for alternative approaches to characterize the physiological and metab...

Enrichment and characterization of ammonia-oxidizing archaea from the open ocean: phylogeny, physiology and stable isotope fractionation

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Bias in assessments of marine microbial biodiversity in fosmid libraries as evaluated by pyrosequencing

“…The use of fosmids to propagate large (B40 kb) genomic inserts with a high degree of fidelity (Kim et al, 1992) has enabled contextual analysis of genes and their genomic neighbourhood; this has led to the identification of particular metabolic pathways, such as RNA helicase in Archaea (Stein et al, 1996), and the discovery of bacteriorhodopsin in marine bacteria (Béjà et al, 2000). Fosmid libraries have also given an insight into the genomic variation within and between mixed marine microbial assemblages in relation to water depth (Suzuki et al, 2004;DeLong et al, 2006) and symbiosis in marine invertebrates (Hughes et al, 1997;Schleper et al, 1998;Campbell et al, 2003).…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

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