First insight into the genome of an uncultivated crenarchaeote from soil

Abstract

Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…These may be major contributors to ammonia oxidation in world soils (Leininger et al, 2006), yet they have only been detected in agricultural soil clone libraries (Ochsenreiter et al, 2003), a fosmid metagenomic library from a soil sample (Quaiser et al, 2002) and in root-extract enrichment cultures (Simon et al, 2005). Cells were disrupted from the soil matrix, isolated with a micromanipulation method for individual FISH probe-positive cells (Ishøy et al, 2006) and used for MDA and DNA sequencing (Kvist et al, 2007).…”

“In‐gel patch electrophoresis:” A new method for environmental DNA purification

“…The "In gel patch electrophoresis" method is more simple, faster and more efficient than other methods usually reported. Under standard electrophoretic conditions, humic substances with phenolic groups co-migrate with nucleic acids [11,12]. The addition of a hydroxyapatite patch to the agarose gel eliminates co-migration by retarding the electrophoretic mobility of humic substances.…”

“…These compounds severely inhibit amplification reactions by PCR, hydrolysis by restriction enzymes, as well as ligation and cloning procedures. 3 Numerous attempts have focused on methods of genomic DNA extraction from a variety of environmental samples [11][12][13]. Laborious and time-consuming protocols involving DNA purification (gradient centrifugation, glass bead extraction, chromatography column, long-termed gel electrophoresis, spin column, agarose plug, two phase system) or extensive sample dilution prior to PCR have been necessary to obtain a PCR amplification from an environmental template.…”

Metagenomics: Future of microbial gene mining

“…Quaiser and co-worker (2002) pression patterns [62][63]. Quaiser and co-worker (2002) [64] studied and analyzed genome of an uncultivable [64] studied and analyzed genome of an uncultivable Crenarchaeote Crenarchaeote using direct genome analysis. Authors have using direct genome analysis.…”