Novel phylogenetic lineages of as yet uncultivated crenarchaeota have been frequently detected in low to moderate-temperature, marine and terrestrial environments. In order to gain a more comprehensive view on the distribution and diversity of Crenarchaeota in moderate habitats, we have studied 18 different terrestrial and freshwater samples by 16S rDNA-based phylogenetic surveys. In seven different soil samples of diverse geographic areas in Europe (forest, grassland, ruderal) and Asia (permafrost, ruderal) as well as in two microbial mats, we have consistently found one particular lineage of crenarchaeota. The diversity of Crenarchaeota in freshwater sediments was considerably higher with respresentative 16S rDNA sequences distributed over four different groups within the moderate crenarchaeota. Systematic analysis of a 16S rDNA universal library from a sandy ecosystem containing 800 clones exclusively revealed the presence of the soil-specific crenarchaeotal cluster. With primers specific for non-thermophilic crenarchaeota we established a rapid method to quantify archaeal 16S rDNA in real time PCR. The relative abundance of crenarchaeotal rDNA was 0.5-3% in the bulk soil sample and only 0.16% in the rhizosphere of the sandy ecosystem. A nearby agricultural setting yielded a relative abundance of 0.17% crenarchaeotal rDNA. In total our data suggest that soil crenarchaeota represent a stable and specific component of the microbiota in terrestrial habitats.

Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

SummaryAcidobacteria have been established as a novel phylum of Bacteria that is consistently detected in many different habitats around the globe by 16S rDNAbased molecular surveys. The phylogenetic diversity, ubiquity and abundance of this group, particularly in soil habitats, suggest an important ecological role and extensive metabolic versatility. However, the genetic and physiological information about Acidobacteria is scarce. In order to gain insight into genome structure, evolution and diversity of these microorganisms we have initiated an environmental genomic approach by constructing large insert libraries directly from DNA of a calcerous grassland soil. Genomic fragments of Acidobacteria were identified with specific 16S rDNA probes and sequence analyses of six independently identified clones were performed, representing in total more than 210 000 bp. The 16S rRNA genes of the genomic fragments differed between 2.3% and 19.9% and were placed into two different subgroups of Acidobacteria (groups III and V). Although partial co-linearity was found between genomic fragments, the gene content around the rRNA operons was generally not conserved. Phylogenetic reconstructions with orthologues that were encoded on two of the six genomic fragments (PurF, PurL, PurB and formamidopyrimidine-DNA glycosylase) confirmed the coherence of the acidobacterial phylum. One genomic fragment harboured a cluster of eight genes which was syntenic and highly homologous to genomic regions in Rhodopseudomonas palustris and Bradyrhizobium japonicum , including a conserved two-component system. Phylogenetic analysis of the putative response regulator confirmed that this similarity between Rhizobiales and Acidobacteria might be due to a horizontal gene transfer. In total, our data give first insight into the genome content and diversity of the ubiquitously distributed but poorly characterized phylum of Acidobacteria. Furthermore they support the phylogenetic inferences made from 16S rRNA gene libraries, suggesting that Acidobacteria form a broad group in the same sense and with a similar diversity as that of many well-studied bacterial phyla.

Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen ∼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of ∼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only ∼930 predicted ‘canonical’ gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also ∼370 ‘non-canonical’ gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.

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.