To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

No abstract

Phylogenetic analysis of the Saccharomyces cerevisiae group based on polymorphisms of rDNA spacer sequencesRobert M on t roc her, M a r i e-C h r ist i ne Verner, JerBme B r io I ay,2 Christian Gautier2 and Roland Marrneissel The phylogenetic relationships between species of yeasts assigned to the Saccharomyces sensu stricto group, which includes Saccharomyces cerevisiae and Saccharomyces bayanus, were studied together with Saccharomyces pastorianus and Saccharomyces paradoxus. The ex per i mental approaches used were RFLP analysis of the PCR-amplified rDNA internal transcribed spacer (ITS) and intergenic spacer, and total ITS sequence analysis. Both RFLP and sequence analyses gave fairly similar results. The gene trees generated with either of the two data sets showed the distribution of the yeasts into two major, wellseparated, phylogenetic clusters called ' cerevisiae ' and 'bayanus '. The 'cerevisiae' cluster included the 5. cerewisiae type strain, together with most of the species (16 out of 23), whereas the 'bayanus' cluster included the remaining seven type strains. Therefore, analysis of rDNA sequences confirmed 5. cerewisiae and 5. bayanus as two well-defined taxa. However, 5. pastorianus and S. paradoxus, the two other usually accepted taxa of the nowdefined Saccharomyces sensu stricto complex, could not be clearly separated from 5. bayanus and 5. cerevisiae, respectively. However, in both PCR-RFLP and ITS sequence analyses, S. paradoxus had the outermost position in the cerevisiae cluster. PCR-RFLP analysis of the ribosomal spacer sequences was also carried out on 26 Saccharomyces strains isolated in various wine-growing regions of France in an attempt to clarify their positions in the Saccharomyces phylogenetic tree. Compared to the diversity of the Saccharomyces type strains, less genetic diversity was detected among these yeasts and several of them exhibited identical RFLP patterns. Most of the wine yeast strains (16 out of 26) were closely related to each other and were found within the 'cerevisiae' cluster. The remaining 10 wine yeast strains branched within the bayanus cluster. PCR-RFLP analysis of ribosomal spacer sequences thus appears to be a useful and appropriate method for the correct characterization of Saccharomyces yeast strains used in food processing.