ALBERT

Notes: Abstract New methods based on PCR amplification of 16S rRNA genes from DNA samples extracted directly from the environment allow the description of microbial diversity in natural ecosystems without the need for cultivation. We have applied this technique to an extreme environment presumed to have very low diversity: the crystallizer ponds of a marine saltern with salinity over NaCl saturation. The molecular methodology has shown that indeed very low diversity can be found here. Prokaryotes belonging to the Bacteria domain are a minor component and only members of a closely related cluster of sequences were found, all relatives of the α-Proteobacteria (ca. 83% to Rhodopseudomonas marina). Halophilic Archaea were as expected the largest component of biomass in this environment. All the clones sequenced corresponded again to a highly homologous cluster (probably members of the same genus). However, all the sequences diverged considerably from the ones of the described genera of halophilic Archaea, in fact the data are consistent with the idea that the 16S rRNA genes directly amplified from the saltern correspond to members of an undescribed genus. This is remarkable since many collection strains sequenced come specifically from this saltern. Furthermore, 16S rDNA obtained from archaeal cultures isolated from the same sample had no homology to the sequences obtained by PCR amplification, instead they appear to be members of the well known genus Haloarcula. However, this concurs with the findings of other authors who obtained different organisms by culture from those detected by the sequences retrieved directly by PCR. A possible explanation is that culturability, in standard media, is the exception rather than the rule. To study the biodiversity gradient present along the salinity gradient found in a multi-pond solar saltern we have also applied a novel molecular strategy. This method is based on the restriction digestion of a population of 16S rDNA sequences directly amplified from an environmental sample. Digested fragments separated by polyacrylamide gel electrophoresis generate characteristic profile data for estimation of diversity and overall similarities between the organisms of different environments. The methodology has been applied to a set of five ponds covering the salinity gradient from about twice that of seawater (6.4%) to NaCI precipitation (30.8%). Bacterial (eubacterial) diversity estimated from the complexity of the banding pattern obtained by restriction of the amplicons from the different ponds decreased with increasing salinity while for Archaea (archaebacteria) the reverse was true i.e. the higher the salinity the higher the number of bands. The similarities in taxonomic composition of the prokaryotic populations present in those ponds were evaluated from the number of restriction bands shared by the different samples. The relationships found among the different environments were independent of the enzyme used for digestion and were consistent with previous descriptions obtained by the study of isolates from the different environments. This technique appears to be promising as a rapid method for microbial biodiversity fingerprinting useful to compare several environments and detect major shifts in species composition of the microbial population.

Notes: Abstract Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.