DESCRIPTION OF PROKARYOTIC BIODIVERSITY ALONG THE SALINITY GRADIENT OF A MULTIPOND SOLAR SALTERN BY DIRECT PCR AMPLIFICATION OF 16S RDNA

New methods based on PCR amplification of 16S rRNA genes from DNA samp les extracted directly from the environment allow the description of m icrobial diversity in natural ecosystems without the need for cultivat ion. We have applied this technique to an extreme environment presumed to have very low diversity: the crystallizer ponds of a marine salter n with salinity over NaCl saturation. The molecular methodology has sh own that indeed very low diversity can be found here. Prokaryotes belo nging to the Bacteria domain are a minor component and only members of a closely related cluster of sequences were found, all relatives of t he cr-Proteobacteria (ca. 83% to Rhodopseudomonas marina). Halophilic Archaea were as expected the largest component of biomass in this envi ronment. All the clones sequenced corresponded again to a highly homol ogous cluster (probably members of the same genus). However, all the s equences diverged considerably from the ones of the described genera o f halophilic Archaea, in fact the data are consistent with the idea th at the 16S rRNA genes directly amplified from the saltern correspond t o members of an undescribed genus. This is remarkable since many colle ction strains sequenced come specifically from this saltern. Furthermo re, 16S rDNA obtained from archaeal cultures isolated from the same sa mple had no homology to the sequences obtained by PCR amplification, i nstead they appear to be members of the well known genus Haloarcula. H owever, this concurs with the findings of other authors who obtained d ifferent organisms by culture from those detected by the sequences ret rieved 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 strate gy. 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 ge nerate characteristic profile data for estimation of diversity and ove rall similarities between the organisms of different environments. The methodology has been applied to a set of five ponds covering the sali nity gradient from about twice that of seawater (6.3%) to NaCl precipi tation (30.8%). Bacterial (eubacterial) diversity estimated from the c omplexity of the banding pattern obtained by restriction of the amplic ons 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 wer e evaluated from the number of restriction bands shared by the differe nt samples. The relationships found among the different environments w ere independent of the enzyme used for digestion and were consistent w ith previous descriptions obtained by the study of isolates from the d ifferent environments. This technique appears to be promising as a rap id method for microbial biodiversity fingerprinting useful to compare several environments and detect major shifts in species composition of the microbial population.