Sequencing of Bacterial and Archaeal 16S rRNA Genes Directly Amplified from a Hypersaline Environment

Abstract

The new methods based on PCR amplification and sequencing 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 (salinity over NaCl saturation). The molecular methodology has shown that indeed very low diversity can be found here. Bacteria (formerly eubacteria) are largely minoritary, and only a cluster of closely related sequences was found, all distant relatives of the α-Proteohacteria (82% to Rhodopseudoinonas marina ). Halophilic Archaea were shown by hybridization to be, as expected, the largest component of biomass in this environment. All the archaeal clones sequenced again were highly similar to each other suggesting that they are probably members of the same genus. However, all the sequences diverged considerably from those of the described genera of halophilic Archaea . In fact the data are consistent with the idea that the 16S rDNA genes directly amplified from the saltern correspond to members of an undescribed genus which seems to be abundant in the sample. This is remarkable since many collection strains sequenced come specifically from this same 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 is in agreement with the findings of other authors who by culture have obtained organisms different from those indicated by the sequences retrieved directly by PCR. Possible explanations are discussed.