Molecular methods applied for identification the microbial diversity in a hypersaline environment

Microbial community represents more than half thebiomass on the Earth. A gram of soil, for example,contains millions of microorganisms of differencegroups (4000 different ones). However most of theenvironmental microorganisms are un-culturable (90-99%) and could not be isolated in laboratory conditions.There are huge biotechnological capabilities in thesegroups of microorganisms since it is necessary toconsider this kind of microorganisms in biodiversitystudies. Prokaryotic diversity in Aran-Bidgol salt lake, athalasohaline lake in Iran, was studied by fluorescencein situ hybridization (FISH), denaturing gradient gelelectrophoresis (DGGE) of PCR-amplified fragmentsof 16S rRNA genes and 16S rRNA gene clone libraryanalysis. Total cell abundances in the lake determined byDAPI direct count was 3-4×107 cells/ml. The proportionof Bacteria to Archaea in the community detectable byFISH, applying archaeal and bacterial specific probeswas unexpectedly high and ranged between 1:3 and 1:2.Genomic DNA was directly extracted from environmentsand 16S rRNA of both domains was PCR-amplified.The PCR products of expected size (1500 bp) were gelpurified (DNA extraction kit, Roche, Germany) ligatedinto pGEM-T cloning vector (Promega, USA) and used totransform E.coli DH5α cells. We constructed total of eightclone libraries. Analysis of inserts of 100 clones fromthese libraries constructed revealed a total of 37 OTUs.A majority (63 %) of these sequences were not relatedto any previously identified taxa. Within this samplingeffort we most frequently retrieved phylotypes relatedto Halorhabdus (16 % of archaeal sequences obtained)and Salinibacter (36 % of bacterial sequences obtained).Other prokaryotic groups that were abundant includedrepresentatives of Haloquadratum, the anaerobic generaHalanaerobium and Halocella, purple sulfur bacteria ofthe genus Halorhodospira and Cyanobacteria.

Keywords:

Molecular methods , Archaea , Bacteria , Halophilic microorganisms , Prokaryotic diversity