Monitoring of uncultured Dunaliella sp. in an Egyptian solar saltern field based on RuBisCO-encoding gene cbbL

Abstract

Culture-independent molecular approach was used to explore and evaluate the diversity of Dunaliella species living at the salt field Malahat El-Max Alexandria, Egypt. Bulk genomic DNA was extracted directly from the collected salt water samples. Specific PCR primers and methodology were designed to amplify the gene cbbL, which encodes the large subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) of only Dunaliella species, from the extracted microbial metagenome. The 700 bp-PCR amplicons were cloned and cbbL clone library was constructed and analyzed by sequencing. Rarefaction curve was saturated at sequence analyses of 23 clones, obtaining 19 phylotypes of Dunaliella cbbL, representing the total composition of Dunaliella in the collected sample. All recorded phylotypes had the known deduced amino acid cbbL motive sequence and catalytic sites. Fingerprint sequence, characterizing Dunaliella cbbL, was recorded. The cbbL phylotypes were grouped into two distinct phylogenetic clusters. The cluster 1, consisting of 18 current cbbL phylotypes was rooted with a cluster containing cbbLs of Dunaliella salina, Dunaliella bioculata, Dunaliella primolecta and Dunaliella tertiolecta. The single phylotype, uncultured Dunaliella ElMax.3, forming cluster 2, showed a unique phylogenetic lineage in the evolution of Dunaliella cbbL. This study introduced the first functional gene markers for exploring Dunaliella species in salt waters without culture.