Phylogenetic Diversity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes from Deep-Sea Microorganisms

Abstract

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.Phylogenetic information on deep-sea microorganisms that has been accumulated relates mainly to the 16S ribosomal DNA (rDNA) sequences (9,33,53). In understanding the microbial contribution to deep-sea primary production, the 16S rDNA-based phylogeny will be better complemented by knowledge of the genes encoding the enzymes relevant to carbon fixation. The genes encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) represent such an enzyme that is involved in autotrophy. RuBisCO is the most abundant enzyme on the globe and has attracted much phylogenetic attention (5). RuBisCO catalyzes the assimilation of carbon dioxide to organic carbon via the Calvin-Benson cycle. The enzyme consists of large and small subunits (42). The site responsible for carbon fixation is in the large subunit (42). More than 20% of the amino acid residues in the large subunit are conserved among the higher plants (32). Generally, RuBisCO has two forms. Form I consists of large and small subunits (L n S n , typically L 8 S 8 ), and form II contains only large subunits (L n ) with 25 to 30% amino acid sequence identity with those of form I (32). The large subunits of form I and form II are coded by genes designated cbbL and cbbM, respectively (37). Some organisms have two genes en...

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