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- Results and discussion
- Supporting Information
Sterols are cyclic isoprenoid lipids present in all eukaryotes. These compounds have been used to determine the composition of algal communities in marine and lake environments, and because of their preservation potential have been used to reconstruct the evolution of eukaryotes. In the last years, there have been major advances in understanding the sterol biosynthetic pathways and the enzymes involved. Here, we have explored the diversity and phylogenetic distribution of the gene coding the cycloartenol synthase (CS), a key enzyme of the phytosterol biosynthetic pathway. We propose a gene-based approach that can be used to assess the sterol-forming potential of algal groups. CS coding gene was annotated in genomes of microalgae using protein homology with previously annotated CS sequences. Primers for the detection of CS gene sequences of diatoms, one of the most dominant groups of microalgae, were designed and evaluated in cultures and environmental samples. A comparison of the phylogeny of the recovered CS sequences in combination with sequence data of the gene rbcL coding for the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) demonstrates the potential of the CS gene as phylogenetic marker, as well as an indicator for the identity of sterol-producing organisms in the environment.
- Top of page
- Results and discussion
- Supporting Information
Biomarker lipids have been extensively used for determining the composition and function of microbial communities in past and modern environments (e.g. Volkman et al., 1998; Hinrichs et al., 1999; Sinninghe Damsté et al., 2002; Kuypers et al., 2003; Talbot et al., 2003; Volkman, 2003; Brocks et al., 2005). Lipids make excellent molecular fossils because of their relative resistance to degradation and because some have structures unique to certain taxonomic groups. The combination of DNA-based diversity studies [mainly based on ribosomal RNA (rRNA) gene taxonomy] and chemotaxonomic characterization of lipids has been shown to be a powerful approach to constrain the diversity of microbial communities (e.g. Stephen et al., 1999; Sinninghe Damsté et al., 2004; Villanueva et al., 2004; Rampen et al., 2010). Some studies have also compared biomarker lipids with functional/metabolic genes to assess both the diversity of certain microbial groups as well as their potential ability to perform an activity (e.g. Ertefai et al., 2008; Pitcher et al., 2011).
Sterols are important lipid biomarkers and are present in all eukaryotic organisms and in some Bacteria such as Methylococcus capsulatus (γ-Proteobacteria; Bouvier et al., 1976), Gemmata obscuriglobus (Planctomycetales; Pearson et al., 2003) and some Myxobacteria (δ-Proteobacteria, e.g. Stigmatella aurantica; Bode et al., 2003). These lipids have been considered as important tools for molecular paleontologists because sterols can be preserved as, e.g. steranes in the fossil record for billions of years and thus provide insight into the evolution of eukaryotes (Summons et al., 1999; Brocks and Pearson, 2005; Peters et al., 2005). Furthermore, their distribution has been used for taxonomic information on the presence of certain microalgae (Moldowan et al., 1990; Brocks et al., 1999; Peters et al., 2005; Kodner et al., 2008). The cyclization of squalene to sterols and some of the following steps in the sterol biosynthetic pathway require molecular oxygen, and the presence of steroids in the fossil record are thus considered as indicators of oxygenation of the atmosphere and oceans (Summons et al., 1999; 2006).
The diversity of sterols and their synthetic pathways has been studied extensively and revealed a wide variety of structures (A-ring and side chain alkylation, cyclopropane rings, unsaturations, etc), some of which can be specific for certain eukaryotic groups (Volkman et al., 1998; Volkman, 2003; Rampen et al., 2010). However, most sterols are usually not exclusive of a specific group or genera, and, in addition, a change in sterol distributions may also result from changes in environmental and growing conditions rather than community composition changes (Shifrin and Chisholm, 1980; Fabregas et al., 1997; Rampen et al., 2009a). In addition, (micro)algal taxonomy has been mostly based on morphology or, more recently, on genetic data based on the 18S rRNA gene and the rbcL gene coding for the plastid-encoded large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which do not necessary reflect the structural diversity of sterols (Moniz and Kaczmarska, 2009; Rampen et al., 2010).
One approach to solve the above issues is to examine the presence and diversity of genes involved in the biosynthesis of biomarker lipids as an evidence of the potential ability to biosynthesize the compound of interest, as well as a phylogenetic marker. For example, Pearson and colleagues (2007; 2009) investigated the phylogeny of the producers of hopanoids, isoprenoid bacterial lipids, by analysing the sequence diversity and distribution of the squalene–hopane cyclase (SHC) gene, concluding that the ability of hopanoid production is not as widespread among bacteria as previously thought. Following the same approach, a study by Welander and colleagues (2010) investigated the genes involved in the synthesis of 2-methylhopanoid and showed that the gene required for the C-2 methylation in hopanoids was also present in bacterial taxa other than cyanobacteria, invalidating the use of 2-methylhopanes as biomarkers of the appearance of oxygenic photosynthesis on Earth (Welander et al., 2010). More recently, Villanueva and colleagues (2013) evaluated the diversity of sulpholipid producers in the environment by targeting a gene involved in the sulpholipid biosynthetic pathway and found that the diversity of potential sulfolipid producers in surface waters and microbial mats was widespread in the Cyanobacteria and Proteobacteria phyla.
In this study, we have made use of recent advances on the phylogenomics of the sterol biosynthetic pathway (Desmond and Gribaldo, 2009) and the growing availability of complete or draft genomes of microalgae, allowing the identification of key genes of the phytosterol biosynthetic pathway. Among all the enzymes of the sterol pathway, oxidosqualene cyclases (OSCs) are one of the most conserved at the sequence level, and homologues have been detected in all species capable of sterol synthesis (Desmond and Gribaldo, 2009). OSCs and SHCs are related in amino acid sequences and probably derived from a common ancestor (Fischer and Pearson, 2007). There are two main types of OSCs based on the end product of the cyclization (Fig. 1): lanosterol synthases (found in animals, fungi, choanozoa, trypanosomatids and dinoflagellates) and cycloartenol synthases (CSs; found in higher plants, red and green algae, amoebozoa, diatoms, euglenids and heterolobosea). Previous studies have also identified conserved active sites and specific amino acid residues responsible for particular steps in the cyclization cascade (see Summons et al., 2006 for a review).
Figure 1. Sterol biosynthetic pathway. Isopentenyl diphosphate (IPP) is the isoprene precursor of squalene. Hopanoids are synthesized from the cyclization of squalene by an SHC in a process independent of oxygen (Summons et al., 2006). For sterol synthesis, squalene is transformed by a squalene monooxygenase (SQMO) that requires molecular O2 (Summons et al., 2006). Squalene epoxide is then cyclized either to lanosterol or to cycloartenol by lanosterol synthase (LS) or CS.
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We targeted the gene encoding the CS because it is the first specific step in the phytosterol biosynthetic pathway (Fig. 1), and because it is possible to detect homologues of this gene in different organisms because of its conservation at the sequence level (Summons et al., 2006). We have focused on the characterization of the CS of diatoms as these unicellular algae are thought to be the most common group of eukaryotic phytoplankton in modern oceans and responsible for approximately 40% of marine primary productivity (Falkowski et al., 1998; Moniz and Kaczmarska, 2009). Thus, they are likely one of the most important steroid-producing organisms in marine environments. The taxonomic distribution of sterols in this group is fully based on culture analysis (e.g. Volkman et al., 1998; Volkman, 2003; Rampen et al., 2010) and thus may not reflect environmental diversity.
In this study, we investigated the evolutionary relationships and diversity of the CS in microalgae to unravel the potential of this sterol biosynthetic enzyme as phylogenetic marker and indicator of sterol-producing organisms. Primers targeting conserved areas in diatom CS gene sequences were developed and tested in cultures and applied to environmental samples. Finally, we compared the diversity of CS sequences recovered from environmental samples with other phylogenetic gene markers and with the distribution of the products of their lipid biosynthetic pathway (i.e. sterols).