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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. 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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. 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

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).

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. Supporting Information

Phylogeny and evolution of CS in microalgal groups

In order to construct the phylogeny of the CS in microalgal groups, we searched for orthologs of previously annotated CSs. The putative CS sequences translated from CS coding genes annotated in microalgae genomes (Table S1) were used to construct a maximum likelihood tree (Fig. 2). Bacterial SCH sequences were used as outgroup. Three distinct eukaryotic CS clusters were defined comprising: (cluster 1) Amoebozoa and Heterolobosea, which have already been described to follow the cycloartenol biosynthesis route (Raederstorff and Rohmer, 1987; Nes et al., 1990); (cluster 2) Stramenopiles; and (cluster 3) Rhodophyta, Haptophyta, Chlorophyta and land plants.

figure

Figure 2. Phylogenetic tree of CS sequences based on CS genes annotated in microalgal genomes, as well as bacterial OSCs. Bacterial SHC sequences were used as outgroup. Accession numbers are indicated between parentheses. The phylogenetic tree was inferred by maximum likelihood with the LG + G + F model of protein evolution. Branch support was calculated with the approximate likelihood ratio test (aLRT) and indicated on the branches (values < 50% are not shown). The scale bar indicates evolutionary distance of 0.5 substitutions per site.

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Bacterial OSCs were also included in the tree showing a distinctive cluster of the OSC sequences of the sterol-synthesizing G. obscuriglobus (Planctomycetes) and M. capsulatus (γ-Proteobacteria and Methylococcales) (Bouvier et al., 1976; Pearson et al., 2003; bacterial cluster 1, Fig. 2). This cluster also included a putative OSC sequence of Plesiocystis pacifica (δ-Proteobacteria, Myxococcales), which has not been reported to produce sterols but has been inferred to produce 7,24-cholestadien-3β-ol based on the annotation in its genome of the enzymes leading to this sterol (Desmond and Gribaldo, 2009). The bacterial OSC cluster 2 comprised the sequence of the sterol-producer Stigmatella aurantiaca (Bode et al., 2003), as well as other OSC sequences of δ-Myxococcales. Finally, the bacterial OSC cluster 3 included several γ-Methylococcales sequences and a putative OSC of Fluviicola taffensis (Bacteroides/Chlorobi group; O'Sullivan et al., 2005), species for which it has not been reported yet that they synthesize sterols. The distribution of bacterial OSCs in phylogenetically distant taxa and the evolutionary relationships between bacterial and eukaryotic OSC are still unclear (Pearson et al., 2003; Chen et al., 2007; Desmond and Gribaldo, 2009). Desmond and Gribaldo (2009) reconstructed an OSC phylogeny based on a more limited set of genomes and also found a close relationship of M. capsulatus, G. obscuriglobus, P. pacifica and S. aurantiaca OSCs with eukaryotic OSC sequences. Here, the extended OSC phylogeny reveals a close relationship between bacterial OSCs of the γ-Methylococcales and F. taffensis and the Stramenopiles CS cluster 2, which could potentially indicate that these bacterial OSCs were obtained from Stramenopiles by horizontal gene transfer (HGT).

Comparison of the CS phylogeny with the rbcL protein-based phylogeny showed a different grouping of microalgal sequences and the distance between rbcL protein sequences was shorter than in between CS sequences (Fig. S1), evidencing CS as a less conserved protein than rbcL protein. The chloroplast (plastid)-rbcL gene has been widely used for plant and algal phylogeny because of its conserved nature (Gurgel et al., 2003; Ranker et al., 2003; Wall and Herbeck, 2003, among others). However, a microalgal rbcL-based phylogeny reflects plastidic evolution, in which eukaryotic algae are clustered into two major groups: a ‘red’ and a ‘green’ lineage. The red lineage includes the Rhodophyta (red algae) derived also from a primary endosymbiotic event, and organisms harbouring secondary symbiosis evolved red plastids (Chromalveolata: Haptophyta, Stramenopiles, Cryptophyta and Alveolata) (Keeling, 2004). The green lineage comprises the Viridiplantae (green algae Chlorophyta and land plants Streptophyta), all of which have primary endosymbionts. On the other hand, the CS-based phylogeny presented here shows a clustering according to a nuclear-coding gene. Recent studies based on multigene-based phylogenomic analyses support a cluster including Rhodophyta, Chrolorophyta and Haptophyta, and another one with Rhizaria, Alveolates and Stramenopiles (Hampl et al., 2009), as observed in the CS-based phylogeny.

To conclude, CS as a phylogenetic marker of microalgae based on CS-based phylogeny presented here has several advantages. First, CS is a nuclear-coded protein which phylogeny is in agreement with multigene-based phylogenies (Hampl et al., 2009). Second, it reflects the sterol acquisition evolution and possible events of HGT between bacterial and eukaryotic members as suggested in this study. Lastly, CS has been previously defined as a conserved protein (Summons et al., 2006), but our study points out to a higher diversity between CS sequences of different genera of the same family (diatom CS sequences as mentioned above; Fig. 2) than other known algal phylogenetic markers (e.g. rbcL protein; Fig. S1). This demonstrates the potential of a CS-based phylogeny to discriminate members of the same algal groups, e.g. members of the Bacillariophyta (diatom) phylum.

CS detection in diatom cultures

The three diatom (Bacillariophyta) CS sequences form a separate cluster within the Stramenopiles CS cluster 2 (Fig. 2; Table S1). The CS sequences of Fragilariopsis cylindrus (Bacillariales) and Phaeodactylum tricornutum (Naviculales), both raphid pennate diatoms, are more closely related to each other than to that of Thalassiosira pseudonana (Thalassiosirales). This diatom sequence association has been previously reported in 18S rRNA gene-based phylogenies (Rampen et al., 2009b; Theriot et al., 2010). However, in the rbcL protein phylogeny (Fig. S1), the clustering of the diatom-derived sequences was different, with the rbcL protein sequences of F. cylindrus and T. pseudonana being more closely related. The fact that the CS and the 18S rRNA gene-based clustering of sequences is in agreement supports the value of CS as marker to reconstruct the phylogeny of diatoms.

We developed primers for detection of CS gene sequences in other diatom genera. These primers were designed to match conserved amino acid sites of CS (Table S2). The forward primers Cycloart_F and CycloF_TPF (Table S2) were designed based on the CS gene sequence coding for the amino acid motifs WLLPNWF and PNW(F/I)PFHP conserved in the T. pseudonana, F. cylindrus and P. tricornutum (Fig. S2, S3). For the reverse primer Cycloart_R and CycloR_TPF (Table S2), an area comprising the amino acid motif GYNGSQC was chosen because it is conserved across different phyla following the cycloartenol branch of the sterol biosynthesis, and also because it includes a conserved tyrosine (Y) amino acid residue involved in the cyclization cascade (Summons et al., 2006).

The designed primer pairs were tested on genomic DNA extracted from 14 cultures of diatoms, including the three diatoms, T. pseudonana, P. tricornutum and F. cylindrus, for which whole-genome data were available and based on which the primers were designed (Table S2). Half of the diatom cultures tested showed positive amplification, including Skeletonema costatum CCMP 1281, Skeletonema subsalsum CCAP 1077/8, Pseudo-nitzschia seriata CCMP1309, Extubocellulus spinifer CCMP 393 and, as expected, the three diatom strains that were used to design the primers, T. pseudonana CCMP 1335, P. tricornutum and F. cylindrus CCMP 1102 (Table S3). Different genera showed positive results, suggesting that the primers may be generally applicable within the diatom group. However, in some other cases, the designed primers failed to amplify the CS gene, even in other members of a genus (i.e. Thalassiosira and Extubocellulus) for which amplification was successful in another species.

Partial gene sequences obtained by polymerase chain reaction (PCR) using the developed primers and the genomic DNA of the diatom cultures as template were translated by submitting them as query sequences in xblast (translated nucleotide query against protein database blast) in NCBI. Introns were removed from the amplified sequences in line with the translated sequences suggested by xblast and by comparison with the reported coding DNA sequence of the putative CS proteins of T. pseudonana, F. cylindrus and P. tricornutum (Table S1). The DNA fragment amplified using our primers from S. costatum, S. subsalsum, E. spinifer and P. tricornutum did not contain any introns. On the other hand, the amplified partial CS gene fragments of T. pseudonana, P. seriata and F. cylindrus contained 2, 3 and 4 introns respectively (Fig. S4). A non-uniform intron/exon structure (also known as ‘intron sliding’) in homologous genes has been previously described in OSC gene sequences (Xue et al., 2012) and also in squalene monoxygenase (Fig. 1) gene sequences of rice (Schäfer et al., 1999). Thus, the lack of amplification of CS gene of half of the diatom cultures tested (Table S3) may be due to the presence of an intron sequence in the target area for the designed primers. Alternatively, it may also be caused by a sequence mismatch, but this seems unlikely because the primers target highly conserved regions of the CS gene (see below).

In order to avoid the possible interference of introns in the primer targeted area, we tested the designed CS primers on complementary DNA (cDNA) obtained from exponential cultures of E. spinifer, T. pseudonana and Skeletonema species. rbcL gene amplifications on cDNA were positive, suggesting that the RNA extracted was intact and that the cDNA was properly obtained. On the other hand, CS gene amplifications failed, suggesting that the CS gene is not actively transcribed in exponential phase or that the expression of the gene is too low to be detected with the conditions applied here.

The translated CS gene sequences from the diatom cultures were aligned with the putative CS sequences of the genomes of T. pseudonana, F. cylindrus and P. tricornutum (Fig. S3). The alignment revealed several amino acid changes between diatom genera, also between closely related species, e.g. in the case of S. costatum and S. subsalsum, four amino acid changes were detected in the 181-amino acid long fragment sequences analysed (residues 208, 235, 249 and 288 of the alignment in Fig. S3). In these cases, the amino acids were replaced by another one with a similar polarity (Fig. S3) [i.e. position 208 in S. costatum, glutamine (Q) was substituted by an arginine (R) both polar amino acids, in S. subsalsum], which suggests that these amino acid changes in the CS sequence between members of the same genus might not imply important structural and/or catalytical changes in the CS.

The percentage of identity between the putative diatom CS sequences revealed the highest percentage of identity between CS sequences of the same genus (S. costatum and S. subsalsum, 95%) (Table 1). CS sequences of diatoms belonging to the same order such as Thalassiosirales (Skeletonema and T. pseudonana) and Bacillariales (P. seriata and F. cylindrus) had a percentage of identity of 86% and 83% respectively. The lowest percentage of identity was observed between P. tricornutum (order Naviculales) and P. seriata/F. cylindrus (Bacillariales; 63%). Thus, although there is a relatively high degree of conservation of CS sequences (Summons et al., 2006), the CS sequence is diverse enough to distinguish species of the same genus supporting its value as a phylogenetic marker.

Table 1. Identity matrix (percentage of identitya) of annotated partial CS in diatoms
  1. a

     Where 100% indicates identical sequences.

Identity matrix      
T. pseudonanaT. pseudonana     
F. cylindrus70.7F. cylindrus    
P. tricornutum67.464.6P. tricornutum   
S. costatum85.071.269.0S. costatum  
S. subsalsum85.670.767.995.5S. subsalsum 
E. spinifer75.670.169.676.274.5E. spinifer
P. seriata68.583.463.571.270.166.2

CS and sterol diversity in the environment

We tested the CS gene primers on several environmental samples for the in situ detection of diatom-related CS gene sequences. These samples were a microbial mat from the island Schiermonnikoog that is characterized by the presence of pennate diatoms (e.g. Navicula, Diploneis, Amphora and Cylindrotheca) as shown using microscopic methods (Dijkman et al., 2010). Furthermore, we also analysed suspended particulate matter (SPM) from North Sea surface water, which has high contents of diatom pigments (diatoxanthin and diadinoxanthin, data not shown) and in which Thalassiosira, Chaetoceros and Skeletonema species were previously observed (Cadée and Hegeman, 2002; Brandsma et al., 2012).

Primer pairs Cycloart_F/R and CycloF_TPF F&R, designed based on the diatom sequences available, gave positive results on both environmental samples. The environmental CS gene sequences were amplified, sequenced, translated (removing introns) and used to construct a maximum likelihood phylogeny together with the CS sequences of diatom cultures. The sequences recovered from the North Sea SPM mainly clustered with the order Thalassiosirales (Skeletonema and T. pseudonana) (cluster 1; Fig. 3), while the majority of the diatom mat CS sequences (cluster 2; Fig. 3) were closer to the representative sequence of E. spinifer (order Cymatosyrales; centric diatom) with other sequences clustering separately (cluster 1.a; Fig. 3) and were more closely related to the Thalassiosirales order cluster.

figure

Figure 3. Phylogenetic tree of CS gene sequences obtained from environmental samples and diatom cultures. The phylogenetic tree was inferred by maximum likelihood with the GTR + G + I model of DNA evolution. Branch support was calculated with the approximate likelihood ratio test (aLRT) and indicated on the branches (values < 50% are not shown). The scale bar indicates evolutionary distance of 0.1 substitutions per site. Diatom orders are indicated in blue.

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As a comparison, we also construct a maximum likelihood phylogeny of an amplified fragment of the rbcL gene (Fig. 4). The majority of the rbcL gene sequences retrieved from the North Sea SPM clustered with the order Thalassiosirales (cluster 1) but also to sequences such as P. tricornutum (order Naviculales) (cluster 2.a) (Fig. 4). The rbcL gene sequences retrieved from the microbial mat were more diverse in comparison with the North Sea SPM rbcL gene sequences and clustered with E. spinifer (cluster 3), as well as with the orders Thalassiosirales (cluster 1), Bacillariales (P. seriata and F. cylindrus; cluster 2.b) and Naviculales (cluster 2.a) (Fig. 4).

figure

Figure 4. Phylogenetic tree of rbcL gene sequences obtained from environmental samples and diatom cultures. The phylogenetic tree was inferred by maximum likelihood with the GTR + G + I model of DNA evolution. Branch support was calculated with the approximate likelihood ratio test (aLRT) and indicated on the branches (values < 50% are not shown). The scale bar indicates evolutionary distance of 0.02 substitutions per site. Diatom orders are indicated in blue.

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Amplified rbcL gene fragments (Fig. 4) were more diverse than the CS gene sequences (Fig. 3), which indicates that CS primers are more limited in their diversity coverage. This is expected on basis of the possible lack of amplification of CS sequences with the primers designed in this study. As mentioned above, lack of amplification by the CS primers designed in this study may be due to the presence of mismatches or by introns in the primer area. Based on our study, the phenomenon of ‘intron sliding’ is highly likely between closely related diatom CS gene sequences, while the amplified rbcL gene sequences is generated primers placed in an exon sequence (Corredor et al., 2004). Taking into account that our target are eukaryotic genes, we acknowledge that environmental amplifications of cDNA rather than DNA could have been more appropriate to avoid PCR-biases because of the presence of introns as discussed above. We performed PCR amplifications on cDNA extracts of the environmental samples tested here. As reported for the cultures of E. spinifer, T. pseudonana and Skeletonema species (see above), amplifications with the designed CS primers failed while rbcL-PCR amplifications were positive (data not shown). This suggests that, like for the cultures, the transcriptional activity of the CS gene is low in the environment and, therefore, difficult to detect.

As a complementary tool, we also characterized the sterols in these samples to obtain an idea of the sterol diversity and potentially identify specific diatom sterols. The main sterols in the diatom mat were cholest-5-en-3β-ol (cholesterol; 21%), 24-methylcholest-5,22E-dien-3β-ol (brassicasterol; 13%), 24-ethylcholest-5-en-3β-ol (β-sitosterol: 12%), 5α-cholest-22-en-3β-ol (10%) and 24-methylcholest-5,24(28)-dien-3β-ol (10%) (Table 2). In the North Sea water SPM, the main sterols were desmosterol (30%) and 24-methylcholest-5,24(28)-dien-3β-ol (31%). Some of the detected sterols (Table 2) have been previously detected in diatom cultures, i.e. 24-methylcholesta-5,24(28)-dien-3β-ol, brassicasterol, 24-methylcholest-5,22E-dien-3β-ol and 24-ethylcholesta-5,22E-dien-3β-ol (stigmasterol) (Rontani and Volkman, 2005; Rampen et al., 2010, among others). However, the presence of these sterols does not provide conclusive evidence of the presence of diatoms as all these sterols are also found in other algae. and their presence not ubiquitous in all diatoms (Volkman et al., 1998; Volkman, 2003; Rampen et al., 2010 and references cited therein).

Table 2. Sterol composition and relative percentage in the North Sea SPM and diatom mat samples
SterolRelative %a
North Sea SPMDiatom mat
  1. a

     Relative percentage based on integrated area of each sterol respect to the total sterol detected in the simple.

  2. nd, not detected.

24-Norcholestane7.4nd
Cholesta-5,22E-dien-3β-ol9.410
5α-Cholest-22-en-3β-olnd1.4
Cholest-5en-3β-ol (cholesterol)1.721.1
5α-Cholestan-3β-ol (cholestanol)1.34.5
Cholesta-5,24-dien-3β-ol (desmosterol)30.55.6
24-Methylcholest-5,22E-dien-3β-ol (brassicasterol/diatomsterol)5.613.2
5α(H)-Cholest-7en-3β-ol1.80.9
24-Methylcholest-5,24(28)-dien-3β-ol3110.5
24-Methylcholest-5-en-3β-ol (campesterol)2.58.5
23,24-dimethylcholesta-5,22E-dien-3β-olnd2.3
24-Ethylcholesta-5,22E-dien-3β-ol (stigmasterol)nd4
24-Ethylcholesta-5-en-3β-ol (β-sitosterol)5.612
24-Ethylcholesta-5,24Z-Zden-3β-ol (isofucosterol)3.54.9
4α, 23, 24-Trimethyl-5α(H)cholest-22-en-3β-ol (dinosterol)nd1.1

The diversity of phytosterols detected in the North Sea SPM was lower than in the diatom mat and only one sterol, 24-norcholestane, was uniquely found in the North Sea water but not in the diatom mat. In addition, the higher diversity of diatom CS gene sequences detected in the microbial mat compared with the North Sea SPM also corresponds to a higher diversity of detected sterols. The detection of sequences homologous to the Cymatosirales and Thalassiosirales orders in the microbial mat suggests that cholesta-5,22-dien-3β-ol (10%) may be also sourced by these diatoms as they are also dominant sterols in cultivated relatives (Rampen et al., 2010). 24-Methylcholesta-5,24(28)-dien-3β-ol has also been found in high abundances in some centric diatoms such as Thalassiosira and Skeletonema genera (Rampen et al., 2010). The presence of CS gene sequences closely related to Thalassiosirales (Fig. 3), and the high relative abundance of this sterol in both samples (especially in the North Sea SPM) supports the idea that diatoms from this order may be the source of the high relative abundance of 24-methylcholest-5,24(28)-dien-3β-ol detected in these environmental settings.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. Supporting Information

Our study based on specific gene searching in genomic databases has allowed the design of primers for the detection of a key enzyme (i.e. CS) of the sterol biosynthetic pathway in microalgae. The comparison between the phylogenetic reconstruction of microalgae CS and rbcL sequences supports the value of CS as marker of the presence and phylogeny of sterol-producing microalgae. However, we have also observed a non-uniform intron/exon structure in CS genes of closely related diatom species that might interfere in the PCR amplifications. Further studies based on the sequencing of CS gene transcripts in diatoms and other microalgae, and the reassessment of CS primers for the application in environmental cDNA extracts are thus needed.

The genomic characterization of enzymes involved in lipid biosynthetic pathways opens a new chapter in organic geochemistry studies. Genomic approaches provide an independent assessment of the organism ability to produce a molecule of interest without extensive screening of cultures. The CS sequence analysis of environmental samples has proven to be informative for surveying the sterol-forming diatom community and has expanded the range of CS sequences available, introduced a quick screening of environmental samples for the diversity of sterol-forming microorganisms and may provide a link between sterols and their main sources. This is also the starting point for other studies involving determination of the abundance and expression of this key gene of the phytosterol biosynthetic pathway in environmental samples. Ultimately, the CS has potential to elucidate the evolutionary placement of certain microalgae groups and as a molecular clock to track the appearance of the sterol biosynthetic pathway.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions
  6. References
  7. Supporting Information
FilenameFormatSizeDescription
emi412106-sup-0001-si.tif14692K

Fig. S1. Phylogenetic tree of rbcL protein sequences annotated in microalgal genomes. Tree is arbitrarily rooted. Accession numbers are indicated between parentheses. The phylogenetic tree was inferred by maximum likelihood with the RtRev + I + G + F model of protein evolution. Branch support was calculated with the approximate likelihood ratio test (aLRT) and indicated on the branches (values < 50% are not shown). The scale bar indicates evolutionary distance of 0.1 substitutions per site.

emi412106-sup-0002-si.tif2684K

Fig. S2. Alignment of the CS DNA fragment comprising the designed primers in the sequences from diatom cultures. Black bars indicate DNA sequence area comprising the Cycloart_F and R primers, and yellow bars indicate DNA sequence area comprising the CycloF_TPF and CycloR_TPF primers (see Table S2 for details).

emi412106-sup-0003-si.tif3635K

Fig. S3. Alignment of the CS protein fragment comprising the designed primers in the sequences from diatom cultures. Black bars indicate protein sequence area comprising the Cycloart_F and R primers, and yellow bars indicate protein sequence area comprising the CycloF_TPF and CycloR_TPF primers (see Table S2 for details). Stars indicate amino acid positions in which amino acid changes have been observed between the CS sequences of S. costatum and S. subsalsum. The amino acid positions in this alignment are numbered according to the CS sequence of T. pseudonana (accession number XP_002287432.1).

emi412106-sup-0004-si.tif2014K

Fig. S4. Distribution of introns/exons in the CS gene fragment comprised by the primers designed in this study. Exon areas are indicated with blue boxes and introns as lines. aa, amino acids; Bp, base pairs.

emi412106-sup-0005-si.doc101K

Table S1. Annotated CS orthologs in microalgal genomes.

Table S2. List of primers used for the detection of CS gene coding sequences.

Table S3. Diatom cultures investigated in this study.

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