An efficient DNA isolation protocol for cyanobacteria of the genus Arthrospira Nicolas Morina,b, Tatiana Vallaeysc, Larissa Hendrickxa†, Leys Nataliea, Annick Wilmotteb
a Expert group for Molecular and Cellular Biology, Belgian Nuclear Research Center
SCK•CEN, Mol, Belgium
b Center for Protein Engineering, Institute of Chemistry, University of Liège, Sart Tilman B6,
Liège, Belgium
c UMR5119 ECOLAG, University of Montpellier 2, Montpellier, France
Corresponding author : Nicolas Morin, Expert group for Molecular and Cellular Biology, Belgian Nuclear Research Center SCK•CEN, Boeretang 200, 2400 Mol, Belgium.Tel. : +32(0) 14 332116. Email: nmorin@sckcen.be
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ABSTRACT
Thanks to their photosynthetic and nutritive properties, cyanobacteria of the Arthrospira genus are of interest as food supplements, as efficient oxygen producing life support system organisms for manned space flight, and for the production of biofuels. Despite these potential
valuable applications, full genome sequences and genetic information in general on Arthrospira remain scarce. This is mainly due to the difficulty to extract sufficient high molecular weight nucleic acids from these filamentous cyanobacteria. In this article, an efficient and reproducible DNA extraction procedure for cyanobacteria of the genus Arthrospira was developed. The method is based on the combination of a soft mechanical lysis with enzymatic disruption of the cell wall. The comparison with other extraction protocols clearly indicates that this optimised method allows a maximal recovery of DNA. Furthermore, the extracted DNA presents a high molecular weight, a reduced degradation and an excellent overall quality. It can be directly used for molecular biology purposes such as PCR.
Keywords : Arthrospira sp.; cyanobacteria; DNA extraction; PCR 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1. Introduction
While DNA isolation seems to be a routine procedure for most organisms including viruses, bacteria, fungi, parasites, insects, mammals and plants (Liu, 2009), it is, for various reasons, a rather difficult one when performed on cyanobacteria (Fiore et al., 2000; Wu et al., 2000). The common problems encountered in DNA isolation from cyanobacteria mainly range from cell lysis efficiency (Billi et al., 1998 ; Fiore et al., 2000), to purification issues (Porter, 1988). Even though several methods for extracting cyanobacterial DNA have already been reported in the literature (e.g. Mazur et al., 1980 ; T. Kallas et al., 1983), their respective efficiencies can greatly vary from one species to another (Mak et al., 1992; Fiore et al., 2000). The broad morphological, metabolic and ecological diversities found within the cyanobacterial phylum might be the main reason for this inherent fluctuating efficiencies. Some species have proven to be particularly difficult to extract DNA from, requiring the development of specific DNA isolation protocols (Billi et al., 1998).
This is the case for members of the genus Arthrospira Stizenberger. This genus includes filamentous cyanobacteria with multicellular cylindrical trichomes arranged in an helicoidal shape, cross-walls visible by light microscopy, and containing gas-vesicles (Castenholz et al., 2001). Arthrospira are naturally growing in marine, brackish water and saline lake
environments of tropical and semi-tropical regions (Ciferri and Tiboni, 1985). In addition to being efficient oxygen producers, members of the Arthrospira genus appear to be rich in proteins, essential fatty acids, essential amino acids, vitamins and iron (Ciferri, 1983).
“Generally Recognised As Safe” by the US Food and Drug Administration (Tarantino, 2003), Arthrospira are commonly sold as a nutrition supplements under the name “spirulina”. Some medical studies claim that consumption of Arthrospira could have therapeutic properties as well, but no conclusive results could confirm its potential use as a drug so far (Ayehunie et al., 1998 ; Karkos et al., 2008). Due to its nutritive properties along with its photosynthetic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
way of life, Arthrospira sp. PCC8005 has even been selected by the European Space Agency as a food complement and oxygen producer within the Micro-Ecological Life Support System Alternative, currently under development for long-term manned space missions (Hendrickx et al., 2006). Furthermore, certain species of Arthrospira have recently been investigated for the production of biomass as a precursor to hydrogen and liquid fuels (Ananyev et al., 2008). Despite all these interests, genetic information on Arthrospira remains scarce. While the rapid development of genome sequencing and molecular biology has lead to remarkable discoveries in life sciences during the last decade, no sequencing projects have been successful in closing the genome of this cyanobacterium so far. One of the reasons for this absence appears to be the difficulty to obtain sufficient high quality DNA material from Arthrospira cultures (ref). Arthrospira cells possess a very low amount of genomic DNA compared to other bacteria. The nucleic acids (DNA and RNA) represent circa 4 % of the dry weight of an Arthrospira cell, while it can reach up to 20 % in fast-growing bacteria like Bacillus subtilis (Ciferri, 1983). Furthermore, Arthrospira cells are notably rich in substances which are difficult to remove from DNA extracts such as polysaccharides and polyphenols (De Philippis and Vincenzini, 1998). Finally, Arthrospira cells possess an important number of restriction endonucleases (Zhao et al., 2006). These enzymes are an important line of defence against the incorporation of foreign DNA, but also represent an additional difficulty for DNA extraction, as they can act as an autodestruction-like mechanism during the extraction process.
In this article, a novel DNA extraction procedure for filamentous cyanobacteria of the genus Arthrospira was developed. The efficiency of the protocol was compared against a selection of DNA extraction methods : (i) The Wizard® Genomic DNA Purification System
(PromegaTM), a universal bacterial DNA extraction method, based on enzymatic lysis and
isolation of the nucleic acids on a silica membrane; (ii) the Miniprep protocol (Ausubel et al., 1995), a universal bacterial DNA extraction method, relying on enzymatic cell wall lysis in liquid cultures; (iii) a Fast-Prep®-based extraction procedure (Feurer et al., 2004), a universal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
extraction DNA procedure, relying on mechanical lysis of the cells by bead beating; (iv) a specific method for Arthrospira sp. (Baurain et al., 2002), relying on several washing steps to remove lipophilic pigments and render the samples more susceptible to enzymatic lysis; (v) a procedure for the isolation of high molecular weight DNA from plant-fungus complexes (Möller et al., 1992), using a combination of both mechanical and enzymatic lysis. The high quantity, quality and suitability of the purified Arthrospira DNA for molecular PCR based reactions was demonstrated.
2. Materials and methods
2.1. Strain and culture conditions
Arthrospira sp. PCC8005 was cultivated in “Zarrouk” medium (Zarrouk, 1966) as 100 ml cultures in 250 ml Erlenmeyer flasks under normal ambient atmosphere. The cultures were kept in a light incubator at 6000 lux, at 30 °C, and shaken at 100 rpm (Braun Certomat BS-1 Shaking Incubator, Sartorius Stedim Biotech). Extraction samples were harvested at one third of the exponential growth phase (OD 750nm ~ 1.0).
2.2. DNA extraction and purification
The following extraction procedures were repeated 4 times, each time on 4 different
biological replicates, to assess the reproducibility of the methods. For each DNA extraction cells were collected by centrifugation of 5 ml from a stock culture (10 min, 3500 rpm) before being treated accordingly to each protocol. DNA samples were stored at -20 °C before further utilisation.
(i) Wizard® Genomic DNA Purification System, PromegaTM. The extraction was
performed according to the instruction of the manufacturer
(http://www.promega.com/applications/dna_rna/productprofiles/a1120/default.htm ). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
(ii) Miniprep of bacterial genomic DNA (Ausubel et al. 1995). Cells were resuspended in 567 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) by repeated pipetting. Lysis of the cells was performed using 30 µl of 10 % SDS and 3 µl of 20 mg/ml proteinase K. The mix was subsequently incubated for 1 h at 37 °C. Selective precipitation of proteins and
polysaccharides was performed using 80 µl of CTAB/NaCl (10 % CTAB, 0.7 M NaCl) in presence of 100 µl of 5 M NaCl. The samples were gently mixed by inversion and incubated for 10 min at 65 °C. Nucleic acids were thereafter isolated by a phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich®) separation, followed by one chloroform:isoamyl alcohol (24:1, Sigma-Aldrich®) separation. DNA was finally recovered by precipitation using 0.6 volume of isopropanol and centrifugation (5 min, 4 °C, 15000 rpm). The DNA pellets were washed with 1 ml of cold 70 % ethanol. Finally, the tubes were centrifuged one last time 5 min (4 °C, 15000 rpm), the supernatant was discarded, and each pellet dried before being resuspended in 100 µl TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
(iii) Fast-Prep® DNA Purification (Feurer et al. 2004). Cells were resuspended into 200 µl TE buffer, and transferred into fresh 1.5 ml tubes. Samples were first pre-treated using
lysozyme (50 µl of 5 mg/ml) and RNAse A (30 µl of 10 mg/ml), and the tubes were incubated 30 min at 37 °C. The mix was transferred into 2 ml screw cap tubes (MO BIO Laboratories, Inc. ) containing ~ 0.2 g of glass beads (212-300 µm, Sigma-Aldrich®), 30 µl 10 % SDS and 200 µl phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich®). Mechanical lysis of the samples was subsequently performed : the tubes were placed in a Fast-Prep®-24
Instrument (MPTM Biomedicals), and mixed two times 40 sec, force 6, with 1 min on ice
between each run. The tubes were then centrifuged 5 min (4 °C, 15000 rpm), and the
supernatant collected. Purification of the DNA was performed by adding 0.1 volume from a 3 M ammonium acetate solution, along with 2.5 volumes of cold 100 % ethanol. The tubes were 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
placed 1 h at -20 °C, then centrifuged for 20 min (4 °C, 15000 rpm). The supernatant was discarded, and the DNA pellets were washed with 1 ml of cold 70 % ethanol, and dried before being resuspended in 100 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
(iv) DNA extraction protocol for Arthrospira (Baurain et al., 2002 ; adapted from Pitcher et al., 1989). Cells were washed one time with 100 % ethanol to remove lipophilic pigments, and two times with 1 ml RS buffer (0.15 M NaCl, 0.01 M EDTA, pH 8.0). Cell lysis was done in presence of 100 µl of 50 mg/ml lysozyme for 30 min at 37 °C. Protein degradation was subsequently performed in presence of 2.5 µl of 10 mg/ml proteinase K, for 1 h at 37 °C. Cell lysis was achieved by a last incubation for 15 min at 37 °C, in presence of 500 µl GES buffer (60 % wt/vol guanidium thiocyanate, 0.1 M EDTA, pH 8.0, 1 % wt/vol sarkosyl). The mix was kept for 10 min on ice, followed by an addition of 150 µl 5 M NaCl and 250 µl cold 7.5 M ammonium acetate. After the tubes were kept on ice for another 10 min, a first
extraction was performed using 500 µl of phenol:chloroform:isoamyl alcohol (25:24:1, Sigma-Aldrich®), followed by a second one with 500 µl of chloroform:isoamyl alcohol (24:1, Sigma-Aldrich®). The nucleic acids were precipitated with 100 % ethanol, collected by centrifugation, (4 °C, 15000 rpm), and dried before being resuspended in 100 µl TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The samples were treated with 1 µl of RNAse (10 mg/ml) for 1 h at 37 °C.
(v) DNA extraction protocol from filamentous fungi, fruit bodies and infected plant tissues (Möller et al., 1992). Cells were collected by centrifugation (10 min, 3500 rpm), and further grinded in a mortar using liquid nitrogen and a pestle. The resulting powder was recovered into 2 ml eppendorf tubes, then resuspended in 500 µl of TES buffer (100 mM Tris, pH 8.0; 10 mM EDTA; 2 % SDS). 5 µl of 50 mg/ml Proteinase K was then added and the tubes are incubated for one hour at 37 °C. Selective precipitation of proteins and
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polysaccharides was performed using 140 µl of 5 M NaCl along with 1/10 vol of 10 % CTAB, followed by 10 min incubation at 65 °C. A unique extraction step was performed by adding 1 volume chloroform:isoamyl alcohol (24:1, Sigma-Aldrich®). The tubes were placed on ice for 30 min, before being centrifuged for 10 min (4 °C, 15000 rpm). The supernatant was transferred to fresh tubes. 225 µl of 5 M ammonium acetate was added, mixed gently and the tubes were placed on ice, before being centrifuged 10 min (4 °C, 15000 rpm). The
supernatant was transfered, 0.5 volume isopropanol was added and the tube was placed for at least one hour at -20 °C to precipitate DNA (to maximize DNA recovery, this step can be performed overnight). The nucleic acids were recovered by centrifugation, 15 min (4 °C, 15000 rpm). The supernatant was discarded and the DNA pellet was washed with 1 ml of cold 70 % ethanol. Finally, the tube was centrifuged one last time 5 min (4 °C, 15000 rpm), the supernatant was discarded, and the pellet dried before being resuspended in 100 µl TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
(vi) Novel optimised DNA extraction protocol. Cells were resuspended into 0.5 ml of 0.15 M NaCl, 0.1 M EDTA, and poured into 2 ml cryogenic vials (Nalgene®). Three freeze-thawing cycles, alternating freezing in liquid nitrogen and freeze-thawing at 37 °C in a water bath, were used to damage the cell walls and render the cells more susceptible to further enzymatic lysis. The cells were collected by centrifugation (10 min, 8000 rpm), resuspended in 0.5 ml TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and transferred to fresh 2 ml tubes for enzymatic cell wall lysis with 100 µl of 50 mg/ml lysozyme for 30 min at 37 °C.
Subsequently, proteins were degraded with 5 µl of 50 mg/ml proteinase K and in 2 % SDS final concentration, for 1 h at 37 °C. Polysaccharides, proteins and cell wall debris were thereafter removed by selective precipitation with CTAB in presence of NaCl : 150 µl of 5 M NaCl was added to the tubes, followed by 0.1 volume of a 10 % CTAB stock solution. The samples were gently mixed by inversion, then further incubated at 65 °C for 10 minutes to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
optimize the formation of CTAB-protein and -polysaccharides complexes. Nucleic acids purification was achieved by extraction in 1 volume of chloroform:isoamyl alcohol (24:1, Sigma-Aldrich®). The tubes were placed on ice for 30 min to allow precipitation of CTAB complexes, before being centrifuged (10 min, 8000 rpm). The supernatant was transferred to a fresh tube, gently mixed with 0.6 volume of isopropanol until DNA precipitated. The DNA pellets were recovered by centrifugation, (10 min, 4 °C, 15000 rpm) and washed with 1 ml of ice cold 70 % ethanol to remove any residual salt. After a final centrifugation (5 min, 4 °C, 15000 rpm), the supernatant was discarded, and the pellets were dried before being
resuspended in 100 µl TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The samples were treated with 1 µl of RNAse (10 mg/ml) for 1 h at 37 °C.
2.3. DNA quantification and purity analysis
The quantity and purity of the DNA extracts was assessed by spectrophotometry using the ND-1000 system (NanoDrop Technologies, Thermo Fisher Scientific Inc.). The absorbances at 230, 260 and 280 nm were measured on 2 µl of each sample. Concentration of DNA was estimated by spectrophotometric quantification at 260 nm. The overall purity was assessed by calculating the absorbance ratios 260/280 and 260/230. High values for both ratio (260/280 > 1.8, 260/230 > 2) are commonly accepted as good indicators for pure DNA (Manchester, 1995). In contrast, a low 260/280 ratio highlights a protein and/or phenol contamination within the sample, while a low 260/230 ratio will indicate the presence of organic compounds such as phenolate, thiocyanates, carbohydrates, or salts in the extract.
2.4. DNA quality analysis
The molecular weight and degradation of the isolated DNA were analysed by gel electrophoresis. 10 µl of sample were loaded on a 1 % wt/vol agarose gel stained with ethidium bromide (final concentration 100µM/L, Sigma-Aldrich®) and submitted to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
electrophoresis at 100V for 1h. A 1 kb+ DNA ladder (InvitrogenTM) was used as a reference to
evaluate the size of the DNA.
2.5. PCR amplification
In order to confirm the suitability of the extracted DNA for molecular biology purpose, a PCR was performed according to the Internally Transcribed Spacer (ITS) detection protocol (Scheldeman et al., 1999; Baurain et al., 2002). Six PCR reactions were carried out, for the two clusters and the four subclusters characterizing the ITS variations of
Arthrospira species. PCR primers and conditions were selected accordingly to the protocol described in (Baurain et al., 2002). The PCR products were subsequently loaded on a 1 % wt/vol agarose gel stained with ethidium bromide (final
concentration 100µM/L, Sigma-Aldrich®) and analysed through electrophoresis at 100V for 1h.
2.6. Statistical analyses
All statistical analyses were performed using the R software (R Development Core Team, 2008). Comparison of the DNA yields and purity of the extracts was performed using non-parametric tests. The Kruskal-Wallis test was first used to compare the methods altogether. This test, intuitively similar to a one-way analysis of variance, assessed if the different methods produced the same amount of DNA with the same quality levels, or if at least one method differed from the others. The Wilcoxon rank sum test was subsequently used to compare the optimised protocol against each other procedure independently. This test, as the non-parametric equivalent of the Student test, helped to determine whether the eventual differences observed between the optimised protocol and the other isolation methods were significant or not. An alpha level of 5 % (p < 0.05) was used for each test to accept or reject the null hypothesis.
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3. Results
3.1. DNA quantity and purity
A significant difference in DNA yield was observed between the methods tested (Figure 1a and Figure 2). While the Promega kit appeared inefficient, the other procedures gave disparate results. Finally, the optimised protocol allowed the recovery of a significantly higher amount of DNA compared to all the other methods (Wilcoxon test : p < 0.05). The amount of DNA isolated through this protocol was more than twice the amount of DNA obtained with the protocol described by Baurain et al. (2002), the second most efficient protocol in terms of yield. As for DNA yields, some significant differences in both wavelength ratios were
observed between the methods tested (Figure 1b). Samples isolated with the Fastprep method showed a significantly compromised purity, with a small protein contamination and an important salts and/or phenolic contamination absorbing at 260 nm. In addition, the extracts from the Baurain et al. (2002) method appeared to contain an important salt contamination, as shown by the very low 260/230 ratio. This might be due to the use of a very high
concentration of guanidine salts in the GES extraction buffer (60 % wt/vol). Finally, the DNA samples isolated with the Miniprep protocol (Ausubel et al., 1995), the plant-fungus
extraction protocol (Möller et al., 1992) and the optimised protocol presented ratio values above the quality thresholds.
3.2. DNA quality
The analysis of the DNA extracts by electrophoresis supported the previous observations (Figure 2). The DNA extracted with the Fast-Prep protocol was very degraded. The mechanical disruption of the cell wall using glass beads is an efficient lysis method, but appeared to have a strong shearing effect on DNA. Interestingly, the DNA extracted with the protocol described by Baurain et al. (2002) showed an incomplete migration pattern. A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
significant fraction of the DNA appeared to be blocked in the wells of the electrophoresis gel. The salt contaminants detected by spectrophotometry (Figure 1b) might have formed
complexes with the DNA, resulting in an alteration of its migration. The DNA extracted with both the method described by Möller et al. (1992) and the optimised protocol presented a normal migration pattern. The DNA isolated with the optimised DNA isolation protocol showed a normal migration, no degradation and the strongest fluorescence intensity, which indicates a higher amount and purity (Figure 1a).
3.3. PCR amplification
Two of the DNA samples extracted with the optimised protocol were used to perform a PCR detection of the ITS. Amplification of PCR products was only positive for cluster I and subcluster IB primer pairs (Fig. 3), with a product size of circa 325 bp, as expected for Arthrospira sp. PCC8005 (Baurain et al., 2002). This result confirmed the fact that the DNA extracted with the proposed procedure is suitable for molecular biology analysis such as PCR.
4. Discussion
As for many filamentous species (Fiore et al., 2000), the efficiency of DNA isolation procedures appeared to be extremely variable for cyanobacteria of the genus Arthrospira sp. PCC8005. The different protocols tested presented striking differences in terms of results. More than just highlighting the efficiency of the new optimised protocol, they also represent a clear illustration of the two major problems encountered in DNA isolation from cyanobacteria : poor cell lysis efficiency (Billi et al., 1998 ; Fiore et al., 2000) and high contamination or poor purity (Porter, 1988).
Cell lysis efficiency clearly appeared as a critical step for DNA extraction from Arthrospira sp. In general, when only enzymatic methods were used, bad results were obtained, such as with the Wizard® Genomic DNA Purification System (PromegaTM) and the Miniprep method
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(Ausubel et al., 1992). Arthrospira cells proved to be very difficult to disrupt only by
enzymatic treatment. The particular filamentous structure of Arthrospira sp., with its complex multi-layered cell wall (Ciferri, 1983) and specific ultrastructures such as the polysaccharidic sheath surrounding the cells (Hoiczyk and Hansel, 2000), certainly represents obstacles for the enzymatic degradation. In contrast, a purely mechanical lysis such as the Fastprep method (Feurer et al., 2004) was powerful enough to induce cell lysis, without any enzymatic help. Bead-beating is indeed known to allow the extraction of nucleic acids from a wide variety of organisms for which lysis can be otherwise difficult, such as plants (Haymes et al., 2004 ; Roberts, 2007) or fungi (Borneman and Hartin, 2000). It was also successfully used to overcome the lysis effects encountered with the desert cyanobacterium Chroococidiopsis (Billi et al., 1998). However, the resulting DNA is strongly sheared by this mechanical lysis, and does not allow to obtain intact high molecular weight DNA.
The combination of a soft mechanical treatment with enzymatic lysis appeared as the best alternative for efficient Arthrospira cell lysis. Such a combination had been reported to be successful with Microcystis aeruginosa samples (Fiore et al., 1999). Hence, Arthrospira sp. cell walls needed to be weakened either chemically or mechanically as a complement to enzymatic lysis. The chaotropic agent guanidium thiocyanate (Baurain et al., 2002) has been used for that purpose. Its protein denaturing properties fulfilled the cell lysis, while preventing the action of DNases (Pitcher, 1989). Nevertheless, the extracted DNA suffered from a strong contamination absorbing at 230 nm, most likely a consequence of the very high concentration of guanidium thiocyanate salts.
The use of a mechanical lysis such as the freeze-grinding method developed by Möller et al. (1992), proved to be more appropriate. This method was developed to isolate nucleic acids from plant-fungus complexes, which present similar difficulties as the one encountered with Arthrospira (i.e. high production of polysaccharides, few high molecular weight DNA). However, although the quality of the extracted DNA was good, this method yielded a reduced 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
and more variable amount of DNA. Indeed, part of the samples can be lost during the grinding process or the following transfer to a fresh test tube, which makes this method less
reproducible. In this paper, a freeze-thawing alternative was developed and appears as a more conservative method. It is sufficient to render the cells susceptible to further enzymatic lysis, while avoiding any degradations due to shearing stress. This method also allows a maximal recovery from the samples, making it more reproducible than freeze-grinding based methods. As for purity of the extracted DNA, the best results were obtained with the Miniprep protocol (Ausubel et al., 1995), the plant-fungus extraction protocol (Möller et al., 1992) and the optimised protocol. The common denominator between these methods is the selective precipitation of proteins and polysaccharides using CTAB in presence of NaCl. The use of CTAB was already known to be a critical step for DNA extraction of plants (Murray and Thompson, 1980). It also appeared as an essential one to remove the important amount of proteins and polysaccharides present in Arthrospira sp. (Ciferri, 1983). The introduction of this step clearly improves the efficiency of the extraction procedure, and the final
quality/purity of the extracted DNA.
The DNA extracted with the optimised protocol presents a high molecular weight, a reduced degradation and an excellent overall quality. The developed procedure is fast (only 3-4 hours to complete a full extraction) and reproducible. It does not require the utilisation of toxic coumpounds such as phenol, which could lead to the production of hazardous waste. Finally, it is suitable for molecular biology techniques such as polymerase chain reaction.
A few articles have reported the isolation of nucleic acids from Arthrospira species among other bacteria (Tripathi and Rawal, 1998; Wu et al., 2000; Liu et al., 2005; Ling et al., 2007). However, it is generally impossible to assess the different DNA yields due to the absence of measurement data in the manuscripts. The method described by Mak and Ho (1992) for DNA isolation of bacteria and cyanobacteria reported the extraction of approximatively 55 µg of nucleic acids from 30 mg of wet weight of Arthrospira platensis. By extrapolation, the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
optimised protocol allowed the recovery of circa 50 µg of genomic DNA from 40-50 mg wet weight (measured from a 5 ml culture at OD 750nm ~ 1.0, data not shown). While the
isolation protocol described by Mak and Ho (1992) could seem slightly more efficient, the apparent higher yield is most likely an overestimation due to the absence of RNAse treatment (i.e. the extraction of total nucleic acids, including DNA and RNA).
5. CONCLUSION
By combining an efficient dual enzymatic and a freeze-thawing lysis step with a selective CTAB precipitation, we have designed a protocol to extract efficiently and reproducibly DNA of high quality from Arthrospira sp. The comparison with the other extraction protocols tested in this article clearly shows that the optimised method allows a maximal recovery of highly pure and intact DNA, suitable for further molecular analysis. Later, the high quality DNA was also effectively used for full genome DNA sequencing (article in preparation). The possibility to obtain high quality DNA will be an important step towards an improvement of our genomic knowledge of Arthrospira and a better understanding of this particular organism.
ACKNOWLEDGMENT
This work is dedicated to the memory of Dr. ir. L. Hendrickx who initiated the study of Arthrospira sp. in space flight conditions and the related sequencing project. This research was partly funded by the Belgian Nuclear Research Center (SCK•CEN) through the PhD AWM grant of N. Morin. We also thank the European Space Agency (PRODEX, ESA-GSTP) and the Belgian Science Policy (Belspo) for the financial support in the Arthrospira sp. PCC8005 sequencing project. We thank C. Lasseur and C. Paillé, both from ESTEC/ESA, for their constant support and advice in the frame of the MELiSSA project.
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LEGENDS
Figure 1 : Average DNA concentration (a) and quality markers (b) obtained with each extraction protocol. As for the Promega kit, the ratios were unreliable due to the absence of DNA after extraction.
Figure 2 : Electrophoresis of the extracted DNA on a 1 % wt/vol agarose gel (1,6,11,16,21,26: 1 kb+ DNA ladder, InvitrogenTM; 2-4: Wizard®; 7-10: miniprep; 12-15: FastPrep®; 17-20:
Baurain et al. (2002) ;22-25: Möller et al.(1992); 27-30: Morin et al.)
Figure 3 : Detection and amplification of ITS sequences using the DNA extracted with the optimised protocol (1,6,15: 1 kb+ DNA ladder, InvitrogenTM; 2,3: cluster I; 4,5: cluster II; 7,8:
subcluster IA; 9,10: subcluster IB; 11,12: subcluster IIA; 13-14: subcluster IIB).
Amplification is positive for cluster I (a) and subcluster IB (b) (~325 kb), as expected for Arthrospira sp. PCC8005 (Baurain et al., 2002)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
FIGURE 1 (a) (b) FIGURE 2 FIGURE 3 0 0 . 5 1 1 . 5 2 2 . 5 F a s t p r e p B a u r a i n M ö l l e r M i n i p r e p M o r i n Q u al it y m ar ke rs 2 6 0 / 2 3 0 2 6 0 / 2 8 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 P r o m e g a M i n i p r e p M ö l l e r F a s t p r e p B a u r a i n M o r i n D N A c o n ce n tr at io n ( n g /µ l) 1 2 3 5 7 9
