J. Phycol. 36, 251–258 (2000) XANTHOGENATE NUCLEIC ACID ISOLATION FROM CULTURED AND ENVIRONMENTAL CYANOBACTERIA1 Daniel Tillett and Brett A. Neilan2 School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, NSW, Australia The isolation of high-quality nucleic acids from cyanobacterial strains, in particular environmental isolates, has proven far from trivial. We present novel techniques for the extraction of high molecular weight DNA and RNA from a range of cultured and environmental cyanobacteria, including stains belonging to the genera Microcystis, Lyngbya, Pseudanabaena, Aphanizomenon, Nodularia, Anabaena, and Nostoc, based on the use of the nontoxic polysaccharide solubilizing compound xanthogenate. These methods are rapid, require no enzymatic or mechanical cell disruption, and have been used to isolate both DNA and RNA free of enzyme inhibitors or nucleases. In addition, these procedures have proven critical in the molecular analysis of bloom-forming and other environmental cyanobacterial isolates. Finally, these techniques are of general microbiological utility for a diverse range of noncyanobacterial microorganisms, including Gram-positive and Gramnegative bacteria and the Archea. lysis, but also interfere with most nucleic acid purification protocols (Porter 1988, Wilkins and Smart 1996). Cyanobacterial cells are also rich in endo- and exonucleases and contain photosynthetic pigments, which can inhibit enzymatic reactions, especially reverse transcription (Cardellina et al. 1993, Lau et al. 1993) and the PCR (Cohen et al. 1994, Giovannoni et al. 1990, Golden 1987, Neilan 1995a). In consequence, the techniques developed to isolate cyanobacterial nucleic acids are often complex and laborious, requiring either (1) mechanical cell breakage (Golden 1987, Jackman and Mulligan 1995, Leff et al. 1995, Luo and Stevens 1997, Reith et al. 1986, West and Adams 1997); (2) enzyme digestions (Cohen et al. 1994, de Lorimer et al. 1984, Giovannoni et al. 1990, Golden 1987, Jackman and Mulligan 1995, Joset 1988, Lotti et al. 1996, Nishihara et al. 1997, Palenik 1994, Porter 1988, Smoker and Barnum 1988); (3) multiple organic solvent extractions (Cohen et al. 1994, Ferris et al. 1996, Giovannoni et al. 1990, Golden 1987, Jackman and Mulligan 1995, Leff et al. 1995, Lotti et al. 1996, Mak and Ho 1991, Neilan 1995a, Nishihara et al. 1997, Palenik 1994, Porter 1988, Smoker and Barnum 1988, West and Adams 1997, Zehr and McReynolds 1989); (4) grinding under liquid nitrogen or dry ice (Kramer et al. 1996, Luo and Stevens 1997, Van der Plas et al. 1989); (5) hot phenol (Bovy et al. 1993, Kramer et al. 1996); (6) or cesium chloride ultracentrifugation (de Lorimer et al. 1984, Giovannoni et al. 1990, Golden 1987, Joset 1988, Leff et al. 1995, Reith et al. 1986). In addition, many of these techniques fail to isolate nucleic acids from all cyanobacterial strains or environmental isolates. Jhingan (1992) introduced a novel method for the extraction of DNA from plant matter based on the use of metal xanthates. The formation of water-soluble polysaccharide xanthates with potassium ethyl xanthogenate was used to disrupt plant cell walls. In the presence of amine groups, these polysaccharide xanthates form insoluble complexes that are selectively precipitated (Carr et al. 1975). In addition, the xanthates bind metal ions, thus potentially inhibiting the activity of DNA degrading enzymes, as well as chelating ionic inhibitors of DNA amplification reactions. This rapid xanthogenate-based method does not require the use of toxic organics, enzymatic digestions, or cesium chloride ultracentrifugation. In addition, it can be performed without mechanical tissue homogenizations. We reasoned, as the Cyanobacteria present many of the same difficulties provided by plants, this Key index words: cyanobacteria; DNA extraction; nucleic acids; PCR; xanthogenate The Cyanobacteria are a diverse and cosmopolitan bacterial phylum and possess a number of unique biological characteristics. All cyanobacteria perform oxygenic photosynthesis and contain chlorophyll a and accessory photopigments, such as phycocyanin and phycoerythrin (Castenholz and Waterbury 1989). Many species are capable of fixing atmospheric nitrogen, some of which differentiate specialized cell types for this process and other functions (Castenholz and Waterbury 1989, Golden 1987). In addition, a number of cyanobacterial species can form symbiotic relationships with a taxonomically wide range of organisms, including animals, plants, fungi, algae, nonphotosynthetic protists, and heterotrophic bacteria (West and Adams 1997). Progress in determining the molecular mechanisms underlining cyanobacterial form and function has been hindered by the difficulty encountered with by many cyanobacteria in isolating high-quality nucleic acids. Many cyanobacterial strains produce copious quantities of mucilaginous polysaccharides, which not only make it difficult to achieve complete cellular 1 Received 29 April 1999. Accepted 27 October 1999. Author for reprint requests; fax ⫹61 2 9385 1591; e-mail b.neilan @unsw.edu.au. 2 251 252 DANIEL TILLETT AND BRETT A. NEILAN technique may provide a safe, rapid, and efficient means to extract DNA from cyanobacteria. Unfortunately, we, together with other investigators (Johns et al. 1997, Ross 1995, Williams and Ronald 1994), found the original protocol to be unreliable, and in our hands most cyanobacterial strains yielded little or no DNA. In this paper we present new protocols, based on the use of xanthogenate, for the rapid extraction of high-quality DNA and RNA from a wide range of environmental and cultured cyanobacteria. In addition, these protocols have proven of general utility for the extraction of DNA and RNA from a diverse range of microorganisms and environmental samples. materials and methods Bacterial and archeal strains. The strains used in this study are listed in Table 1. Cyanobacterial strains with designations AWT, NIES, or PCC were obtained from Australian Water Technology (Sydney, Australia), the National Institute for Environmental Studies (Tsukuba, Japan) (Natl. Inst. for Environmental Studies 1991), and the Pasteur Culture Collection (Paris, France) (Rippka and Herdman 1992), respectively. Microcystis incerta HINDAK 1965/17 was obtained from the Institute of Botany, Czech Academy of Science. Escherichia coli JM109 (Yanish-Perron et al. 1985) was obtained from Promega (Madison, WI). Methanococcoides burtonii (Franzmann et al. 1992), Vibrio angustum S14, the Rhodococcus sp. (Paje et al. 1997), Helicobacter pylori SS1 (Lee et al. 1997), and the Acetobacter sp. (Bernardo et al. 1998) were kind gifts of T. Thomas, S. Srinivasan, C. Svenson, B. Burns, and E. Bernardo respectively. The cyanobacterial blooms samples were kindly supplied by P. Hawkins. The cyanobacterial strains were maintained in either JM (Natl. Inst. for Environmental Studies 1991) or BG-11 (Castenholz and Waterbury 1989) media at 25⬚ C with a light intensity of approximately 20 mol⭈m⫺2⭈s⫺1. E. coli JM109 was grown at 37⬚ C in LB media (Sambrook et al. 1989). M. burtonii was grown at 22.5⬚ C in liquid methanogen growth media under anaerobic conditions (Franzmann et al. 1992). V. angustum S14 was grown at 25⬚ C in LB media supplemented with 20 g⭈L NaCl. The Rhodococcus strain was grown at 25⬚ C in PAS media (Paje et al. 1997). H. pylori SS1 was grown at 37⬚ C under mi- Table 1. Bacterial and archeal strains used in this study. Strain Microcystis aeruginosu PCC 7806 Microcystis wesenbergii NIES 107 Microcystis viridis NIES 102 Microcystis incerta HINDAK1965/17 Lyngbya sp. AWT 211. Pseudanabaena sp. AWT 210, Aphanizomenon flos-aquae NIES 81 Nodularia spumigena PCC 73104 Anabaena circinalis AWT 006 Nostoc punctiforme PCC 73102 Escherichia coli JM109 Acetobacter sp. Vibrio angustum S14 Helicobacter pylori SS1 Rhodococcus sp. Methanococcoides burtonii a croaerophilic condition on CSA media (Lee et al. 1997). The Acetobacter strain was grown at 30⬚ C in coconut water medium (Bernardo et al. 1998). DNA extraction. DNA was isolated using the methods of Neilan et al. (1993, 1995), Porter (1988), Jhingan (1992), or the following xanthogenate-SDS (XS) DNA extraction protocol. Briefly, 1 mL volumes of mid to late logarithmic growth phase bacterial cell cultures were harvested by centrifugation and the cell pellets resuspended in 50 L of TER (10 mM TrisHCl, pH 7.4; 1 mM EDTA, pH 8; 100 g⭈mL RNase A). Alternatively, small amounts of environmental samples, approximately 10–20 mg wet weight, were resuspended in 50 L of TER. To each cell suspension in a 1.5-mL microcentrifuge tube was added 750 L of freshly made XS buffer (1% potassium ethyl xanthogenate [Fluka, Buchs, Switzerland]; 100 mM Tris-HCl, pH 7.4; 20 mM EDTA, pH 8; 1% sodium dodecylsulfate; 800 mM ammonium acetate) and the tubes were inverted several times to mix. The tubes were incubated at 70⬚ C for 10 to 120 min in a waterbath, with the time dependent on the microorganism under investigation. After incubation, the tubes were vortexed for 10 s before being placed on ice for 30 min. Precipitated cell debris was removed by centrifugation at 14,000 rpm for 10 min and the supernatants carefully transferred to fresh eppendorf tubes containing 750 L of isopropanol. Samples were incubated at room temperature for 10 min and the precipitated DNA pelleted by centrifugation for 10 min at 12,000 g. The DNA pellets were washed once with 70% ethanol, air-dried, and finally resuspended in 100 L of TE (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8). To ensure that the DNA samples were free of exo- and endonucleases, samples were incubated at 37⬚ C for 2 h in 1⫻ HinfI restriction enzyme buffer before gel electrophoresis. RNA extraction. RNA was extracted using the following xanthogenate-SDS-phenol (XSP) protocol. Briefly, cells were pelleted and resuspended in 50 L of culture medium or TE. The concentrated cells were added to 1.3 mL of preheated (65⬚ C) XSP buffer (1:1 volumes of XS buffer and phenol). The tubes were incubated at 65⬚ C for 5 min. During this incubation, the tubes were inverted several times to facilitate the mixing of the organic and aqueous phases, important in ensuring efficient cell lysis. The tubes were vortexed for 10 s, and 200 L of chloroform:isoamyl alcohol (24:1) was added to each tube. The phenol and aqueous phases were separated by centrifugation at 14,000 rpm for 5 min. The aqueous phases were carefully transferred to fresh eppendorf tubes containing 500 L of phenol:chloroform:isoamyl alcohol (25:24:1). The tubes were incu- Phyluma Kingdom DNAb RNAc Cyanobacteria (Chroococcales) Cyanobacteria (Chroococcales) Cyanobacteria (Chroococcales) Cyanobacteria (Chroococcales) Cyanobacteria (Oscillatoriales) Cyanobacteria (Oscillatoriales) Cyanobacteria (Nostocales) Cyanobacteria (Nostacales) Cyanobacteria (Nostacales) Cyanobacteria (Nostacales) Purple Bacteria (Gamma) Purple Bacteria (Alpha) Purple Bacteria (Gamma) Purple Bacteria (Epsilon) Gram positive (High G⫹C) Methanogen (Group V) Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Archea 21 16 25 9 11 8 14 8 10 5 ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ 42 44 50 14 ND 10 ND 15 11 ND ⫹ ND ND ⫹ ND ND Phylum (order) of strain as based on 16S rRNA sequence. Plus indicates DNA isolated using XS protocol (g⭈mg dry cell weight). Noncyanobacterial strains were not quantified during this study. c Plus indicates total RNA isolated using XSP protocol (g⭈mg dry cell weight). ND, not determined. Noncyanobacterial strains were not quantified during this study. b 253 XANTHOGENATE DNA EXTRACTION FROM CYANOBACTERIA bated at 65⬚ C for 2 min, vortexed briefly, then centrifuged for 4 min at 14,000 rpm. The aqueous phases were once again carefully transferred to fresh tubes. The phenol–chloroform extractions were repeated until no opaque interface was visible between the two phases; in most cases two extractions were sufficient. After the final phenol–chloroform extraction, the aqueous phases were transferred to fresh tubes and 1 volume of isopropanol was added. The tubes were inverted several times and the RNAs allowed to precipitate on ice for 15 min. Total RNA was isolated by centrifugation at 14,000 rpm for 10 min. The RNA pellets were washed once with 70% ethanol, air-dried, and finally resuspended in 50 L of DEPC-treated water (Sambrook et al. 1989). To ensure that the RNA samples were free of ribonucleases, samples were incubated at 42⬚ C for 16 h before gel electrophoresis. PCR amplification. Amplification of the phycocyanin intergenic spacer region (PC-IGS) and 16S rRNA gene were performed as described previously (Neilan et al. 1995a, 1997). Briefly, the PC-IGS PCR contained 2 L of 10⫻ PCR buffer (Biotech International, Perth, Australia), 2 L of 25 mM MgCl2, 0.5 L of 10 mM of each deoxynucleotide triphosphate, 5 pmol of each of the two PC-IGS primers (Table 2),] 10 ng of genomic DNA, 1 unit of Taq polymerase (Biotech International, Perth, Australia), and water to 20 L. The PC-IGS PCRs were subjected to 30 cycles of 94⬚ C for 10 s, 50⬚ C for 20 s, and 72⬚ C for 40 s in a Perkin-Elmer 2400 PCR thermocycler. The cyanobacterial 16S rDNA gene PCR amplification was performed as described previously, except that primers 27F and 408R (Table 2) were used (Neilan et al. 1995a). The cyanobacterial 16S rDNA PCR thermal cycling conditions consisted of 30 cycles of 94⬚ C for 10 s, 60⬚ C for 20 s, and 72⬚ C for 40 s. Restriction enzyme digestion. Approximately 200 ng of XS isolated DNA was digested with 5 units of the restriction enzyme HinfI in 15 L of 1⫻ buffer (Boehringer, Mannheim, Germany). Reaction mixtures were incubated overnight at 37⬚ C prior to analysis by agarose gel electrophoresis. results XS DNA isolation from cultured cyanobacteria. The XS DNA extraction protocol was tested on a range of cultured cyanobacteria from the orders Chroococcales, Oscillatoriales, and Nostocales (Table 1). High-quality DNA was isolated from all strains tested, including Microcystis aeruginosa PCC 7806, M. wesenbergii NIES 107, M. viridis NIES 102, M. incerta HINDAK 1965/17, Lyngbya sp. AWT 211, Pseudanabaena sp. AWT 210, Aphanizomenon flos-aquae NIES 81, Nodularia spumigena PCC 73104, Anabaena circinalis AWT 006, and Nostoc punctiforme PCC 73102 (Fig. 1A). The DNA yield varied between 5 g⭈mg⫺1 and 25 g⭈mg⫺1 of dry cell weight, depending on the strain studied. The quality of the extracted DNA for successive enzymatic reactions was assessed by restriction enzyme digestion and PCR (Fig. 1B and 1C). All DNAs were found to be free of active DNases and were efficiently cleaved by the restriction enzyme HinfI. Because many cultures of filamentous cyanobacterial strains Table 2. contain contaminating heterotrophic bacteria, the cyanobacterial phycocyanin intergenic spacer PCR was performed to ensure that cyanobacterial DNA had been extracted (Fig. 1C). PCR amplicons were obtained from all cyanobacterial strains tested, indicating that not only was cyanobacterial DNA isolated, but the isolated DNA was free from PCR inhibitors. XS DNA isolation from environmental cyanobacterial blooms. A large toxic cyanobacterial bloom event occurred in the Botany Ponds, Sydney, Australia, over the late summer and autumn months of 1996. This mixed bloom was dominated by species of the genera Microcystis and Anabaena, and underwent a number of complex population successions as assessed by microscopy and toxin data (Baker et al. 1998). Bloom samples had been collected on a weekly to monthly basis and frozen stocks were stored. We undertook to study the genetic population structure by analyzing the phycocyanin intergenic spacer and 16S rDNA region (Neilan et al. 1995a, 1997). Initial attempts to obtain DNA free of PCR inhibitors from the Botany Ponds bloom samples proved difficult. A range of techniques were tried, including the methods of (Neilan et al. 1993, 1995a, Porter 1988). Many of the Botany Ponds bloom samples were highly pigmented and the DNA extracted from these samples failed to allow PCR amplification. This difficulty in isolating enzyme inhibitor-free DNA has been commonly observed with environmental samples (Giovannoni et al. 1990, Golden 1987, Neilan 1995a). We investigated the ability of the XS DNA extraction protocol to isolate PCR inhibitor-free DNA from the Botany Ponds bloom samples. This technique provided DNA free of PCR inhibitors with PCR amplicons of the PC-IGS and cyanobacterial 16S rDNA regions obtained from all eight examined bloom samples (Fig. 2). These included samples that were originally highly pigmented and had not provided amplifiable template when other DNA extraction methods were used (Neilan 1995a, Neilan et al. 1993, Porter 1988). XS DNA isolation from other microorganisms. The XS DNA extraction protocol was tested on a range of representative noncyanobacterial strains, to assess its general microbiological utility. A range of Gram-positive and Gram-negative bacteria and an archea were examined, including E. coli JM109, an Acetobacter sp., V. angustum S14, a Rhodococcus sp., H. pylori SS1, and Methanococcoides burtonii (Table 1). High-quality DNA was isolated from all Gram-negative and Gram-positive bacteria and the archea examined (Fig. 3), including strains that have previously proven recalci- Oligonucleotide primers used in this study. Primer Sequence Reference 27F 408R PC␤F PC␣R AGAGTTTGATCCTGGCTCAG TTACAA(C/T)CCAA(G/A)(G/A)(G/A)CCTTCCTCCC GGCTGCTTGTTTACGCGACA CCAGTACCACCAGCAACTAA Neilan et al. 1997 Neilan et al. 1997 Neilan et al. 1995 Neilan et al. 1995 254 DANIEL TILLETT AND BRETT A. NEILAN Fig. 1. DNA isolated from cultured cyanobacteria using the XS DNA extraction method. (A) 2 L of genomic DNA electrophoresed on a 0.65% agarose gel in 1⫻ Tris–Acetate–EDTA (TAE) buffer with 100 ng of lambda HindIII DNA marker (lane 12). (B) HinfI restriction enzyme digest of XS isolated genomic DNA electrophoresed on a 1.5% agarose gel in 1⫻ TAE with 50 ng of lambda HindIII DNA marker (lane 12). (C) Amplification products from the cyanobacterial phycocyanin intergenic spacer PCR electrophoresed on a 3% agarose gel in 1⫻ TAE with 100 ng of Phi-X 174 HaeIII DNA marker (lane 12). All gels were stained with ethidium bromide and photographed under UV transillumination. trant with other methods (Bernardo et al. 1998, Paje et al. 1997). The DNA quality and yield obtained from noncyanobacteria was assessed by restriction enzyme digestion (Fig. 3). All DNA samples were digested with the restriction enzyme HinfI and were observed to be free of extraneous nucleases, including the H. pylori strain, which had been previously found to contain high levels of exonucleases (de Ungria et al. 1998). XSP RNA extraction from cyanobacteria and other microorganisms. In an effort to study the transcriptional regulation of the microcystin synthetase gene in Microcystis aeruginosa PCC 7806 (Dittmann et al. 1997 and Tillett and Neilan unpublished data) we required a robust RNA extraction protocol suitable for cyanobacteria. Previous investigators have also noted difficulties in RNA isolation from cyanobacteria and other polysaccharide-rich samples (Bugos et al. 1995, Luo XANTHOGENATE DNA EXTRACTION FROM CYANOBACTERIA Fig. 2. Isolation of PCR inhibitor-free DNA from environmental cyanobacterial blooms using the XS DNA extraction method. Phycocyanin intergenic spacer pyc PCR and cyanobacterial 16S rDNA (16S) were performed on XS-extracted DNA from the Botany Ponds cyanobacterial bloom samples collected in 1996 on the dates indicated. Both PCR products from each sample were pooled, and a total of 4 L run on a 2% agarose gel in 1⫻ TAE with 100 ng of Phi-X 174 HaeIII DNA marker. The gel was stained with ethidium bromide and photographed under UV transillumination. and Stevens 1997, Wilkins and Smart 1996). We reasoned that a combination of the XS buffer with the hot phenol method (Giovannoni et al. 1990, Kramer et al. 1996, Sambrook et al. 1989, Wilkins and Smart 1996) might enable high-quality RNA to be isolated rapidly from cyanobacteria in general and from M. aeruginosa PCC 7806 in particular. The XSP RNA extraction protocol was tested on a range of cyanobacterial and noncyanobacterial strains, 255 including M. aeruginosa PCC 7806, M. wesenbergii NIES 107, M. viridis NIES 102, M. incerta HINDAK 1965/17, Nodularia spumigena PCC 73104, Pseudanabaena sp. AWT 210, Anabaena circinalis AWT 006, E. coli JM109, and H. pylori SS1 (Table 1). Total RNA yield varied among the strains examined, ranging from 10 g⭈mg⫺1 to 50 g⭈mg⫺1 of dry cell weight (Fig. 4). We regularly obtained ⬎2 g of total RNA per milliliter of M. aeruginosa PCC 7806 cell culture. This compares favorably with the method of Luo (Luo and Stevens 1997), who obtained 82 g of total RNA from 2 L of cyanobacterial culture. All XSP-extracted RNA samples were observed to be free of extraneous RNases, with no degradation visible after incubation at 42⬚ C for 16 h (Fig. 4). In addition, RNA isolated from M. aeruginosa PCC 7806 and H. pylori SS1 using the XSP method has been used successfully for primer extension and nuclease protection analysis (Tillett and Neilan 1999a). discussion The advent of cyanobacterial molecular investigations has created the need for rapid and efficient techniques for the extraction of nucleic acids from both cultured and environmental isolates. This paper presents novel methods for the extraction of highquality nucleic acids from a wide range of environmental and cultured cyanobacteria, as well as from other bacterial and archeal microorganisms. XS DNA isolation. The XS DNA extraction method rapidly provided high-quality DNA of high molecular weight from a diverse range of cyanobacteria and other microorganisms. The DNA was free of enzymatic inhibitors and was suitable for restriction enzyme digestion, cloning, and PCR (Tillett and Neilan 1999b). The XS buffer contains no hazardous chemicals and is of low cost. In addition, this technique re- Fig. 3. DNA isolated from noncyanobacterial microorganisms using the XS DNA method. Lane 1: 50 ng of lambda HindIII DNA marker. Lanes 2, 4, 6, 8 and 10: 2 L of genomic DNA isolated using the XS protocol. Lanes 3, 5, 7, 9, 11: 7.5 L of HinfI-digested, XS-isolated genomic DNA. Lane 12: 100 ng Phi-X 174 HaeIII DNA marker. DNA samples were electrophoresed on a 1.5% agarose gel in 1⫻ TAE, stained with ethidium bromide, and photographed under UV transillumination. 256 DANIEL TILLETT AND BRETT A. NEILAN quires minimal handling of the microbial sample and does not require mechanical homogenization. This not only reduces the risk of sample contamination, but minimizes the production of aerosols, of particular importance if the sample contains possible human pathogens (Ross 1995). This technique is routinely used in our laboratory and, to date, the XS protocol has been used successfully on ⬎200 cyanobacteria strains and other microorganisms (Tillett and Neilan unpublished data). While we have found there is generally a good correlation between cyanobacterial culture density and DNA yield, it should be noted that the DNA yield is generally greater and of better quality from cells extracted in mid-log phase. Finally, the XS DNA extraction method provided PCR inhibitorfree microbial DNA from a range of environmental samples, including cyanobacterial blooms, river sediment, worm castings, and marine biofilms (Thompson 1997 and Tillett and Neilan unpublished data). Important steps in the XS DNA extraction procedure should be noted. First, the XS buffer should be freshly prepared. We routinely make the XS buffer from stored premade stock solutions and potassium ethyl xanthogenate powder. Second, the XS buffer should not be overloaded with biomass by attempting to extract too much sample, as this results in DNA of poor quality. We therefore recommend using 750 L of XS buffer per 1–2 mL of log-phase cell culture. Third, the cell pellet should be fully resuspended in TER buffer before the addition of the XS buffer. Fourth, the 70⬚ C incubation time required varies Fig. 4. Total RNA isolated from cyanobacteria and noncyanobacteria using the XSP method. Two L of XSP isolated RNA samples were electrophoresed on a nondenaturing 2% agarose gel in 1⫻ TAE with 100 ng of Phi-X 174 HaeIII DNA marker. The gel was stained with ethidium bromide and photographed under UV transillumination. from bacterial strain to strain, and it is advisable to determine the ideal time empirically. We have found that Gram-negative bacteria and archea require 15 min or less, unicellular cyanobacteria 30 to 60 min, and filamentous cyanobacteria and environmental samples up to 2 h. Fifth, it is recommended the samples be vortexed for at least 10 s after the 70⬚ C incubation step. Without vortexing, chromosomal DNA remains associated with other cellular components and is lost during the debris precipitation step. Sixth, the samples should be left on ice for a minimum of 15 min, or until the XS buffer becomes opaque and viscous. This ensures that proteins and other cellular debris have aggregated before centrifugation. XSP RNA isolation. We found the combination of hot phenol and XS buffer (XSP) to be a rapid and effective method to isolate total RNA from cyanobacteria and other microorganisms. This protocol allows the isolation of greater than 100 g of nuclease-free total RNA in ⬍2 h. RNA isolated using the XSP protocol has been used for reverse transcriptions, nuclease protection assays, primer extensions, and Northern blotting (Tillett and Neilan 1999a). This technique does not require expensive chemicals such as guanadinium salts, grinding under liquid nitrogen, or cesium chloride ultracentrifugation. In addition, it has proven to be of general utility with a range of noncyanobacterial microorganisms. The important steps in the XSP RNA extraction procedure should also be noted: First, the XSP buffer should be preheated to ensure the immediate inacti- XANTHOGENATE DNA EXTRACTION FROM CYANOBACTERIA vation of RNases upon cell lysis. Second, inversion and vortexing of the tubes is important in ensure efficient cell lysis and separation of the RNA from other cellular components. Third, heating the sample with phenol–chloroform. The failure of cold phenol–chloroform to remove RNases is enigmatic, however, the use of hot phenol–chloroform proved critical for the removal of endogenous RNases from certain cyanobacterial strains. Interestingly, no cautions were taken to exclude exogenous RNases beyond the wearing of gloves and DEPC treatment of the RNA resuspension water. This suggests that the major problems encountered with RNA isolations are from endogenous and not exogenous sources. Denaturation of genomic DNA by the hot phenol in the XSP extraction technique results in a relatively low level of DNA contamination, from ⬍1 ng to 20 ng of DNA per microgram of total RNA (Fig. 4). Depending on the organism, this method enriches the RNA fraction of total nucleic acids by 20- to 100-fold (Table 1). This level of DNA contamination should not prove problematic for many applications (e.g. Northern blots). If required, however, the DNA can be easily removed by enzymatic DNase treatment (Sambrook et al. 1989). We have successfully used a post-XSP DNase treatment step to remove contaminating DNA in the development of an RT-PCR-based transcript mapping protocol (Tillett and Neilan 1999a). Finally, the XSP extraction technique has proven of general utility in the extraction of DNA from nuclease-rich samples. To date, it has been used to extract DNA from DNaserich Vibrio vulnificus cultures (Weichart et al. 1997), plasmids from clinical Helicobacter isolates (de Ungria et al. 1998), as well as many cyanobacterial strains (Tillett and Neilan unpublished data). The work described here presents novel nucleic acid extraction protocols for cultured and environmental microorganisms. In addition, these protocols may be of utility to other fields, as they have also proven of use in the extraction of nucleic acids from a diverse range of sources, including rocks, fungi, blood, mouse gastrointestinal tissue, and plant matter. 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