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Applied and Environmental Microbiology, October 1998, p. 4053-4056, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Method for DNA Extraction from the Desert Cyanobacterium Chroococcidiopsis and Its Application to Identification of ftsZ

Daniela Billi,¹ Maria Grilli Caiola,^1,* Luciano Paolozzi,¹ and Patrizia Ghelardini²

Dipartimento di Biologia¹ and Centro Acidi Nucleici del CNR c/o Dipartimento di Biologia,² Università di Roma "Tor Vergata," I-00133 Rome, Italy

Received 30 April 1998/Accepted 6 July 1998

	ABSTRACT

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A method was developed for extraction of DNA from Chroococcidiopsis that overcomes obstacles posed by bacterial contamination and the presence of a thick envelope surrounding the cyanobacterial cells. The method is based on the resistance of Chroococcidiopsis to lysozyme and consists of a lysozyme treatment followed by osmotic shock that reduces the bacterial contamination by 3 orders of magnitude. Then DNase treatment is performed to eliminate DNA from the bacterial lysate. Lysis of Chroococcidiopsis cells is achieved by grinding with glass beads in the presence of hot phenol. Extracted DNA is further purified by cesium-chloride density gradient ultracentrifugation. This method permitted the first molecular approach to the study of Chroococcidiopsis, and a 570-bp fragment of the gene ftsZ was cloned and sequenced.

	TEXT

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Many attempts have been made to adapt the genetic and molecular biological methods devised for the study of eubacteria for research in cyanobacterial biology, but few have been successful. Genetic techniques are available for only a few cyanobacterial strains, which are used as paradigms for cyanobacterial research (25).

To date, no molecular approach has been developed for the analysis of Chroococcidiopsis, a desiccation-tolerant cyanobacterium colonizing hypolithic and cryptoendolithic habitats in extremely hot and cold deserts worldwide (9). In these severe environments, like the Ross Ice Shelf in Antarctica, metabolic processes are so slow that time scales of biological and geological processes overlap, and these rock-inhabiting communities might be the oldest living organisms in existence on earth (17). In addition, the morphological characteristics of Chroococcidiopsis and its resemblance to certain Proterozoic fossils have suggested that it may be the most primitive living cyanobacterium (10).

The cytology and ultrastructure of field- and laboratory-desiccated Chroococcidiopsis cells, as well as their ability to survive prolonged nutrient limitation and starvation, have been reported (3, 4, 11, 12), but the mechanisms that contribute to the desiccation resistance of this cyanobacterium are poorly understood, as are those in most other prokaryotic cells (20). For example, the ability of Nostoc commune to withstand dehydration reflects a complex array of factors at every level of cell structure and function (21).

The molecular biology of Chroococcidiopsis is particularly difficult to study. Its growth rate is relatively low, with a generation time of about 16 days (4), although some fast-growing strains, with a generation time of a few days, have been isolated and deposited at the Culture Collection of Microorganisms from Extreme Environments, Florida State University, Tallahassee.

In addition, Chroococcidiopsis cells are surrounded by a mucilaginous envelope, whose ultrastructural and chemical complexity increases with age (2). The production of large quantities of polysaccharides causes difficulties in DNA purification (19). The envelope also compounds the contamination problems caused by the slow growth of Chroococcidiopsis; fast-growing bacteria and fungi are a serious problem, especially heterotrophic bacteria, which can grow within the polysaccharides of the cyanobacterial envelope (8). The presence of these heterotrophs can make cyanobacteria easier to culture, but they can be very difficult to remove.

The aim of the present study was the development of a strategy for extraction of high-quality DNA from Chroococcidiopsis despite these difficulties.

The newly developed method was tested in an attempt to identify ftsZ, a gene essential for eubacterial cell division and found in all bacterial species investigated (16). The gene has also been found in Arabidopsis thaliana, as a nucleus-encoded protein localized in the stromal compartment of the chloroplast (18).

Cyanobacterial strains and growth conditions. The following fast-growing Chroococcidiopsis strains (mean generation time, 3 to 4 days) were used: (29)N6904, crypotendolithic; (48)N6911B and (034)N6909B, hypolithic, from the Negev desert (Israel); and (568)G91-19, hypolithic, from the Gobi desert (Mongolia). The Anabaena cylindrica Lemm strain was from the National Institute for Environmental Studies collection (University of Tsukuba, Japan).

Chroococcidiopsis strains and Anabaena cylindrica were grown in BG-11 medium and nitrogen-free BG-11 medium, respectively (22), at 27°C under 90 µmol of photon m-² s-¹ provided by fluorescent cool-white bulbs (19-h-8-h light-dark cycle). In all procedures 50-ml samples of a culture of Chroococcidiopsis cells grown for 3 or 8 months were used.

Evaluation of bacterial contamination. Chroococcidiopsis cells were counted in a Bürker chamber under a light microscope. Bacterial contamination was evaluated from counts of CFU on Luria-Bertani plates at 37°C. Bacterial contamination was found in about 10 to 20% of the total Chroococcidiopsis cells after three or more months in culture. Electron microscopic analysis indicated that no more than three bacteria are present in the envelope of Chroococcidiopsis.

Elimination of bacterial contamination. We developed a method of differential lysis based on the observation that aged Chroococcidiopsis cells are characterized by thick envelopes (Fig. 1A) and resistance to lysozyme. The method, as summarized in Fig. 2, consists of lysozyme treatment followed by osmotic shock, which generally disrupts bacterial cells.

View larger version (94K): [in this window] [in a new window]   FIG. 1.   Electron micrographs of ultrathin sections of Chroococcidiopsis strain (29)N6904 stained with periodic acid-thiosemicarbazide-silver proteinate for 2 h, for detection of polysaccharides (26). (A) Chroococcidiopsis cell from a 4-month-old culture surrounded by a thick electron-dense envelope with a division septum visible in the cytoplasm. (B) Chroococcidiopsis cell from an 8-month-old culture showing a thick multilayered envelope with a bacterium inside (arrow). Bars, 0.5 µm.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Protocol for DNA extraction from Chroococcidiopsis.

Three preliminary washes of a 50-ml sample of the Chroococcidiopsis culture (10⁸ cells/ml) with sterile water and centrifugation at 200 × g for 5 min reduced the bacterial contamination to about 1% of the total Chroococcidiopsis cells, suggesting that most bacteria were present in the outer layers of the Chroococcidiopsis envelope. Indeed, the presence of bacteria within the Chroococcidiopsis envelope has been observed (Fig. 1B).

Chroococcidiopsis cultures were then resuspended in 1/15 initial volume of SM (0.1 M NaCl, 16 mM MgSO₄, 50 mM Tris hydrochloride [pH 8.0]) containing 10 mg of lysozyme (Sigma Chemical Co., St. Louis, Mo.) per ml and incubated at 37°C for 1.5 h. Cells were pelleted by centrifugation at 200 × g and subjected to the osmotic shock of three washings with sterile water. This treatment reduced contamination to about 0.01% of the total Chroococcidiopsis cells.

The integrity of Chroococcidiopsis cells was evidenced by the absence of phycobiliproteins in the absorption spectrum of the aqueous supernatant recovered after the osmotic shock, whereas cell lysis occurred in Anabaena cylindrica, used as a control (Fig. 3). After this treatment Chroococcidiopsis cells exhibited their typical morphology under the light microscope.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Absorption spectra of the supernatants recovered from about 108 cells of Chroococcidiopsis strain (29)N6904 and Anabaena cylindrica Lemm after lysozyme treatment and osmotic shock. The absence of phycobiliproteins () indicates the cell integrity of Chroococcidiopsis; lysis occurred in Anabaena cylindrica (---). Supernatant recovered from A. cylindrica was diluted 15-fold.

In order to eliminate DNA from the bacterial lysate, we resuspended Chroococcidiopsis cells in 1.5 ml of 4 mM MgSO₄ containing 50 µg of DNase I (Sigma) per ml and incubated them at 37°C for at least 45 min. The efficiency of this treatment was evaluated with a 4-month-old Chroococcidiopsis culture labeled with [³H]thymidine (1 µCi/ml) for 2 days at 37°C.

After DNase I treatment the remaining radioactivity that could be precipitated with ice-cold 10% trichloroacetic acid (TCA) was about 2% of the initial value. This value included ³H-labeled DNA of Chroococcidiopsis, extracted as reported below. Briefly, after labeling, 100-µl samples were incubated with 0.9 ml of 10% TCA on ice for 45 min. Tritiated materials were then filtered onto membrane filters, the filters were washed once with ice-cold 1% TCA and three times with 95% ethanol, and radioactivity was counted in 5 ml of scintillation fluor.

DNA extraction. Chroococcidiopsis cells were lysed by the method of Hoffman and Winston (13), modified as follows: 1.5 ml of phenol saturated with 0.1 M Tris hydrochloride (pH 7.4) and glass beads (20% [vol/vol], 0.5-mm diameter) were added to 1.5 ml of DNase I-treated cells. Four 2-min cycles of heating at 65°C and vortexing for 30 s were performed. Then 1/5 volume of TE buffer (1 mM EDTA [pH 8.0], 10 mM Tris hydrochloride [pH 7.4]) was added to the mixture.

Cell debris and glass beads were eliminated by centrifugation at 12,000 × g for 5 min, and the organic phase was extracted once with TE. The pooled aqueous phases were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). Nucleic acids were precipitated with cold ethanol from the final phase, after the addition of sodium acetate (pH 4.5) to a 0.3 M final concentration, by overnight incubation at 20°C. The yield was about 100 µg of DNA from 10¹⁰ Chroococcidiopsis cells.

DNA purification. Genomic DNAs obtained from Chroococcidiopsis strains were a good substrate for PCR, as reported below, but were resistant to a wide range of restriction endonucleases.

A further DNA purification was achieved by cesium chloride (CsCl) density gradient ultracentrifugation according to standard procedures (23). The density of DNA was 1.7 g/cm² (24). The gradient was collected from the bottom in 0.4-ml fractions, and those containing DNA were identified by spotting of 2-µl samples from each fraction on 1% agarose gel containing ethidium bromide (0.5 µg/ml) and examined under UV light. The DNA-containing fractions, corresponding to the fluorescent samples, were pooled and dialyzed against TE.

As shown in Fig. 4A, after purification, DNA was successfully digested with EcoRI and HindIII for 4 h at 37°C, and similar results were obtained with PstI, SalI, and XbaI in all strains (data not shown).

View larger version (107K): [in this window] [in a new window]   FIG. 4.   (a) HindIII (lane 1) and EcoRI (lane 2) digestions of CsCl-purified DNA from Chroococcidiopsis strain (29)N6904. Lane 3, 1-kb DNA ladder (Gibco BRL). (b) Southern transfer of gel in panel A. The DNA blot was hybridized with the 570-bp fragment of ftsZ. Lanes: 4, 6-kb EcoRI fragment; 5, 1-kb HindIII fragment.

Detection of the ftsZ gene in Chroococcidiopsis. A set of degenerate oligonucleotide primers (5'-AATGCYGTTAACCGSATGATT-3' and 5'-GCCYKYACRTCWGCAAARTC-3') from the conserved regions of the Anabaena sp. strain 7120 and Arabidopsis thaliana genes (EMBL database accession no. T22504 and Z31371) were used to carry out PCR from genomic DNA of Chroococcidiopsis strains (48)N6911B, (29)N6904, and (568)G91-19.

Amplifications were done in a Hybaid Omn-E thermal cycler under the following conditions: 3 min of denaturation at 94°C followed by 5 cycles of amplification with a 1-min denaturation at 94°C, 1 min of annealing at 40°C, and 1 min of extension at 72°C. Then 25 cycles of amplification with a 1-min denaturation at 94°C, 1 min of annealing at 53°C, and 1 min of extension at 72°C were performed. An extra extension step of 5 min at 72°C was added after completion of the 25 cycles.

As a control for PCR conditions, the same oligonucleotides were used to amplify the 570-bp fragment of ftsZ from DNA of A. cylindrica extracted as described by Porter (19).

In A. cylindrica, a fragment of about 570 bp was amplified, and one major DNA fragment of the expected size was amplified in each of the Chroococcidiopsis strains (48)N6911B, (29)N6904, and (568)G91-19. The same PCR products resulted from DNAs purified by CsCl density gradient ultracentrifugation (data not shown).

The 570-bp PCR product obtained from Chroococcidiopsis strain (29)N6904 was cloned into the HincII site of the pUC18 vector according to standard procedures (23). Both strands of the fragment were sequenced with the ABI PRISM dye terminator cycle-ready reaction kit and ABI PRISM 310 genetic analyzer (Perkin-Elmer).

DNA and protein sequence comparisons were done with University of Wisconsin Genetics Computer Group programs (7), and sequence alignments were optimized with the FASTA program (15). The deduced amino acid sequence of the 570 bp of the ftsZ gene of Chroococcidiopsis showed identities of 88.7% with that of Anabaena sp. strain PCC 7120 and 63.9% with that of Arabidopsis thaliana.

Southern hybridization. Because of the DNA sequence homology between the PCR product obtained in Chroococcidiopsis and ftsZ, we expected to detect this gene in Chroococcidiopsis by using the 570-bp fragment as an [-³²P]ATP-labeled probe in Southern analysis.

DNA was extracted from Chroococcidiopsis strain (29)N6904, purified by CsCl density gradient ultracentrifugation, digested with EcoRI and HindIII, and transferred onto Hybond-N filters. The filters were incubated for 4 h at 42°C in prehybridization solution (50% [wt/vol] formamide, 5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.5% [wt/vol] sodium dodecyl sulfate [SDS], 5× Denhardt solution, 0.1 mg of salmon sperm DNA ml¹). The filters were then incubated in the same solution after the addition of the heat-denatured ³²P-labeled DNA probe (30 ng). After hybridization, the filters were washed twice in 2× SSC-0.5% (wt/vol) SDS and then twice in 0.2× SSC-0.1% (wt/vol) SDS for 15 min at 65°C. Filters were exposed to X-ray films, and two bands, corresponding to the 6-kb EcoRI fragment and 1-kb HindIII fragment, were detected (Fig. 4B).

Conclusions. The protocol for DNA extraction described here is based on the resistance of Chroococcidiopsis to lysozyme and represents a simple and efficient DNA purification method that overcomes problems of bacterial contamination and the difficulty of lysing cyanobacterial cells. Bacterial contamination was reduced at least by 3 orders of magnitude by lysozyme treatment followed by osmotic shock. Then DNase treatment was performed in order to eliminate DNA from the bacterial lysate.

Resistance to lysozyme is a feature shared by some unicellular and filamentous cyanobacteria and is associated with envelope thickness (1, 24). For example, our method caused Anabaena cylindrica cells to lyse; Anabaena vegetative cells, unlike heterocysts, lack additional envelope layers and are susceptible to lysozyme (5).

Our method uses phenol and glass beads to overcome the resistance of Chroococcidiopsis to complete cell lysis. It overcomes the purified DNA's resistance to hydrolysis by further purification, i.e., ultracentrifugation on a cesium-chloride density gradient. Successful hydrolysis with different restriction endonucleases suggested that unlike N. commune DNA (14), the DNA of Chroococcidiopsis might be not highly methylated.

The development of this method permitted, for the first time, the identification and cloning of a gene fragment from Chroococcidiopsis. The identified fragment showed a significant homology with ftsZ, and its presence in this cyanobacterium lends support to the suggestion that the Z ring is a cytoskeletal element used by prokaryotes to carry out cell division, despite their morphological and evolutionary diversity (16).

This work represents a first step in meeting the formidable challenge of elucidating the molecular biology of Chroococcidiopsis, and the method developed here may further investigations of this difficult-to-study cyanobacterium.

	ACKNOWLEDGMENTS

We thank E. I. Friedmann for providing us with Chroococcidiopsis strains.

This work was supported by a grant from the Ministero dell' Università e della Ricerca Scientifica e Tecnologica (Murst 60%).

	FOOTNOTES

^* Corresponding author. Mailing address: Dipartimento di Biologia, Università di Roma "Tor Vergata," Via della Ricerca Scientifica, 00133 Rome, Italy. Phone: 39-6-72594344. Fax: 39-6-2023500 E-mail: maria.grilli{at}uniroma2.it.

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Applied and Environmental Microbiology, October 1998, p. 4053-4056, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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