A simple and efficient method for releasing DNA from Dermatophilus congolensis

Journal of Microbiological Methods 33 (1998) 197–202

Journal of Microbiological Methods

A simple and efficient method for releasing DNA from Dermatophilus congolensis a, b A.A. Makinde *, C.L. Gyles a

b

National Veterinary Research Institute, Vom, Plateau State, Nigeria Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario, Canada N1 G 2 W1 Received 10 October 1997; received in revised form 19 March 1998; accepted 19 March 1998

Abstract A modification of a combination of various techniques for extracting DNA from Actinomycetes and other Gram-positive bacteria was adapted for releasing DNA from Dermatophilus congolensis. This was based on pre-treatment of whole cells of Dermatophilus congolensis with acetone followed by further treatment in a solution containing achromopeptidase, lysozyme and ethylenediamine tetracetic acid (EDTA) before the final exposure to sodium dodocyl sulphate (SDS) for complete lysis. This protocol resulted in good lysis leading to sufficient yield and satisfactory recovery of chromosomal DNA from Dermatophilus congolensis. Agarose gel electrophoresis of DNA revealed one to seven discrete bands of extrachromosomal DNA which were observed in six isolates. This procedure which provides recovery of good quality total genomic DNA should pave the way for studies involving genetic analysis of Dermatophilus congolensis including genotypic typing.  1998 Elsevier Science B.V. All rights reserved. Keywords: DNA extraction; Recovery of total genomic DNA; Dermatophilus congolensis

1. Introduction Dermatophilus congolensis is a typical gram-positive actinomycete which forms branching hyphae at some stage of its development and also produces spore-bearing mycelia. It is of major veterinary and economic importance in tropical climates, where it causes chronic epidermatitis (dematophilosis) in livestock such as cattle, sheep, and goats and other domesticated animals including horses and donkeys. Although there have been several reports of differences among the species with respect to cultural, morphological, biochemical properties (Ellis et al., 1993a), genetic diversity (Skalka and Pospisil, 1992; *Corresponding author.

Faibra, 1993; Trott et al., 1995), pathogenicity (Ellis et al., 1992, 1993b) and immunological reactions (Makinde and Majiyagbe, 1982; Makinde and Ojo, 1986, 1987; How and Lloyd, 1988, 1990; Ellis et al., 1991; Sutherland et al., 1991; Skalka and Pospisil, 1993) there is still need for proper characterization of isolates of the genus Dermatophilus. Morphological polymorphism exhibited by this bacterium coupled with its wide host range, variable growth rates and morphology in liquid or solid media, and its ubiquity in various geographical zones of the world suggest the need for its proper phenotypic and genotypic characterization as a pre-requisite for vaccine development. Dermatophilus congolensis possesses a chemotype III cell wall which it shares with Nocardioform

0167-7012 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0167-7012( 98 )00034-7

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actinomycetes (Goodfellow and Pirouz, 1982). Although studies by Gordon (1964), Richard et al. (1967) and Roberts (1970), based on morphological features and cultural properties suggest that Dermatophilus congolensis is a relatively homogeneous species, recent studies have consistently indicated the heterogenicity of the organism (Ellis et al., 1992, 1993a,b; Skalka and Pospisil, 1992, 1993; Faibra, 1993; Trott et al., 1995). With the advent of new technologies, it is now desirable to explore other methods of characterization. Lack of a procedure for release of high molecular weight DNA from any organism can hinder its genetic studies hence this study was aimed at developing a suitable technique for isolating deoxyribonucleic acid (DNA) from Dermatophilus congolensis, particularly since difficulties are often encountered in the extraction of DNA from Actinomycetes generally (Crameri et al., 1983; Dobritsa, 1984; Loeffelholz and Scholl, 1989). This is an essential first step in studies aimed at characterizing isolates by restriction endonuclease fingerprinting and in exploring association of surface antigens and exported bacterial products with the genes which encode them.

subjected to acetone pre-treatment as described by Heath et al. (1986). About 200 mg of acetone-treated cells were suspended in 5 ml of buffer containing 0.15 M Tris (pH 8.0), 0.45 M sucrose, 8.0 mM EDTA and 10 mg / ml (w / v) of achromopeptidase (Sigma Chemical Co. St. Louis, Mo.) and 10 mg / ml (w / v) of lysozyme (Boehringer Mannheim Gumbtl, Biochemical, Germany) and incubated at 378C with continuous shaking for 2 h as described by Barsotti et al. (1987). The mixture was then subjected to sodium dodecyl sulphate (SDS) lysis based on the method adopted by Serwold-Davis and Groman (1986) which involved the addition of 0.8 ml of 50 mM Tris-0.25 mM EDTA (pH 8.0) and 0.4 ml of 20% SDS in 50 mM Tris-20 mM EDTA, pH 8.0; mixing by several gentle inversion of tubes; and finally incubation at 608C for 1 h. Proteinase K (70 mg / ml in 1 mM EDTA) was added to each tube and the contents were incubated overnight at 378C with continuous shaking. The mixture was then centrifuged and the supernatant (lysate) collected and divided into two equal volumes. One volume of lysate was subjected to techniques for chromosomal DNA extraction while the second was used for extrachromosomal DNA extraction.

2. Materials and methods

2.3. Chromosomal DNA preparation

2.1. Organisms and conditions of growth

Chromosomal DNA was purified by the technique described by Loeffelholz and Scholl (1989). The lysate was divided into 1 ml aliquots and to each was added 0.3 ml of 5 M sodium perchlorate, followed by equal volume of chloroform–isoamyl alcohol (24:1). The mixture was shaken gently on a Nurator shaker (Adams, BDC, NJ) for 15 mins at room temperature and then centrifuged for 2 min at 12,000 rpm in a microcentrifuge (Brinkman Instruments, NY). The aqueous phase was collected and extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1). The nucleic acids were precipitated by the addition to the aqueous phase of 2.5 volumes of cold 95% ethanol and the mixture was kept at 2708C for 1 h. The precipitate was pelleted by centrifugation at 12,000 rpm, washed with cold 70% ethanol and air-dried. The dried pellet was suspended in 200 ml of 10 mM Tris HCl buffer, pH 7.5 containing 1 mM EDTA (Tris EDTA), then treated with RNase (0.2 mg / ml) and protease (70

Twenty isolates of Dermatophilus congolensis, twelve from Nigeria, three from Canada, two from the United States of America and three from Australia were investigated. Each isolate was cultured in 50 ml of Brain heart infusion broth (Difco, Detroit, Mi.) at 378C in 5% CO 2 under humidified conditions for 72 h. The cultures were harvested by centrifugation at 70003g for 15 min, washed twice with sterile distilled water and sedimented by centrifugation.

2.2. Lysis of Dermatophilus congolensis Preliminary studies compared several routine techniques for DNA extraction from different bacteria and a protocol was designed for maximum yield of total DNA from Dermatophilus congolensis as follows: Washed whole cells of Dermatophilus congolensis isolates from broth cultures were initially

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mg / ml) for 1 h at 378C. The DNA preparation was extracted once with phenol–chloroform–isoamyl alcohol, then once with chloroform–isoamyl alcohol, and finally precipitated by addition of 95% ethanol as described earlier. The precipitate which contained large quantity of chromosomal DNA was dissolved in 50 ml of Tris EDTA and stored at 2208C.

2.4. Extrachromosomal DNA preparation To purify extrachromosomal DNA the second volume of lysate was treated as described by Serwold-Davis and Groman (1986). A 5 ml volume of the mixture was treated with the addition of 0.4 ml of freshly prepared 3 N NaOH and the tubes gently shaken for 10 min before the addition of 0.5 ml of 2 M Tris pH 7.0 followed by further mixing for another 5 min. To each tube 1 ml 5 M NaCl was added and the contents were mixed for 1 min before cooling on ice for 1 h. Following cooling the mixture was centrifuged at 70003g for 5 mins to remove chromosomal DNA precipitate. The supernatants were divided into 0.5 ml aliquots and an equal volume of phenol with 0.1% 8-hydroxyquinoline saturated with 3% NaCl was added. The tubes were mixed gently for 5 min and centrifuged at 12 000 rpm for 2 min. An equal volume of chloroform– isoamyl alcohol (24:1) was added to the plasmidcontaining aqueous phase and the contents of the tube were mixed for 5 min and centrifuged at 12 000 rpm. The aqueous phase was collected and one-tenth volume of 3 M sodium acetate added followed by one volume of isopropyl alcohol. The mixture was mixed gently and kept at 2208C overnight for precipitation of plasmid DNA. The precipitate was removed by centrifugation at 12,000 rpm for 2 min and allowed to air-dry before suspension in 50 ml Tris EDTA (Tris 5 mM, EDTA 0.5 mM, pH 8.0).

2.5. Restriction enzyme digestion of DNA from Dermatophilus congolensis Chromosomal DNA samples from selected isolates were digested with the Restriction endonuclease EcoRI under the conditions specified by the Manufacturer (Bethesda Research Laboratories Inc., Burlington, Ont.). Extrachromosomal DNA preparations were heat-treated for 2 min at 1008C in the presence

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of 0.1% Sarkosyl solution followed by fast cooling on ice as described by Goyal (1992) before digestion with the enzyme. Chromosomal DNA from all isolates and those from selected isolates were subjected to electrophoresis in 0.8% (w / v) Agarose (Sigma Chemical Co. St. Louis, MO) in standard Tris-borate buffer. Lambda Hind III restriction fragments and plasmids of E. coli V517 (Marcina, 1978) served as molecular weight markers for chromosomal and plasmid DNA respectively. The gels were stained in 0.5 mg / ml ethidium bromide for 15 min and DNA bands were visualized by observation on a UV transluminator (Spectroline TR-302, Ultra-Violet Products Inc. Ca). Gels were photographed with a Polaroid MP-3 Land Camera through a Kodak No 22 orange filter.

3. Results Dermatophilus congolensis was found to be resistant to lysis by either lysozyme or achromopeptidase when these were applied singly or even following pretreatment of cells by acetone. Dermatophilus congolensis however, became more susceptible to lysozyme after their pre-treatment with acetone and achromopeptidase. Certain isolates were, however, more susceptible to lysis than others particularly those that showed mucoid and less deep-seated colonies on solid media. All 20 isolates of Dermatophilus congolensis showed characteristic dense accumulation of chromosomal DNA. Six of the isolates showed additional bands interpreted to be extrachromosomal DNA bands. Typical chromosomal DNA bands are shown in Fig. 1 while Fig. 2 shows the appearance of two isolates of Dermatophilus congolensis following electrophoresis of EcoRI digested DNA. Fig. 3 shows the presence of plasmid DNA in selected isolates.

4. Discussion Actinomycetes, the group of bacteria to which Dermatophilus congolensis belongs, are known to be among the most difficult organisms from which to extract DNA because of their resistance to lysing

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Fig. 1. Agarose gel electrophoresis of chromosomal DNA isolated from eight (8) representatives of Dermatophilus congolensis. The isolates in each lane are as follows: 1,V-5; 2, V-18; 3, NY-1; 4, Sp-2; 5, NY-3; 6, V-19, 7, V-14 and 8, G-2. C denotes chromosomal DNA bands while arrows indicate extrachromosomal DNA.

enzymes and agents and their high level of nuclease activity. (Coykendall and Munzenmaiez, 1979; Crameri et al., 1983; Dobritsa, 1984; Kudo et al., 1988; Loeffelholz and Scholl, 1989). It was therefore not surprising when it was found that routine techniques used for lysing Gram-positive bacteria and even some Actinomycetes did not prove satisfactory with Dermatophilus congolensis. Robinson et al. (1979) and Rush et al. (1975) showed that treatment of Staphylococcus aureus cells with acetone and then with alcohol did not affect any of the biochemically and biologically relevant properties of DNA. Heath et al. (1986) also observed that electropheretic analysis of such treated cells revealed the presence of chromosomal DNA and at least six discrete bands of extrachromosomal DNA following the release of

Fig. 2. Agarose gel electrophoresis of EcoR1 restriction endonuclease digest of DNA extracted from Dermatophilus congolensis isolates A,V-2 and B, V-5. E. coli V157 fragments as molecular weight standards are shown in Lane C.

DNA from lysozyme-digested cells after acetone extraction. Restriction endonucleases analysis (REA) of the DNA from different isolates of Dermatophilus congolensis was recently used to resolve them into 4 distinct groups for the enzyme Apa I, 5 groups for Bam HI and 6 groups for Pvu II (Ellis et al., 1993a). Masters et al. (1995) also used REA to distinguish 3 isolates of Dermatophilus chelonae sp. nov from 30 isolates of Dermatophilus congolensis while Faibra (1993) identified 6 ribotypes of Dermatophilus congolensis based on hybridized DNA patterns. Thus any technique that would lead to increase yield of genomic DNA from Dermatophilus congolensis

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Fig. 3. Agarose gel elctrophoresis of extrachromosomal DNA isolated from six (6) isolates of Dermatophilus congolensis; 1, NY-3; 2, V-18; NY-1; 4, SP-4; 5, G-2 and 6, V-5. Arrows indicate plasmids while C indicates chromosomal DNA.

would definitely enhance the development of a DNA for Dermatophilus congolensis. Presently DNA probe diagnostic kits for a number of organisms including mycobacteria, mycoplasma, Staphylococcus aureus, Epstein–Barr virus and Herpes virus etc. are available in the market (Pasternak, 1988). In this study it was found that pre-treatment of Dermatophilus congolensis cells with acetone (Heath et al., 1986) followed by treatment with achromopeptidase and digestion by lysozyme prior to lysis with SDS as described for Actinomycetes (Barsotti et al., 1987) and Streptomycetes and Micromonospora (Ogawa et al., 1983) dramatically improved the yield of DNA from Dermatophilus congolensis. Isolation of purified DNA was carried out by purification steps described by Loeffelholz

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and Scholl, 1989). This successful attempt at isolation of DNA from Dermatophilus congolensis should encourage further studies on genomic relationship within the genus, Dermatophilus. The detection of extrachromosal DNA following the adopted extraction process further confirmed the suitability of this technique as previously adopted methods showed that extrachromosomal DNA was not always readily recognizable in Actinomycete DNA preparations (Crameri et al., 1983). The presence of extrachromosomal DNA in many isolates of Dermatophilus congolensis indicated that the Genus, Dermatophilus, may possess plasmids which could prove very useful for its characterization. However, failure to observe plasmid bands in some isolates may, however, be due to either absence of plasmids in these isolates or due to other yet unidentified factors. These factors may include nuclease activities associated with actinomycetes (Dobritsa, 1984) which may differ from isolate to isolate, the number of passages of the isolates in culture, storage and even treatment during preparation (Okanishi et al., 1980). The presence of the extra-chromosomal DNA was not related to either the geographical or host sources of the Dermatophilus congolensis isolates. Extrachromosomal genetic elements in Dermatophilus congolensis may prove useful in determining a range of properties ascribed to other Actinomycetes such as antibiotic resistance, antibiotic production, fertility, cellular differentiation and melanin production (Dobritsa, 1984). Of even greater interest is the possibility that these plasmids may encode virulence determinants in Dermatophilus congolensis.

Acknowledgements We wish to thank Jinru Chen, John Dennis and Jutta Hammermueller for their technical assistance and Paul Huber for his assistance with photography. The Dermatophilus congolensis isolates sere kindly supplied from Australia by S.S. Sutherland (SP-2 / 129 and Sp-4 / 429), from Canada by D.A. Barnum (G2 / 32CI, G-3 / 5C3 and G5 / 32D1) and from the United States of America by W.A. Samsonoff (NY1 /A-21 and NY-3 /A-33). The Nigerian isolates were provided by Dr. A. A. Makinde of the National

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Veterinary Research Institute, Vom, Nigeria. This work was carried out in the Department of Veterinary Microbiology and Immunology University of Guelph, Guelph Ontario, CANADA N1G 2W1.This study was supported by a grant from CIDA / NSERC, Canada. The release of Dr. A. A. Makinde by the Director, National Veterinary Research Institute, Vom to utilize the Research Associateship is highly appreciated.

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