A rapid method for the base ratio determination of bacterial DNA

ANALYTICAL,

BIOCHEMISTRY

81, 461-466

A Rapid Method Determination

t 1977)

for the Base Ratio of Bacterial DNA

A rapid. simple, and inexpensive procedure is described for the isolation and base analysis of bacterial deoxyribonucleic acids (DNA) in samples of 0.2 mg. The isolation of DNA utilized hydroxyapatite and Sephadex columns. Following nuclease digestion of DNA samples, the 5’-deoxymononucleotides were separated on a high-pressure Dowex 50 column; base ratios of the Dowex eluate were determined with a recording spectrophotometer equipped with a peakintegrating device. Total processing time per bacterial genome. from cell harvesting to base ratio quantitation. was IO- 12 hr.

In establishing the taxonomic position of bacterial isolates of unknown status, the determination of the genomic A + T/G + C ratios is a \:$uable parameter. Although DNA base ratios have been used widely as an analytical probe, none of the reliable methods meet the need to determine the base ratios of several hundred strains of bacteria conveniently. We have developed a simple, rapid, and accurate procedure, appropriate for numerous determinations over a relatively short time period. Methods currently used for DNA base analyses require either undenatured moderately intact molecules of DNA prepared by the somewhat lengthy Marmur method (1). costly instrumentation and time-consuming procedures at the base-analysis stage, or a combination of both. Melting profiles (2), while relatively rapid, require very careful preparation of native DNA: traces of proteins, RNA, polyamines, inorganic cations (2). or counterion-type polysaccharides from various bacterial strains (3,4) may significantly distort T,, values. The use of CsCl densitygradient determinations, on the other hand, requires costly instrumentation (5) and is not easily applicable to a large number of samples. Methods involving high-pressure liquid chromatography (6,7) while rapid and sensitive, are also costly in instrumentation. In our method, rapid isolation and purification of bacterial DNA is accomplished by a slight modification of Britten’s hydroxyapatite column procedure (8) as adapted by Meinke et al. (9) and Markov and Ivanov (10). Separation and quantitation of the 5’-deoxyribomononucleotides produced by nuclease digestion of the DNA is achieved by the procedure of Blattner and Erickson (II), as modified by Junowicz and Spencer (12), which uses Dowex 50 columns. By these procedures, the determination of the base composition of one bacterial strain. from cell harvest to final results, can be accomplished in lo- 12 h, and a number of strains can be carried through each phase of processing simultaneously. 461

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MATERIALS

The strains of bacteria used either were reference strains obtained from the American Type Culture Collection (ATCC) or were isolated from the Saint John River and tenatively identified by numerical taxonomy (13). Other bacterial DNAs used as standards and 5’-deoxymononucleotides were from the Sigma Chemical Co., St. Louis, MO.; hydroxyapatite (Bio-Gel HTP) powder and ion-exchange material (AG SOW-X4, minus 400 mesh) were from Bio-Rad Laboratories; pancreatic DNase I and snake venom phosphodiesterase were from Worthington Biochemical Corp.; Trypticase Soy Broth (TSB) was from Baltimore Biological Laboratories (used at a concentration of 15g/liter, half the normal strength). METHODS

The TSB was dispensed inlOO-ml aliquots in 500-ml erlenmeyer flasks and was inoculated with 10 ml of a 16-hr culture of each organism which had been grown in the same medium. Each flask was incubated for 6 hr at 3o”C, with agitation at 300 rpm, in a New Brunswick Co. Psychrotherm controlled-environment incubator. Preparution oj’bacterial cell suspension. Cells from each culture (110 ml) were harvested by centrifugation at 4100g at 5°C for 10 min, and the pellet was resuspended in 10 ml of MUP buffer: 8 M urea, 0.12 M Na,HPO,, and 0.12 M NaH,PO, (8). The cells were disintegrated in a French Press (American Instrument Co. 19099-5) at 20,000 psi and then were examined by phase-contrast microscopy to determine the number of intact cells. If required, the suspension was treated a second time in the French Press. Preparation oj’ hydroxyapatite column urrd suspension. A column of hydroxyapatite (Bio-Rad DNA-grade Bio-Gel HTP) was prepared by suspending 0.8 g of the Bio-Gel in 9 ml of 0.01 M sodium phosphate buffer (0.005 M Na,HPO,, 0.005 M NaH,PO,) at pH 6.8 in a SO-ml erlenmeyer flask and allowing it to settle for 10 min. The clear upper layer was decanted. The process was repeated, and the precipitate was resuspended and allowed to settle. The clear fluid was decanted and resuspended in 9 ml of the phosphate buffer. A 20-ml plastic syringe was fitted with tubing at the needle attachment, and the opening was plugged with glass wool. The hydroxyapatite suspension was poured into the syringe which was clamped in the vertical position. The column was washed with 10 ml of MUP and was then ready for use. A suspension of hydroxyapatite was prepared in the same manner as the column by suspending 1.5 g of the Bio-Gel HTP twice in 18 ml of the phosphate buffer. This suspension was decanted, resuspended in 30 ml of MUP, and Preparation

of bacterial

culture.

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decanted again. The bacterial cell-free extract was added to this preparation of hydroxyapatite. Zsolntion of DNA. The cell-free extract-hydroxyapatite mixture was kept at room temperature for I hr and was swirled occasionally for the first half-hour. The suspension was allowed to settle, and the supernatant was decanted. The hydroxyapatite was poured into columns and was washed first with 30 ml of MUP, followed by 30 ml of 0.014 M sodium phosphate buffer (0.007 M Na,HPOJ. 0.007 M NaH,PO,, pH 6.8), and the DNA was eluted with 15 ml of 0.40 M phosphate (0.20 M Na,HPOA, 0.20 M NaH,PO,, pH 6.8). The solution was concentrated on a flash evaporator to a small volume (ca. I ml) and was desalted rapidly on a Sephadex G-SO column (medium, 2 x 33 cm, equilibrated with aqueous ammonia. pH 8). The DNA-containing fractions were reconcentrated on a flash evaporator to a concentration of about 2 mgiml and were subjected to enzyme digestion. Alternatively, when multiple DNA samples were worked up simultaneously and the DNA was to be stored for a long time, the DNA eluted from the hydroxyapatite was treated as follows. The DNA solution was dialyzed in distilled water for 18 hr at 4°C. and the quantity of DNA was determined at 260 nm. The mixture was adjusted to 0.2 M with respect to NaCl, and the DNA was precipitated with 2 vol of absolute ethanol at -20°C. The DNA was centrifuged at 10,OOOg at 4°C. The precipitate was washed with ether and dried under vacuum. Digestion of DNA. The digestion mixture, a total volume of 0.5 ml containing 1 mg of DNA, 36 mM sodium acetate, pH 6.5, 15 mM MgCl,, and 200 pg of pancreatic DNase. was incubated at 37°C for 2 hr: the pH of the pancreatic DNase digest was adjusted to 8.5 with a 1% NH, solution, snake venom phosphodiesterase (SVPD) was added to a concentration of 53 pg/ml, and the mixture was incubated for 2 hr at 37°C. The digest was either immediately applied to the ion-exchange column (ca. 0.2 mg of DNA) or, alternatively, was divided into 0.2-mg aliquots and was frozen. The SVPD was pretreated according to Sulkowski and Laskowski ( 14). DNA analysis. A column of cation-exchange resin (AG 5OW-X4, minus 400 mesh) was prepared according to Junowicz and Spencer (12); the column measured 45 x I. I cm (i.d.) and was washed at the rate of 1 ml/min with 0.1 M NH,HCO, (pH 3.2) buffer. The column was calibrated with 5’-deoxyribomononucleotide standards. The digest aliquctapplied to the column contained 0.2 mg of DNA. The water-jacketed column was maintained at a constant temperature of 20°C with a Lauda K-2RD Thermostatic water pump. RESULTS

Table common

AND DISCUSSION

1 shows the G + C content of the DNA from a number of bat erial strains the base compositions of which have already

464

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1

BASE RATIO DETERMINATIONS OF BACTERIAL DNAs G + C (%) Bacterium Escherichia co/i E. coli Micrococcus /.vsodeikricus Clostriditcm perfringens Pseudomonas Jluorescens’ P. aeruginosa’ P. alcaligenes’ P. putidap P. fluorescens Biotype P. jluorescens Biotype P. stuzerid

Bd E”

Literature”

Experimental*

49.8-52.5 49.8-52.5 71.2-72.0 26.5-30.9 60.5 57.1-68.4 66.3 62.5 59.4-61.3 63.6 60.07-66.3

51-52.5 50-52.2 73.7 23 60-61.5 68.8 64.8-65.2 61.8-62.9 57.58-64.55 60.2 60.08

U Handbook of Microbiology (1973) (Laskin. A. I.. and Lechavalier. H. A.. eds.), Vol. 2. CRC Press. Cleveland, Ohio. * Values based on three to eight determinations; less than 2%) variation between samples was generally observed. r ATCC strains. d Natural isolates identified by numerical taxonomy.

been determined by a variety of other well-established chemical and physical methods. In all cases, good agreement is found between the literature values and those determined by the present method. Table 1 also shows some preliminary base ratio determinations from a number of bacterial strains characterized by numerical taxonomy. Chart 1 provides a summary of the sequential steps involved in our base-analysis method together with an indication of processing time. In CHART 1 SEQUENCE OF STEPS AND TIME REQUIRED FOR THE DETERMINATION OF BACTERIAL

BASE RATIOS

Cell harvest and lysis: 2 hr DNA isolation with hydroxyapatite:

3 hr

5 DNA desalting with sephadex: 2 hr DNA digestion with pancreatic DNase and SVPD: 3 hr Deoxyribomononucleotide

1 separation and quantitation:

1.5 hr

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practice, multiple samples were processed at each stage in the sequence. The procedure was especially advantageous at the ion-exchange column stage, as it was observed that the base ratio data was most reproducible if the column was run on a continuous schedule. The type of column used is particularly suitable for continuous use, since there is no requirement for gradient elution and the subsequent need for regenerating the ionexchange resin. Under normal operating conditions, such a column may be run for 2 months without repacking. Another advantage of this type of column is that all four deoxynucleotides are measured and, hence, serve as internal checks against each other. In contrast, other chemical methods of base ratio determination only measure purines (3) or pyrimidines plus guanine (15). Apart from reducing random experimental error in the calculation of G + C base ratios, the monitoring of all four bases could be significant because the commercial preparations of pancreatic DNase I and/or venom phosphodiesterase can be contaminated with adenosine deaminose (6), in which case the 5’-deoxyinosinic acid produced could be mistaken for 5’-deoxyguanylic acid in some assay systems.’ In view of the difficulty of directly applying the Britten method for DNA isolation (8) to the isolation of a particular plant DNA, it appears from our own experience that no substantial problems are involved in adapting this very facile technique to bacterial cells. By avoiding intermediate precipitation steps [in contrast to Marmur’s method ( 1)], the Britten method (8) insures that a representative fraction of the entire bacterial genome is being isolated rather than some arbitrary subfraction(s) (8). ACKNOWLEDGMENTS The authors wish to thank Dr. John Spencer. McGill University. Montreal. for his suggestion that the Blattner column might be useful and for allowing Dr. Cashion to observe it in his laboratory. This work was supported by a grant from the Inland Waters Directorate of the Department of the Environment. Canada.

REFERENCES I. Marmur. J. (1961) J. Mol. Biol. 3, 208. 11. Mandel. M.. and Marmur. J. (1968) in Methods in Enzymology (Grossman, L.. and Moldave. K.. eds.), Vol. 12. p. 194. Academic Press, New York. 3. Broughton, W. J., Dilworth. M. J., and Passmore. I. K. (1972) And. Bioch~m. 46, 16‘G 172. 4. DeLey. J.. Cattoir. H., and Reynaerts. A. (1970) E(dr. J. Bioc1renf. 12, 133. 5. Mandel, M.. Schildkraut, C. L.. and Marmur. J. (1968) in Methods in Enzymology (Grossman, L.. and Moldave. K.. eds.). Vol. 12. pp. 184-195, Academic Press, New York. 6. Duch, D. S.. and Laskowski. M.. Sr. (1971) Antrl. Biocl~enz. 44. 42-48. 7. Breter. H. J.. and Zahn, R. K. (1973) AM/. Bir~hrm. 54, 346-353.

’ P. Cashion.

unpublished

observations

466

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8. Britten,

R. J., Pavich.

M.,

and

COMMUNICATIONS Smith,

J. (1969)

Carnegie

Inst.

Washington

Yearb.

68,400-402. 9. 10. 11. 12. 13. 14. 15.

Meinke. W.. Goldstein, D. A.. and Hall, M. R. (1974) Anal. Biochem. 58, 82-88. Markov. G. G., and Ivanov, I. G. (1974) Ann/. Biochem. 59, 555-563. Blattner. F. R.. and Erickson, H. P. (1967) Anal. Biochrm. 18, 220-227. Junowicz, E., and Spencer, J. H. (1969) J. Chronzatogv. 44, 342-348. Kaneko, T.. Holder-Franklin, M. A.. and Franklin, M., manuscript in preparation. Sulkowski. E.. and Laskowski. M.. Sr. (1971) Biochim. Biophys. Acta 240, 443-447. Wang, S. Y. (1968) in Methods in Enzymology (Grossman, L.. and Moldave. K.. eds.). Vol. 12. pp. 178-184, Academic Press. New York.

P. CASHION M. A. HOLDER-FRANKLIN J. MCCULLY M. FRANKLIN Microbiology Department Uni\lersity Fredericton. Receiteed

Research Laborcrtoyv ofBiology of New Brunswick News Brrrns~vick. E3B 5A3 Cunada November 22. 1976; accepted April

18. 1977